This study was aimed to edit myostatin (MSTN) gene of Mongolian bovine fetal fibroblasts using CRISPR-Cas9 system,build an unmarked Mongolian bovine cell line which its MSTN gene had been knocked out,and provide experimental material for cultivating muscular Mongolian bovine.Skin fibroblast cell lines were established on 45 day of gestation in Mongolia bovine fetus by tissue adherent method,at the same time,a gRNA target site in exon 2 region was selected,and three high scores sgRNAs sequences which designed in online software were chosen,unmarked Cas9/gRNA vector was constructed by kit method,its activities was tested by T7E1 digestion,the active vector was transfected into Mongolian bovine fetal fibroblasts by electric transfer,and seed transfected single cells in 96-well plates by infinite dilution method and pipette method,which DNA was extracted,and obtained positive cells by PCR amplification and sequencing verification in the end.The results showed that we constructed three unmarked CRISPR-Cas9 vectors and found that only one vector was active.A total of 96 single cell strain were obtained by screening single cells which after electrical transfection,and only one single cell strain was found that occur single-base mutation after sequencing analysis,which made MSTN gene could not encode normal protein.This study established successfully a MSTN gene inactivated cell line,which could be used as a donor cell for subsequent somatic cell nuclear transfer for the production of unmarked Mongolian bovine with MSTN gene knockouted.It was of great significance to cultivate Mongolian bovine with high meat production rate and biosecurity.

In order to reveal the pathogenesis of brucellosis and develop a new molecular vaccine of brucellosis,the ovine lymphocyte antigen DRB1 gene was designed to be displayed on yeast cell surface using yeast surface display system,explored a platform for screening antigen peptides and developing a vaccine against sheep brucellosis.A pair of primers were designed according to the published sequences of sheep MHC Ⅱ DRB1 gene in GenBank.DRB1 gene was cloned using sheep cDNA extracted from spleen tissue as template,the PCR product was inserted into the cloning vector pEASY-T1 by double enzyme digestion,then named pEASY-T1-DRB1.Ligated into the yeast surface display plasmid vector pYD1 by double enzyme digestion,the recombinant plasmid pYD1-DRB1 was successfully constructed on the surface of yeast.Point mutation was made at the end of DRB1 gene exon 2 in order to make restriction enzyme cutting site using pEASY-T1-DRB1 as a template and the specific primers of exon 2 was designed according to mutated sheep DRB1 gene sequence.DNA pooling of 500 sheep samples was made and amplified the sequence of DRB1 gene exon 2 which was digested by double enzyme,then connected to surface display restructuring mutation carriers pYD1-DRB1-TB by the same double enzyme digestion,the yeast surface display libraries were constructed.E.coli DH5α competent cells were transformed and library capacity was calculated.It was transformed into yeast EBY100 cell,after galactose induced,it was detected that DRB1 gene library on the yeast cell surface under the fluorescence microscope by immunofluorescence assay.The results showed that DRB1 gene was successfully integrated into yeast genome,the library capacity of librariy was more than 10⁵,and DRB1 gene library was successfully displayed on the surface of yeast cells.The results suggested that this library could be used for screening of Brucella antigen peptide.

In order to analyze the characteristics of Cofilin2 gene and its expression in tissues of Sansui ducks,the specific primers were designed according to the sequence of Gullus Cofilin2 gene in GenBank,and amplified for Cofilin2 gene in Sansui ducks,then the Cofilin2 gene characteristic was analyzed using bioinformatic softwares,and the mRNA level of Cofilin2 gene in different tissues of Sansui ducks were detected with Real-time quantitative PCR.The results showed that the length of Cofilin2 gene was 498 bp in Sansui ducks,which encoded 166 amino acids.The identities of Cofilin2 gene in Sansui ducks were 94.4%,91.6%,91.6%,87.6%,77.7%,70.5% and 69.5% shared with that of Gallus,Macaca mulatta,Bos taurus,Mus musculus, Salmo salar,Homo sapiens and Sus scrofa,respectively.The phylogenetic tree analysis result showed that Cofilin2 gene maintained a highly conservative among different species.The secondary sructure of this encoding protein was consistes of alpha helix,extended strand,beta turn and random corn,and the curved spiral in tertiary structure.Real-time quantitative PCR detection results showed that there were different degrees expression of Cofilin2 gene in heart,liver,spleen,lung,kidney,brain,thymus,duodenum,leg muscle and bursa of Fabricius,the expression of Cofilin2 gene was the highest in heart,and that was the lowest in bursa of Fabricius.The results provided some basic data for the molecular mechanism of Sansui duck diseases.

This study was aimed to establish a prokaryotic expression system of mink growth hormone (mGH),explore a scale production method,and get specific anti-mGH serum from the immune rabbit.In this study,the prokaryotic expression vector pET28b-mGH was transferred into Rosetta(DE3)^TM pLysS competent cells and the inclusion body protein was got successfully which induced by IPTG.The protein was dissolved and refolded with 8 mol/L urea solution,the recombinant protein was obtained by Ni-NTA resin affinity chromatography purification.The recombinant protein was confirmed by Western blotting and MALDI-TOF-MS methods,the purity and concentration of mGH fusion protein were determined by SDS-PAGE and BAC methods,respectively.In the case of refolding mGH protein,the immune antigen was prepared with a complete fluoride adjuvant or incomplete fluoride adjuvant.Three New Zealand White rabbits were immunized by subcutaneous multipoint injection the immune antigen,the first dose was 600 μg per rabbit,the second dose was 600 μg per rabbit,the third dose was 400 μg per rabbit.We took the rabbits' blood sampling on 10 day after the third immunization,and then separated antibody serum,the anti-mGH serum specificity and antibody titer were identified by Western blotting and indirect ELISA,respectively.The result showed that the purified protein was mGH fusion protein,SDS-PAGE results showed that the purity of the mGH fusion protein was more than 90%,and the BCA result showed that the concentration reached to 669 μg/mL.The rabbit anti mGH serum was specificity,the anti-mGH sera with a titer of over 1:256 000.The results showed that the prokaryotic expression vector pET28b-mGH could express efficiently mGH fusion protein in Rosetta (DE3)^TM pLysS cells,the expressed protein was immunized with New Zealand White rabbits after denaturation,renaturation and purification,which could be used to prepare high titer specific antiserum.

In order to further reveal the regulation function of PDK4 gene in intramuscular preadipocyte cells,this study collected the longissimus dorsi from the 5-day-old Congjiang Xiang pig.Type Ⅱ collagenase was adopted to digest the cells,and intramuscular preadipocyte cells were separated and cultured,and then the morphology were observed.According to the cloning PDK4 sequence,online software was used to design and synthesize three pairs of specific single siRNA and a pair of negative control sequences,annealing products were cloned into pLVX-shRNA2-Puro vector directly.The recombinant plasmids were verified by double enzyme digestion and sequencing.The constructed interference plasmids were transfected into the intramuscular preadipocyte cells of Congjiang Xiang pig.The most effective interference sequences were screened by Real-time PCR.The results showed that the primary culture of intramuscular preadipocyte cells started adherent in about 4 h,and the cell morphology was uniform after 24 h,the cells were firmly adherent.On the 5th day,the adherent cells were monolayer.The results of double enzyme digestion and sequencing showed that the four pairs of siRNA interference sequences designed and synthesized were correctly connected with the vector plasmid,indicating that the PDK4 gene interference vectors were constructed successfully,and the transfection of the intramuscular preadipocyte cells was successful.The results of Real-time PCR showed that the interfering vector could effectively reduce the mRNA expression of PDK4 gene in intramuscular preadipocyte cells with the best interference efficiency of 81.90%.In this study,we successfully cultured the intramuscular preadipocyte cells and constructed the interference vector of PDK4 gene,which laid the foundation for further research on the regulation mechanism of PDK4 gene on lipid metabolism.

To establish a sandwich ELISA for quantitative detection of porcine reproductive and respiratory syndrome virus (PRRSV) soluble receptors,porcine sialoadhesin (Sn) soluble receptor Sn4D-Fc was purified from the recombinant adenovirus-transduced PK-15 cell and used as the standard antigen,mouse anti-porcine Sn serum was used as the first antibody and biotin-labeled rabbit anti-porcine Sn polyclonal antibody as the second antibody.The established sandwich ELISA was validated by detection of the soluble receptor in the serum samples of rAd-Sn4D-Fc-injected pigs.The results showed that the Sn4D-Fc standard antigen was purified to a purity of 94.3%,and was able to be recognized by anti-Sn serum.By using 5 μg/mL of the first antibody and 1:4 000 of second antibody,the detection limit of established sandwich ELISA was up to 0.4 ng/mL without cross reaction to control antigen.The Sn4D-Fc soluble receptor could be detected in the rAd-injected pigs with the highest expression level of 6.54 ng/mL and the longest duration of 15 d.These data suggested that the established sandwich ELISA was usable for quantitative detection of PRRSV soluble receptors in vitro and in vivo.

In order to obtain strains with higher cellulose-degrading ability isolated from Sika deer (Cervus nippon) rumen,the fresh rumen liquid was collected from Sika deer,and CMC-Na medium was used to isolate and screen strains,and the morphological,physiological and biochemical characteristics and 16S rDNA gene sequence were analyzed to identify the taxonomic position of the strains.Moreover,the cellulose producing conditions of the strain was preliminarily studied by DNS method,and the characteristics of cellulose production with different substances as substrates were studied in order to provide theoretical basis for the application of strain.The results showed that N-11 strain was a highly efficient cellulose-degrading bacteria,which was identified as Bacillus cereus.The properties of the cellulose production showed that when the fermentation temperature ranged from 30 to 45℃ (optimum at 40℃),pH was 6.0 to 7.0 (optimum at 6.0),and carbon source content was 2% to 5% (optimum at 5%),it had the higher enzyme activity.In addition,when cultivated for 36 h,the peak of cellulose production was reach which CMC cellulose activity of N-11 strain was 12.563 U/mL and FPA cellulose activity was 12.414 U/mL.The relative cellulose activity still retained above 80% at 20 to 50℃ or pH 6.0 to 8.0 when cultivated for 1 h.These results indicated that N-11 strain had a high cellulose productivity,and it might have some potential for industrial applications.

The biological characteristics of eleven Bacillus strains from rumen were studied,including the activity of produced enzyme,the endurance to acid and bile salt,the activity of antibacteria and the compatibility.3,5-dinitrosalicylic acid colorimetric method (DNS method) was used to determine the enzyme activity,acid resisting test and bile salt resisting test were conducted by plate spreading method,antibacterial activity was determiend by Oxford cup method,and while indoor compatibility experiment was studied by filter paper method.The results showed that the activity of cellulase produced by T-2,T-3 and T-10 were higher;T-2,T-5,T-7,T-8 and T-9 had stronger acid resistance;T-4,T-5,T-9 and T-11 had stronger ability of bile salt-tolerance;T-4 and T-7 had stronger antibacterial activity against Vibrio parahaemolyticus,and T-7 had strongest antibacterial activity against Aerogenes,T-5,T-9 and T-10 had stronger antibacterial activity against Escherichia coli and T-3 had strongest antibacterial activity against Sarcina lutea.Indoor compatibility experiments showed that there were antagonistic effects among some strains of eleven Bacillus strains.The combinations of the selected strains for bacteriostat were T-3,T-4,T-5;T-3,T-4,T-10;T-4,T-5,T-10;T-3,T-7,T-10.The combinations of the selected strains for higher cellulase activities were T-2,T-3,T-5;T-2,T-3,T-10;T-2,T-5,T-10;T-3,T-5,T-6;T-3,T-5,T-10.The results could provide some references for the research and development of animal feed.

The objective of this study was to investigate the effect of fermented corn gluten meal (FCGM) on in vitro fermentation characteristic and microorganism population.Three Holstein cows cannulated permanent ruminal fistula with a body weight of (600±25) kg were used to provide rumen fluid.The substrate was total mixed ration (TMR),and FCGM was added to buffered rumen fluid with concentrations of 0,0.3,0.6 and 0.9 g/L,respectively (dry matter basis) with three repetitions per group.Gas production was recorded at 12,24,36 and 48 h of incubation.Moreover,the pH,in vitro dry matter disappearance (IVDMD),cellulase activity,NH₃-N,volatile fatty acids (VFA) and microbial protein concentration of fermentation liquor were determined after 12,24 and 48 h of incubation.After incubation for 24 h,the relative abundance of microorganism was determined.The results showed as followed:①Compared with the control group,adding different levels of FCGM could significantly or extremely significantly improved the gas production (except 12 h),slow gas production,potential gas production and the effective gas production rate (P < 0.05;P < 0.01).②Compared with the control,adding different levels of FCGM could significantly or extremely significantly decreased the pH of the culture medium and increased the content of bacterial protein,VFA and NH₃-N,cellulose enzyme activity and IVDMD (P < 0.05;P < 0.01),and that of 0.9 g/L FCGM group were highest.③The relative expression of the Ruminococcus albus,Ruminococcus flavefaciens,Fibrobacter succinogenes,Streptococcus bovis,Prevotella,Butyrivibrio fibrisolvens,Ruminobacter amylophilus,fungus and protozoa in 0.6 and 0.9 g/L FCGM groups were significant increased (P < 0.05),with that in 0.9 g/L FCGM group were highest.However,the relative abundance of methanogens was significantly decreased compared with the control group (P < 0.05) and that was the lowest in 0.9 g/L FCGM group.In conclusion,the results demonstrated that FCGM could improve gas production,VFA,NH₃-N and microbial protein concentration,increase the relative abundance of cellulolytic bacteria,starch degradation bacteria,proteolytic bacteria and protozoa,improve rumen fermentation,promote the growth of some microbes in the rumen,decrease the relative abundance of methanogens and regulate the population structure of rumen microbe.The optimum dose of FCGM was 0.9 g/L.

This experiment was conducted to investigate the effect of TMF with different fermentation agents on in vitro fermentation characteristics of rumen microorganisms in Yanbian Yellow cattle.The experiment was divided into four experimental groups:CON group (control group),T1 group (adding NFK),T2 group (adding RJ) and T3 group (adding CT-10) with three replicates per group.The TMR with different fermentation agents was loaded into the fermentation bag and sealed at room temperature for 7 d.And then,the ruminal DM disappearance rate was determined by Nylon Bag Technique and the fermentation characteristics were measured by in vitro gas production technique.The results showed that:With the prolonging of fermentation time,the different fermentation agents could significantly affect the rumen DM disappearance rate,pH,gas production,gas composition,volatile fatty acid (VFAs) and microbial protein content (MCP) in different extent.At 8 h,compared with the control group,the rumen DM disappearance rate of T1 and T3 groups were significantly increased (P < 0.05),but there was no significant difference among the groups at the other time periods (P > 0.05).The pH of culture medium in each experimental group decreased gradually,and the pH in T2 group was significantly lower than that in control group when cultivated for 1 h (P < 0.05).The content of VFAs in each experimental group showed an upward trend.At 12 h,the content of NH₃-N in T3 group was highest (5.79 mg/dL),while T2 group was lower than the control group (P > 0.05).The content of MCP increased first,then decreased and then increased.At 1,3 and 12 h,the MCP contents of the experimental groups were significantly lower than that in control group (P < 0.05),but there was no significant difference at 6 and 9 h (P > 0.05).After 1 h cultivation,different fermentation agents had no significant effect on gas production (P > 0.05).At 9 and 12 h,the control group had the highest gas production,and significantly higher than experimental groups (P < 0.05).The changes of H₂ production in group tended to be consistent,showing the fluctuation state.With the prolongation of incubation time,the content of CH₄ and CO₂ showed a rising trend and were significantly affected by different fermentation agents (P < 0.05).In conclusion,we found that the DM disappearance rate of T3 group was higher,and the ferment effect of CT-10 was equivalent with T2 group adding RJ which could efficiently improve the rumen fermentation characteristics,so CT-10 could be used into IMF of Yanbian Yellow cattle.

This experiment was conducted to investigate starch degradation characters and analyze the effective degradation rate and real-time degradation rate in the rumen of 5 silages,including alfalfa silage,whole wheat silage,oat silage and 2 whole corn silages,in order to estimate starch degradation rate according to degradation rate of a single time and update the relevant data of starch in silages for Chinese Dairy Cattle Nutrient Requirements.4 Holstein cows with permanent ruminal cannulas were selected and nylon-bag technique was used to determine the dynamic and effective degradation rate of starch.The time points of nylon-bag technigue were 4,8,12,24,30,36,48 and 72 h in the rumen of dairy cows with 4 parallels for each cow.The results showed as follows:①The degradability of starch increased gradually over time,and tended to be steady after 24 h.The effective degradability of starch of whole wheat silage was significantly higher than that of oat silage,alfalfa silage and whole corn silage (P < 0.05),which was decreased in turn.②There was a strong correlation between effective degradability and dynamic degradability of starch at 8 h with R² was 0.9876.It was viable that rumen degradability of starch at 8 h could be used to determine starch degradability of silage in the rumen of dairy cows.The results of the test had reference value for designing diet of dairy cows.

The purpose of this experiment was to study the effects of aqueous extract of fermented Astragalia and Glycyrrhiza on growth performance,immune organ indexes and meat quality of 817 broilers.90 one-day-old healthy 817 broilers were chosen and randomly divided into three groups by one-factor completely random design with 3 repetitions each group and 10 broilers per repetition.The broilers in control group were fed basal diet + fresh water,that in groups Ⅰ and Ⅱ were fed basal diet + unfermented or fermented Astragalia and Glycyrrhiza with a dilution ratio of 1:2 000,respectively.On the 21 and 42 d,the average daily gain,F/G,the immune organ indexes and the meat quality such as muscle nutrient content,the meat color and drip loss were all measured.The results showed that during 1 to 42 days,compared with control group, the average daily gain of groups Ⅰ and Ⅱ increased by 5.09% (P < 0.05) and 12.72% (P < 0.05) and F/G was reduced by 10.84% (P < 0.05) and 22.09% (P < 0.05),respectively.At the age of 21 days, compared with control group,the spleen and bursa of Fabricius indexes of groups Ⅰ and Ⅱ were raised by 4.40% (P > 0.05),13.19% (P > 0.05) and 32.39% (P < 0.05),37.09% (P < 0.05),respectively.At the age of 42 days,the spleen and bursa of Fabricius indexes were raised by 31.97% (P < 0.05),50.82% (P < 0.05) and 16.25% (P > 0.05),38.13% (P < 0.05),respectively.The liver index of groups Ⅰ and Ⅱ at 21 and 42 days were higher than control group.At the age of 42 days, compared with control group,the moisture content (P < 0.05), crude fat content (P < 0.05), a^* value and inosinic acid content of breast muscle in groups Ⅰ and Ⅱ were all raised, b^* value and drip loss were declined.The moisture content,crude protein content,a^* value and inosinic acid content (P < 0.05) of thigh muscle in groups Ⅰ and Ⅱ were higher,both b^* value and drip loss (P < 0.05) were declined.The differences in the breast and thigh muscle of L^* values among the groups was not significant (P > 0.05).In conclusion, aqueous extract of fermented Astragalia and Glycyrrhiza could improve the growth performance,immune indexes and meat quality of 817 broilers,and the effect was better than the unfermented group.

This experiment was conducted to investigate the effect of Chinese herbal-probiotics-bile acids compound on production performance,egg quality and immune indexes of laying hens.160 495-day-old Hy-Line Brown hens in healthy were chosen and divided into 2 groups with 4 replicates per group and 20 hens per replicate.The hens in control group were fed a basal diet and that in the experimental group were fed the basal diets supplemented with 0.2% Chinese herbal-probiotics-bile acids compound (Astragalus,Angelica ainensis,Lactobacillus,bile acids,etc).The preliminary trial lasted for 1 week,and the experimental period lasted for 3 weeks.The results showed that the laying rate, average egg weight and average daily feed intake of the experimental group were increased by 4.97%,1.71% and 0.49% than that of the control group and feed/egg of the experimental group was decreased by 6.33% than that of the control group.The experimental group of protein height,yolk color and Haugh units were higher than the control group by 9.87%,6.64% and 3.94%.The IgA and IgG of experimental group were increased by 2.38% and 1.51% than that of the control group,respectively.The quantities of Lactobacillus in feces of the experimental group were significantly higher than that in the control group (P < 0.05),while the E.coli in the experimental group were significantly lower (P < 0.05).In conclusion,the addition of Chinese herbal-probiotics-bile acid compound preparation for the laying hens could improve the production performance and egg quality,and have a certain effect in improving immunity and disease resistance.

To evaluate the possibility of subtituting in-feed antibiotics with fermented Chinese herb residues (CHR) as feed additive,120 three-way cross piglets weaned at 21 days of age were randomly assigned into 4 treatment groups with 6 replicatins with 5 piglets each replication,representing control group,CHR group,fermented CHR group and antibiotic (colistin sulfate) group.At the 28th day,1 pig was randomly selected from each replication,and the blood samples were collected from the anterior vena cava in the morning before feeding.The serum was isolated for testing the biochemical parameters,antioxidant and immunological indexes.The results showed that feed gain ratio (F/G) in fermented CHR group were significantly lower than that of the control and antibiotic groups (P < 0.05); Total protein and albumin of the fermented CHR group were significantly higher than that of the antibiotic group (P < 0.05); Triglyceride and total cholesterol of the fermented CHR group was significantly lower than that of the control group (P < 0.05); Glutathione contents,catalase and superoxide dimutase activities of fermented CHR group were significantly higher than that of the control group (P < 0.05); The contents of immunoglobulin G(IgG),IgA and IgM of CHR and fermented CHR groups were significantly higher than those of control and antibiotic groups (P < 0.05).These findings suggested that the fermented CHR could present certain roles in regulating plasma lipid concentration and improved the antioxidant capacity and immunity in weaned piglets,it could be used as the substitute for antibiotics in piglets' feed.

The experiment was aimed to analyze the difference of meat quality traits between Simmental beef cattle and Wagyu hybrid population,and provide theoretical instructions of biological mechanisms of meat traits and beef cattle breeding process.Phenotypic data on meat quality traits of Simmental beef cattle and Wagyu hybrid population were collected,including eye muscle area,muscle shear force,water-holding capacity,pH,meat color,fat color and marbling score,the correlation analysis and comparison analysis between these two breeds were conducted.The results showed that fat color,water-holding capacity and muscle shear force existed highly correlation among each other,as well as meat color with fat color and pH,there was significant correlation between meat color and pH (P < 0.05).By contract,water-holding capacity showed a negative correlation with pH,meat color and marbling score.We conducted comparisons in traits between two populations,and found that marbling score,eye muscle area,and pH value of Wagyu hybrid cattle was extremely significant higher than those of Simmental beef cattle (P < 0.01).In terms of gender,the marbling score,pH and muscle shear force of Simmental beef cow was extremely significantly or significantly higher than those of Simmental beef cattle (P < 0.01;P < 0.05),the meat quality and muscle shear force of Wagyu hybrid cow was significantly or extremely significantly higher than those of Wagyu hybrid cattle (P < 0.05;P < 0.01).As a conclusion,Wagyu hybrid cattle performed better than Simmental beef cattle in meat quality traits.This study gives instruction of exploration of meat quality traits,and references of beef cattle breeding.The meat quality of Simmental beef cow was better than that of Simmental beef cattle,and the meat quality of Wagyu hybrid cow was worse than that of Wagyu hybrid cattle.

This study was aimed to investigate the SNP of eukaryotic translation elongation factor 1 delta (EEF1D) gene in Chinese Horstein cow and it's correlation analysis with milk production traits.Sequenom MassARRAY SNP genotyping method was applied to detect the polymorphisms of EEF1D gene in 1 252 Chinese Holstein cows,and the association of EEF1D genotype and genotype combination with milk production traits were analyzed.The results showed that two SNPs were identified,including EEF1D-1 and EEF1D-3 in 5'-flanking region.Two genotypes were found in EEF1D-1 and three genotypes were found in EEF1D-3.Chi-square test showed that EEF1D-1 locus deviated from Hardy-Weinberg equilibrium in Chinese Holstein cow (P < 0.05),but EEF1D-3 locus did not deviate from Hardy-Weinberg equilibrium in Chinese Holstein cow (P > 0.05).Polymorphic information content (PIC) in EEF1D-1 and EEF1D-3 loci were 0.10 and 0.28,showing low polymorphism and moderate polymorphism.In the EEF1D-1 locus,it had extremely significant and significant effect on milk fat percentage and milk protein percentage (P < 0.01) and 305 d milk yield traits (P < 0.05),respectively.In the EEF1D-3 locus,it had extremely significant effects on 305 d milk yield traits,milk fat percentage and milk protein percentage (P < 0.01).The GG-AG and GG-GG genotypes combination in Chinese Holstein cow had significantly greater milk fat percentage than those with GG-AA (P < 0.05).Therefore,EEF1D gene might be useful as molecular markers for milk yield traits in Chinese Holstein cow breeding.

The study was aimed to analyze the gene polymorphism of bone morphogenetic protein receptor-1B (BMPR-1B) gene in six sheep population,which would provide a theoretical basis for marker-assisted selection and breeding for prolificacy in sheep.The blood samples of a total of 381 individuals from six sheep breeds (Xinji Fine wool sheep,Northeast Fine wool sheep,Chinese Merino (Mulley type) sheep,Dorper×Han crossbred sheep (F1),Dorper×Han crossbred sheep (F3) and White Dorper sheep) were used to study the polymorphism of BMPR-1B gene by PCR-RFLP.Secondary structures of proteins were predicted using DNAStar,gene frequency,genotype frequency,Hardy-Weinberg equilibrium and genetic diversity parameters (heterozygosity (He),homozygous (Ho),effective allele (Ne),polymorphic information content (PIC)) were calculated by Excel 2003 software.The results showed that there were no polymorphism in Xinji Fine wool sheep,Northeast Fine wool sheep,Chinese Merino (Mulley type) sheep and White Dorper sheep.The polymorphism in Dorper×Han crossbred sheep (F1) and Dorper×Han crossbred sheep (F3) existed three kind of genotypes (++,B+ and BB).According to χ² text,Dorper×Han crossbred sheep (F1) were not in Hardy-weinberg equilibrium,and Dorper×Han crossbred sheep (F3) were in Hardy-weinberg equilibrium.The PIC were 0.311 and 0.264 in the Dorper×Han crossbred sheep (F1) and Dorper×Han crossbred sheep (F3),which both meaned moderate diversity.The BMPR-1B gene might be a major gene which affected the prolificacy trait of Dorper×Han crossbred sheep (F1) and Dorper×Han crossbred sheep (F3).

This study was aimed to investigate the relationship between genetic variations of PRR5L gene with fat traits and carcass traits in chicken.Yellow Dwarf chicken were chosen as research material and primer was designed according to DNA sequence of chicken PRR5L gene (accession No.XM_015287209.1),the single nucleotide polymorphisms(SNP) of PRR5L gene 5'UTR region was detected by PCR direct sequencing.The results showed that there were three polymorphic sites of C480G,G514A and A579G,and all polymorphic sites showed three genotypes.The Chi-square test results showed that the C480G and A579G loci were in Hardy-Weinberg equilibrium (P > 0.05),the G514A locus was not in Hardy-Weinberg equilibrium (P < 0.05).In the fat traits,there were no significant differences among all genotypes of three polymorphic sites of PRR5L gene in abdominal fat weight,abdominal fat rate and intramuscular fat content (P > 0.05).In the carcass traits,there was significant difference between AG and GG genotypes in the G514A locus (P < 0.05).There was also no significant difference of all genotypes in the measured indexes at the C480G and A579G loci (P > 0.05).Therefore,the G514A locus of PRR5L gene 5'UTR might be a site that affected the growing development of chicken breast,however,we needed to further explored whether this site could be used as a marker for the identification of carcass traits in chicken.

This study was aimed to investigate the polymorphism of bone morphogenetic protein receptor-ⅠB (BMPR-ⅠB) gene and its correlation with the reproductive traits in Large White pigs.The polymorphism of BMPR-ⅠB gene was analyzed by high-throughput sequencing technologiy,the association of different genotypes of BMPR-ⅠB gene with the total number of births,alive births,deaths and deformities using the least square method by SPSS statistical software.The results showed that the polymorphism was not detected in BMPR-ⅠB gene 746A > G and 852G > A loci,in the 804G > C locus,the frequency of GG genotype was dominant genotype,and G was the dominant allele; In the 960C > T locus,the frequency of genotypes CC,TT and CT were higher in American,British and Canadian Large White pigs,respectively.The results of χ² test showed that the 804G > C and 960C > T loci were fit with Hardy-Weinberg equilibrium in the different Large White pigs lines (P > 0.05).There were no significant differences in the effects of 804G > C locus on the number of total births,alive births,still births,deformity and other reproductive traits of the three different lines of Large White pigs (P > 0.05).The number of stillbirths with TT genotype in American Large White pigs were significantly lower than that of CC genotype in 960C > T locus (P < 0.05),which was decreased by 1.027; The total litter size of the British Large White pigs with TT genotype was significantly higher than that of CC genotype (P < 0.05),and extremely significantly higher than that of CT genotype (P < 0.01),which was in-creased 1.976 and 2.118,respectively; The number of live births in Canadian Large White pigs with TT genotype was significantly higher than that of CC genotype (P < 0.05),the stillbirth and deformity of TT genotype were significantly lower than that of CC genotype (P < 0.05),which were decreased 0.707 and 0.337,respectively.The TT genotype in 960C > T locus could be used as the marker genotype for reproductive traits of three different lines of Large White pigs.

Baoshan pig,Diqing Tibetan pig,Gaoli Gongshan pig,Lijiang pig,Mingguang Small-ear pig,Diannan Small-ear pig,Saba pig,Dahe pig,Zhaotong pig and Dahe Black pig are ten local pig breeds with excellent breed characteristics in Yunnan province,China.To assess their genetic diversity and genetic structure,a total of 549 unrelated ear tissue samples from the ten indigenous pig breeds were genotyped using 15 fluorescence-labeled microsatellite DNA markers of 14 chromosomes in the ArkDB database.The number of alleles (Na),effective allele number (Ne),Shannon's information index (I),apparent heterozygosity (Ho),expected heterozygosity (He), F-statistics(Fis,Fit,Fst),gene flow (Nm),population genetic distance and other related parameters and polymorphic information content (PIC) were determined,cluster tree was built by NTsys 2.10,and STRUCTRUE 2.3.3 software was used to evaluate the genetic structure of the population.The results showed that the Na of 15 microsatellite loci was 293,ranging from 5 to 38 alleles per locus.The mean number of alleles and effective number of alleles per locus were found to be 19 and 8.3682,respectively.The average Shannon's information index,observed heterozygosity,expected heterozygosity and polymorphism information content of 10 indigenous pig populations ranged from 1.4374 to 2.0317,0.6389 to 0.7756,0.7021 to 0.8281,0.6647 to 0.7993,respectively.The average inbreeding coefficient within accessions (Fis) and overall inbreeding coefficient (Fit) per locus ranged from -0.0678 to 0.4594 and 0.0618 to 0.5567,respectively.The moderate degree of genetic differentiation index (Fst) ranged from 0.0713 to 0.1801 with an average of 0.1101,which demonstrated that approximately 11.01% of genetic variations were among populations and 88.99% of genetic differences were within populations.The high level of gene flow at each locus was discovered from 1.6392 to 3.2551,with a mean Nm of 2.0214.The phylogenetic relationships of pig breeds were constructed using UPGMA method based on Nei's genetic distance.The UPGMA dendrogram showed that the Gaoli Gongshan pig and Diqing Tibetan pig jointed as one cluster.And then joined with Baoshan pig,Lijiang pig and Mingguang Small-ear pig breeds as a big group.STRUCTURE analysis confirmed the analysis result observed in the UPGMA tree.The study indicated that the genetic diversity was abundant in ten Yunnan local pig breeds.There were close inbreeding phenomenon in the ten breeds.The level of genetic differentiation among populations was moderately,with a certain amount of gene flow.

Nucleotide binding oligomerization domain-like receptors (NLRs) family is similar to Tol-like receptors (TLRs) family,which is well conserved,exists not only in plants but also in animals,and plays important roles in the innate immune system.NLRs family comprises many subfamilies,in which NACHT,LRR and PYD domains-containing protein 7 (NLRP7),a member of NLRP subfamily, which is expressed in a variety of immune cells and immune organs,and participates in cell growth,proliferation,differentiation,apoptosis and immune response(inflammation).NLRP7 was not only expressed in the immune system but also expressed in the mammalian testis and ovary of the reproductive system as well as early embryos,and NLRP7 mutations usually cause the human family recurrent hydatidiform mole disease,resulting in pregnancy failure.Recent studies have found that NLRP7 is a maternal imprinted gene,and the abnormal imprinting status of NLRP7 is closely related to the abnormal embryonic development.Although the hydatidiform mole disease has not been found in large animals such as pigs,cattle and sheep,many unexplained abortion,stillbirth and so on may be related to the mutation of NLRP7.NLRP7 has degraded in mice and other rodents during the evolution process,so the current studies of NLRP7 mainly focus on the primates.This paper reviews the latest research progress about NLRP7 in order to provide reference for the exploration of mammals reproductive disorders and other diseases.

In this study,the body weight and body size traits were measured and analyzed by principal component analysis of 5-week-old different feathers Kirin hens to understand the inherent relationship between the body weight and body size traits of different feather Kirin hens and provide the basic information for their selection and breeding.50 one-day-old White feathers Kirin hens and 50 one-day-old Yellow feathers Kirin hens were selected in this experiment and were divided into 2 groups with 5 replicates per group,10 hens per replicate,the body weight and body size were measured at 5 weeks old under the same feeding conditions.The results showed that the variation coefficient of tibia length of White feather Kirin hens and tibia girth of Yellow feather Kirin hens were large which were breeding potential traits.The correlation between body weight and tibia length of White and Yellow feather Kirin hens were both extremely significant (P < 0.01) with the correlation coefficient was 0.931 and 0.944,respectively.And there were significant correlation between body weight and sternal length,tibia girth,tibia length and sternal length (P < 0.05).The 7 body measurement traits of different feathers Kirin hens could simplified to 4 principal components that occupied 94.784% in White feathers Kirin hens and 94.809% in Yellow feathers Kirin hens of total information amount,and each of 4 principal components for information were not identical and could reflect Kirin hens' bodily form feature.In conclusion,there was a significant correlation between body weight and body size traits of different feathers Kirin hens under free feeding conditions.And it suggested that the selected 4 principal components could reflect Kirin hens' bodily form feature and provide the basic information for selecting and breeding in different feathers Kirin hens.

In order to understand the virulence factor,gene mutation and evolution of Streptococcus suis serotype 9 strain from Guangdong,the cerebrospinal fluid,joint fluid,spleen,liver,lung and lymph node were collected for bacterium culture and biochemical identification,and PCR method was used to identify its serotype and the partial virulence genes.cps9D,gdh,gapdh and orf2 genes were sequenced and analyzed,and phylogenetic trees were constructed.The results showed that the isolate was gram-positive chain cocci,β-hemolysis in blood agar plate,and could ferment most sugars,and amplify cps9D gene successfully,including important conservative gene gdh and virulence genes gapdh and orf2,which proved that it was Streptococcus suis serotype 9.The nucleotide homologies of cps9D,gdh,gapdh and orf2 genes of the isolate with different strains at home and abroad in GenBank were 95.9% to 100.0%,98.6% to 99.8%,98.7% to 99.6% and 95.5% to 99.9%,respectively.It showed that the isolate had higher nucleotide homology with other Streptococcus suis serotype 9 strains at home and abroad,which was basically consistent with the epidemic strains at home and abroad,and there was not much variation.Phylogenetic tree showed that the isolate had high homology and close relationship with Streptococcus suis isolates from different sources at home and abroad,and they were closely related.

In order to investigate porcine circovirus type 2 (PCV2) infection and molecular biological characteristics from pig farms in Tianjin and its surrounding areas,the PCR method was used to investigate clinically suspected PCV2 infection case and isolate PCV2 using dulac cells,and the ORF2 gene of PCV2 were cloning,sequencing,and comparing with ORF2 gene of the PCV2 reference strains published in GenBank.The results showed that the positive rate of PCV2 was 25.0% to 38.1% in different regions,the positive rate in summer was obviously higher than that in the other three seasen,reaching 44.0%,and the positive rate of fattening pig was the highest,while that of milking sows was the lowest among different ages pigs.24 strains PCV2 were isolated successfully,the homology of ORF2 sequence was 87.8% to 99.2% with the reference strains,and the homology of 24 isolates strains was 89.4% to 100.0%.The phylogenetic tree analysis showed that 24 isolates belong to two branches.Propagation characteristics of a PCV2 isolate (the Y16155-1 strain) in vitro showed that the virus DNA could be detected at 6 h post inoculation and its numbers reached peak at 72 h post inoculation,the viral titer was 10^-4.3 TCID₅₀/0.2 mL.This result laid a theoretical and scientific foundation for further study molecular epidemiology of PCV2 and the prevention and control of porcine circovirus related diseases in this area.

This study was aimed to understand the contamination of Escherichia coli at the process of beef cattle slaughter in the region of Yili,Xinjiang,and to detect the infection of non-O157 pathogenic Shiga toxin-producing E.coli (STEC).The fecal samples of beef cattle and swab samples of carcass surface in a certain designated slaughterhouse in Yili were collected,and the samples were performed with an isolated identification of E.coli,PCR detection of virulence genes (eae,stx1 and stx2), O157 identification (rfbE),ERIC-PCR genotyping and mouse pathogenicity test.The results showed that 42 strains of E.coli were isolated and identified out from 45 samples,and the isolation rate was 93.3%.Among them, the virulence gene of eae was not detected,and two E.coli strains simultaneously programmed the virulence genes of stx1 and stx2,and the detection rate was 4.8%.All the identified strains were non-O157 type.Genotyping test of ERIC-PCR revealed that the two strains of non-O157 STEC were very similar and closely related to each other.The mice were injected with intraperitoneal injection,and started to die after 6 h of bacterial attack.It showed that there was a presence of intestinal hemorrhage,its liver,spleen and kidney were found to have obvious hemorrhage and enlargement,meanwhile the anatomical control mice showed no sign of anomaly,indicating that the strains had a certain pathogenicity.In conclusion,E.coli contamination was exist in the process of slaughtering of beef cattle.The non-O157 STEC in the faeces had contaminated the carcass,and the environmental health of key link of slaughter and processing of beef cattle should be strengthened.

The objective of this trial was to determine the growth curve of Enterococcus faecalis that caused encephalitis in lambs and find a rapid and accurate method to determine the number of Enterococcus faecalis at different growth stages,and objectively evaluate its toxicity and effect on the brain tissue of mice.Plate counting method and D_λ value method were used to analyze the growth curve of Enterococcus faecalis. The relationship between the absorbance (D_{600 nm}) and the live bacteria (CFU) measured by plate colony counting method at the appropriate time period was explored.According to the data analysis,mice were infected with Enterococcus faecalis,and the death of mice was recorded.Finally,the Karber method was used to calculate the median lethal dose (LD₅₀) of the mice infected with Enterococcus faecalis.The mice were infected with the dose of LD₅₀,the brain tissues of the dead mice were collected in time and all the mice without death were sacrificed 72 h after infection,and the brain tissues were collected,too.Some of them were stained with smear and the pathological sections were made.Pathological changes were observed.PCR was used to identify bacteria.The results showed that the growth curves of Enterococcus faecalis were basically the same in both methods,the growth of Enterococcus faecalis was rapid at 2 to 8 h for logarithmic growth phase,the growth rate was slow for 8 to 14 h for stable period,the number of deaths increased after 14 h for decline phase.The relationship between D_{600 nm} and CFU of Enterococcus faecalis was studied,and the regression equation was established,y=20.769x-1.3422,R²=0.997.The LD₅₀ for the infection mice was 7.77×10¹¹ live bacteria.With this dose to infect mice,we could see gram-positive cocci in both brain tissue smear and staining.PCR result showed that there was a size of 112 bp band.Morphological observation of the brain tissue of mice found that the bacteria could lead to brain tissue congestion,bleeding,the formation of micro-thrombosis,meningeal congestion.The results of this study revealed that the establishment of the relationship between the growth curve and the D_{600 nm} and CFU could provide a Real-time monitoring of the number of Enterococcus faecalis,and provide an important theoretical basis for further study on the mechanism of Enterococcus faecalis crossing the blood-brain barrier.

For the diagnosis,prevention and treatment of Apis cerana's bacterial disease in Guizhou,a pathogen was identified in this paper by isolation and purification,morphological observation and biochemical test.The bacterial 16S rRNA gene universal primers were used for amplification and sequencing.The results of the sequencing were analyzed on NCBI.16S rRNA sequences of 30 bacteria of 5 genera with high homology were selected to analyze the nucleotide homology and construct the phylogenetic tree,and animal regression test and drug sensitivity test of the isolate were carried out.The results showed that the isolate was short gram-negative bacilli,and the biochemical tests were initially identified as Hafnia alvei.16S rRNA sequences shared the highest homology with Hafnia,ranging from 97.0% to 99.6%.The phylogenetic tree indicated that the isolate belonged to the branch of Hafnia.The animal regression test showed that the isolate had some pathogenicity to Apis cerana,indicating that the bees were infected by Hafnia alvei.Antibiotic sensitive test showed that the isolate was resistant to penicillin,erythromycin and clindamycin,and was sensitive to amikacin,sulfamethoxazole and ofloxacin.The results of this study provided a reference for the study of honeybee Hafnia infection in Apis cerana and provided reasonable drug advice for the bee farms.

To detect the drug resistance characteristics of Aeromonas sobria to aminoglycosides and tetracyclines antibiotics,10 strains of Aeromonas sobria isolated from different fish were used for detection and analysis.Four aminoglycosides resistance genes (aph(3')-Ⅱa,ant(3″)-Ⅰa,aac(6')-Ⅰb and aac(3)-Ⅱa) and three tetracyclines resistance genes (tetA,tetC and tetM) were detected by PCR.The drug resistance phenotype of six antibiotics were analyzed by Kirby-Baner disc diffusion method.The results showed that the positive rate of the resistance genes of aminoglycosides aph(3')-Ⅱa,ant(3″)-Ⅰa,aac(3)-Ⅱa and aac(6')-Ⅰb in 10 strains of Aeromonas sobria were 20%,30%,0 and 20%,respectively.The positive rate of the resistance genes of tetracyclines tetA,tetC,tetM in 10 strains of Aeromonas sobria were 70%,20% and 60%,respectively.The analysis of drug resistant phenotype results showed that the strains were intermediary to streptomycin,and sensitive to gentamicin,kanamycin,doxycycline,minocycline,while the strains were resistant to tetracycline.The result implied that the identification of Aeromonas sobria had a certain degree of resistance to aminoglycoside and tetracycline antibiotics,which provided a reference for benefits further research of the resistance mechanism of Aeromonas sobria.

To establish the model of pregnant mice infected by Neospora caninum (N.caninum) and understand the pathogenicity of N.caninum on pregnant mice,female BALB/c mice were inoculated intraperitoneally with different doses of N.caninum tachyzoites purified from Vero cells.Then these female BALB/c mice were maintained with male BALB/c mice in the same cage the next day.The clinical symptom and morbidity were observed daily.Basing on N.caninum Nc5 gene,PCR was performed to detect parasitic DNA in brain,liver,spleen and placenta from N.canium-infected mice,placenta wet weight and placental coefficients of pregnant mice were measured,too.The results revealed that the optimal dose to infect mice was 10⁵ tachyzoites.Pregnant mice behaved mental disorders,ataxia,and other clinical symptoms,even death simultaneously.Pathological changes such as congestion,hemorrhage,and enlargement occurred in the major organs of brain,liver,and spleen of model mice.Nc5 gene was detected successively in brain,liver,spleen and placenta of pregnant mice infected with parasites.With the increase of post-infection days,the weight of placenta and the fetus were increasing and the placental coefficient decreased gradually following infection.Significant differences were observed in placental weight and placental coefficient between model group and control group on 12,14 and 16 d (P < 0.05),respectively.The results indicated that the model of pregnant BALB/c mice infected by N.caninum was successfully established which laid the foundation for the study of pathogenesis of N.caninum.

Propionibacterium freudenreichii (P.freudenreichii) is a gram-positive strain,mesophilic,aerotolerant and pleomorphic rods.And it is a safe strain with propionic acid as the final product of fermentation.P.freudenreichii is traditionally used as a starter for aroma production and holes of Swiss cheeses.It can produce vitamin B₁₂ and propionic acid which humans and animals need,and promote the bifidogenic growth and has the anti-inflammatory properties.This paper introduced the safety,unique metabolic pathways and physiological characteristics of P.freudenreichii,and described its application in aroma formation of cheese,probiotic effects and protectants of food and feed in detail.Besides,the paper emphatically introduced the mechanism of aroma in cheese,and summarized the biogenic characteristics from the aspects of regulating the balance of intestinal flora,producing conjugated linoleic acid,probiotic metabolic activity,immunomodulatory effect and Helicobacter pylori antagonism.All of above provide the theoretical basis for full use of the biodiversity of P.freudenreichii.

Brucella is a facultative intracellular parasitic bacterium.It has strong pathogenicity and often causes chronic persistent infections,although there is no typical virulence factor.Brucellosis is listed as a serious zoonosis in the world and directly causes serious losses to the economic income of animal husbandry.It not only hinders social development,but also threatens human health and public health security.The target cells of Brucella is mainly macrophages,it develops a higher strategy to escape the immune cell killing by the immune system,even multiply within the cell and weakens the function of macrophages.The killing and antigen presenting functions of macrophages are partially lost,thereby establishing long-term persistent infection in host cells.This article focuses on the mechanism of Brucella cell viability,and analyzes the regulation of different polarization types of macrophages in the process of Brucella infection,and the role of related inflammatory pathways in the development of inflammation.Brucella intracellular survival not only accommodates the different immune microenvironments during persistent infection,but also accommodates the differences in nutrient availability of target cells during infection.It confirms the key role of immune evasion and interaction with host cell metabolism during chronic infection.It is also explained that NF-κB pathway is a key factor in regulating the balance status of M1/M2 subtype macrophages.In conclusion,the continuous infection of Brucella in host cells is a huge challenge for domestic and foreign scholars.The mechanism and pathogenesis of immune evasion in Brucella need to be studied more specifically.

In order to explore in vivo therapeutic efficacy of antimicrobial peptide NZ2114 against Streptococcus suis (S.suis),the evaluation was performed on the mice model,in which NZ2114 and S.suis serotype 2 CVCC 3928 were used as objects of this study.36 female ICR mice were randomly divided into 6 groups:Blank control group (no infection,intraperitoneal injection of 0.2 mL PBS),negative control group (infection,intraperitoneal injection of 0.2 mL PBS),test groups Ⅰ and Ⅱ (infection,intraperitoneal injection of 0.2 mL 2.5 and 5.0 mg/kg NZ2114,respectively),positive control groups Ⅰ and Ⅱ (infection,intraperitoneal injection of 0.2 mL 7.5 and 15.0 mg/kg ceftriaxone sodium,respectively).The results showed that the survival rates of blank control group and 5.0 mg/kg NZ2114 test group were both 100%,while that of 15.0 mg/kg ceftriaxone sodium positive control group was only 50%.The bacteria load in liver and lung decreased 59.41% (P < 0.01) and 69.19% (P < 0.01) in 5.0 mg/kg NZ2114 test group,and those of 15.0 mg/kg ceftriaxone sodium positive control group decreased 43.94% (P < 0.01) and 19.60% (P < 0.05),respectively.Compared with negative control group,the levels of TNF-α and IL-1β in mice serum decreased 85.83% (P < 0.01) and 56.20% (P < 0.05) in 2.5 mg/kg NZ2114 test group,those of 5.0 mg/kg NZ2114 test group decreased 84.02% (P < 0.01) and 43.86% (P < 0.05),and those of 15.0 mg/kg ceftriaxone sodium positive control group decreased 24.49% and 8.82%,respectively.Moreover,after treatment of 5.0 mg/kg NZ2114 for 1 d,the diffuse interstitial infiltration of inflammatory cells and other inflammatory symptoms in lung tissue obviously reduced,swelling and vacuoles changes produced by liver cells were inhibited and splenic nodules returned to normal,and the basic clinical symptoms were recovered to normal after treatment for 7 d.These results suggested that NZ2114 could effectively increase the survival rate of mice,decrease the bacterial translocation in lung and liver,inhibit the release of TNF-α and IL-1β,and relieve the lung,liver and spleen from acute injury induced by S. suis,which were better than ceftriaxone sodium and indicated that NZ2114 had a good potential as an antibiotic substitute for the treatment of S. suis serotype 2 disease.

In order to help the cow pasture owners at grass-roots level,raise the consciousness of diagnosis for dairy cow foot and hoof disease and improve the treatment level,the paper adopted the uncertainty of knowledge expression method with threshold and applied confidence coefficient and many value logic to make evaluation upon knowledge fuzziness through simulating the veterinary clinical diagnostic thinking.Based on C# development language,ASP.NET operation platform and SQL Server 2008r2 database management tool,the researcher figured out the dairy cow foot and hoof disease auxiliary diagnosis specialist system based on the B/S structure,which helped realize the forward diagnosis,reverse diagnosis and differential diagnosis for dairy cow foot and hoof disease,as well as medical records administration,knowledge query and other functions.With the help of this new system,the diagnosis and treatment level towards dairy cow foot and hoof disease of cow pasture owners at grass-roots level could be improved effectively.

In order to investigate the freshness changes of retailed hot fresh beef at different storage temperature,Four Jinjiang cattle was selected and the longissimus dorsi were placed at 5,15,25 and 35℃ with relative humidity (RH) 80%.The change of pH,L^*,a^*,b^*,aerobic plate count,thiobarbituric acid reactive substances (TBARS),total volatile basic and nitrogen(TVB-N) value during different storage time (0,6,12,24,48,72,96 and 120 h) were analyzed.The results showed that,with the extension of storage time,the pH was decreased first and increased later.L^*,a^* and b^* values were increased first and decreased later,the number of aerobic plate count,TBARS and TVB-N values all increased during the whole storage period at the diffident storage temperature,and the higher the storage temperature,the faster the variation trend of the related indexes would be.In conclusion,in order to ensure the freshness and safety of the retailed hot fresh beef,the shelf life of fresh beef placed at 5,15,25 and 35℃ should not be exceed 96,72,24 and 12 h,respectively.

The study was aimed to develop a rapid and simple enzyme-linked immunosorbent assay (ELISA) method for the detection of milk κ-casein (κ-CN) content and provide technical services for solving the problem of adulterated milk protein.The indirect competition ELISA method and the indirect ELISA method were respectively established by taking the milk κ-CN as the envelope antigen and the enzyme labeled antibody (HRP-IgG) as the detection antibody,and the two detecting methods were analyzed and compared.The results showed that the κ-CN antigen concentration was 2.5 μg/mL,the linear range was 62.5 to 1 000.0 ng/mL,the coefficient of variation was less than 2%,the recovery rate was 98.46% to 101.68% when using the indirect competitive ELISA method; The κ-CN antigen concentration was 1.56 μg/mL,the linear range was 0.098 to 3.125 μg/mL,the coefficient of variation was less than 1%,the recovery rate was 99.10% to 101.06% for the indirect ELISA method.Comparing these two methods,the indirect competitive ELISA method took shorter time and the amount used of antibody was less.The indirect ELISA method did not need to cover the higher cost of protein,and the correlation coefficient was higher.The latter was difficult to form ELISA kit system because of its more components and samples need to be coated,so it was not suitable for field testing.The indirect competition method could be used for the large-scale preparation of ELISA kit system for rapid detection of protein content,because of its low dosage of antibodies,cost savings,fast and simple operation,was better for practical applications.

In recent years,the number of goat farming in Southern China has increased year by year,and the intensive degree is becoming higher and higher.However,the climatic characteristics of high temperature and high humidity in Southern China can easily lead to the heat stress response of goats,which leads to the reduction of production performance and hinders the rapid development of sheep industry in the South.At present,in the field of heat stress regulation,most domestic and foreign scholars focus on the research and development of nutritional additives for relieving heat stress.And there is a lot of research on the effects of heat stress on the rumen microbial and rumen structure damage of goat,but it is still worthy of further research.In the future,it is necessary to carry out systematic research on the evaluating indicators of whether the goat is in the form of thermal stress and the degree of heat stress,and carry out the research on the molecular response mechanism,cytokines and HSPs under the heat stress condition of goat.The authors focuses on the effects of heat stress on goat behavior and physiological indexes,the molecular regulation mechanism of goat during heat stress period,and the prevention and control measures of heat stress.The aim of this paper is to provide references for carrying out large-scale breeding of goat in Southern China and the summer heat stress regulation.