In order to clarify the effect of LPIN1 gene on the fat deposition,reproductive performance and lactation regulation of buffalo,LPIN1 gene was cloned and analyzed by bioinformatics.cDNA was extracted from buffalo ovary,and then LPIN1 gene CDS region was cloned and sequenced.The physical and chemical properties,secondary structure and tertiary structure of lipin1 protein were predicted and analyzed by bioinformatics software.The results showed that the coding region of buffalo LPIN1 gene was 2 793 bp,which encoded 930 amino acids.The buffalo LPIN1 gene nucleotide sequence shared 99.6%,97.9%,97.7%,97.5%,97.4%,97.1%,89.9%,89.8%,86.2% and 83.5% of homology with that of Bubalus bubalis(predicted),Bos mutus,Bos taurus,Capra hircus,Pantholops hodgsonii,Ovis aries,Sus scrofa,Camelus bactrianus,Homo sapiens and Mus musculus,respectively.The buffalo lipin1 protein shared 99%,99%,99%,99%,94%,94% and 90% of similar amino acids sequence with that of Bos taurus,Bos mutus,Capra hircus,Pantholops hodgsonii,Camelus bactrianus,Sus scrofa and Homo sapiens.The results of phylogenetic tree constructed by Mega 5.0 showed that LPIN1 gene in buffalo had a closest relation with Bos taurus,followed by Ovis aries and Capra hircus,indicating LPIN1 gene was highly conserved in different species.The secondary structure of the lipin1 protein was formed with α-helix,β-turn,T-turn and random coil.lipin1 protein was weakly acidic,which mianly located in the nucleus,and had Lipin_N,LNS2 and AF1Q domain and no signal peptide.The Lipin_N and LNS2 were the conservation domain.

This study was aimed to screen the polymorphism of myocyte enhancer factor 2D(MEF2D) gene in Qiabbei Ma goat,and analyze the genetic structure by bioinformatics method.Primers were designed according to Capra hircus MEF2D gene sequence in GenBank (accession No.:NC_030810),the mixed DNA pool and PCR products combined with direct sequencing were used to obtain the polymorphisms of exons 2,3,4,5 and 6 of MEF2D gene,the phylogenetic tree of MEF2D gene,physicochemical properties,hydrophobicity,transmembrane structure,signal peptide and secondary structure of MEF2D protein in Qianbei Ma goat were analyzed using bioinformatics analysis software.The results showed that three SNPs were screened,namely Exon3-G17617A,Exon5-C20180A and Exon5-C20259T.Phylogenetic tree analysis showed that the genetic relationship were most closed between Qianbei Ma goat and Bos taurus,and were more closely related to Sus scrofa and Homo sapiens than Rattus norvegicus and Mus musculus.Protein physical and chemical properties analysis showed that the theoretical isoelectric point of MEF2D protein was about 7.74,the minimum value of hydrophilicity was -2.867,the maximum value of hydrophobicity was 1.933,which was not a transmembrane protein and no signal peptide existed.The secondary structure showed that the increase of α-helix and extended chain,the decrease of β-turn and random coil of MEF2D protein Exon5-C20180A in Qianbei Ma goat.This result indicated that the polymorphism of MEF2D gene in Qiabbei Ma goat was successfully screened,which provided a foundation for the study of molecular genetic marker-assisted selection in breeding of Qianbei Ma goat.

Horses and donkeys are the important herbivorous livestock in China,which play an important role in the historical changes and production in human life.Prior to the mechanization of agriculture,human used horses and donkeys for riding and carrying goods.With the progress of mechanization degree and transportation,the service function of horses and donkeys have gradually decreased.The modern horse industry mainly focuses competition,leisure and entertainment,and by-product processing,while the donkey industry concentrates on the development and utilization of skin,meat,milk and biological products.The economic traits of horses mainly include body size,coat color,race ability,disease,extreme environmental adaptability,etc.On donkey,growth,skin and lactation traits are more concerned.By means of genomics and bioinformatics,we can effectively and accurately identify genes related to main economic traits in horses and donkeys.The association genes with body weight and size are located in the LCORL/NCAPG gene region.The MSTN gene is associated with the development of skeletal muscle,which in turn affects horse racing performance.The genes related to dwarf traits include HMGA2 and TBX3 genes in Shetland and Debao ponies,and the mutation of the ACAN gene is the causal mutation for the dwarf symptoms of the Shetland pony.The DMRT3 gene mutation affects the alternate gaits.KIT gene is associated with the white spot phenotype in coat color,MC1R gene has been shown to be the main gene responsible for chestnut color,ASIP gene is associated with black hair.The EDNRB gene mutations cause lethal white foal syndrome.EPAS1 gene and mitochondrial NADH6 gene play a key role in the adaptive evolution of the plateau.The authors review the research progress of functional genes related to main economic traits in horses and donkeys,and the future research of Equus genomics are discussed,so that the studies of molecular genetics and breeding in horses and donkeys are better provided to more researchers.

In order to understand the genetic evolution of the major virulence genes of equine herpesvirus 1 (EHV-1) of Xinjiang (XJ2015 strain) and construct TK gene-deleted strain,we cloned,sequenced and bioinformatics analyzed the full-length of TK,gI and gE genes of XJ2015 strain in this study.The DNA of the XJ2015 strain was used as a template,the TK gene recombination arms TKL and TKR were amplified to construct plasmid pUC-TKLR,and then inserted the amplified EGFP (CMV+polyA) into the pUC-TKLR plasmid.The results showed that it had higher homology between TK,gI and gE genes of XJ2015 strain and foreign EHV-1 isolates,which were 99.8% to 100.0%,99.6% to 100.0% and 99.9% to 100.0%,respectively,and had the lowest homology with foreign EHV-3 isolate,which were 72.9%,59.4% and 62.1%,respectively;Genetic evolutionary analysis showed that all three genes were genetically related to foreign EHV-1 isolates,the evolutionary relationship with EHV-3 was far away,but it was closely related to EHV-9 and EHV-4.There were no significant difference,no obvious regional features,the functional genes were conserved,the evolution was slow and homologous genes had the same or similar functions of TK,gI and gE genes of XJ2015 strain between foreign EHV-1 strains.After enzyme digestion,sequencing and transfection,the plasmid pUC-TKLR-EGFP for TK gene deletion targeting was successfully constructed.Through the analysis of EHV-1 main virulence genes and the construction of TK gene deletion targeting vector,it provided a theoretical basis for the epidemiological investigation and analysis of the TK gene deletion strain in Xinjiang.

In order to construct the prokaryotic expression vector of tetramer precursor chain of SLA-3-HB01 gene in Hebao pig and express the SLA-3-HB01 protein,using SLA-3-HB01/pMD18-T as a template,one pair of primers was designed to amplify the tetramer precursor chain named as SLA-3-HB01-BSP by PCR.The SLA-3-HB01-BSP was cloned into pMD19-T simple vector,the positive clones were double digested by NdeⅠ and XhoⅠ followed by sequencing.The target gene was further ligated into the expression vector pET-21a (+) and transformed into Escherichia coli BL21(DE3).After induction with IPTG,the size of expressed protein was detected by SDS-PAGE,and then inclusion bodies of SLA-3-HB01-BSP protein were extracted and detected.The results showed that SLA-3-HB01-BSP was amplified successfully,the size of product was about 896 bp.The double digestion with NdeⅠ and XhoⅠ demonstrated that the target gene was successfully cloned into pMD19-T simple vector with the inserted size of 876 bp.After sequencing and analyzing,it was proved that the target gene was consistent with the primary gene in sequence and a BSP tag was identified on the 3' end of the target gene.The recombinant expression vector SLA-3-HB01-BSP/pET-21a(+) was successfully constructed.SDS-PAGE detection result showed that the molecular weight of the target protein was about 33.5 ku.The molecular weight of inclusion body was about 33.5 ku,which was consistent with that of the target protein in mycoproteins.Scanning by UVP in Gel Imaging system,it was shown that the purity of inclusion body of protein was about 90%,which was suited for the requirements to study the related structure and function of the protein.In this study,the recombinant expression vector pET-21a (+) for SLA-3-HB01 tetramer precursor chain was successfully constructed,and inclusion body of protein with a certain purity was obtained.

The aim of the trial was to optimize the prokaryotic expression system of the σB protein of the new-type duck reovirus (NDRV) XX strain and evaluate its immunogenicity.Based on the measured σB protein sequence of NDRV-XX,the whole gene of σB protein was optimized,synthesized and ligated into pET-32a(+) plasmid according to E.coli codon bias without changing the amino acid sequence.The prokaryotic expression recombinant plasmid pET-32a(+)-sσB was constructed and transformed into E.coli BL21(DE3),induced by IPTG,and its expression conditions were optimized.The SDS-PAGE results showed that the optimal induction time,temperature and IPTG concentration were 3 h,32℃ and 0.25 mmol/L,respectively.Soluble analysis showed that the recombinant protein existed mainly in the form of inclusion bodies.The sσB soluble protein with purity higher than 90% was obtained after ultrasonication,denaturation,renaturation,and Ni²⁺ column affinity chromatography.The expression of sσB recombinant protein on E.coli BL21(DE3) was increased by 14.6% compared with σB protein.It accounted for 32.3% of the total bacterial protein.Western blotting results showed that sσB recombinant protein possessed NDRV antigen immunogenicity.The prokaryotic expression system of σB protein in NDRV-XX strain was successfully constructed and optimized,the expression of σB protein was increased,and recombinant σB protein with good NDRV antigen immunoreactivity was obtained.The results of this study laid the foundation for subsequent in-depth study of NDRV σB protein function and its application and the development of genetic engineering vaccine.

The aim of this study was to evaluate the effects of zinc sulfate on immune function and egg quality by adding different levels zinc sulfate into the basal diet of Jinghong laying hens.Five hundred forty 20-week-old healthy Jinghong laying hens were randomly allotted to six treatments,with 6 replicates per treatment and 15 layers per replicate.These birds were feed with six diets,the basal diet (control group,T1,Zn content was 25 mg/kg),and the diets added 25,50,75,100 and 125 mg/kg zinc sulfate (calculated by Zn) respectively on the basis of the basal diet (T2,T3,T4,T5 and T6 groups).The pre-feeding period was 2 weeks and the experimental period was 24 weeks.The rate of broken and crack egg was calculated every 4 weeks.At 33 weeks and 45 weeks of age,the egg quality,antioxident and immune parameters were observed.The results showed that:Compared to the control group,the supplementation of different levels zinc sulfate did not affect broken and crack egg,eggshell thickness and strength,egg albumin height,Haugh units and yolk color,the serum activities of ALP and LDH,the contents of IgM,IgA and Newcastle disease antibody titer,as well as immune organ weight,organ index and the avtivity of CuZn-SOD in the liver(P > 0.05),but increased the serum content of IgG at 33 weeks extremely significantly (P < 0.01).In conclusion,the recommended optimal addition amount was 75 mg/kg Zn from zinc sulfate in the diet.

The experiment was aimed to study the effects of 1,25-dihydroxycholecalciferol (1,25-(OH)₂-D₃) on growth performance,bone mineralization and duodenal phosphate transport gene expression.A total of 180 broilers (1 day old)were divided into 3 treatments with 6 replicates per treatment of 10 chickens per replicate.Three levels of 1,25-(OH)₂-D₃ were added to the basic diet:0,5 and 10 μg/kg,respectively.Broilers were slaughtered at 14 days of age,and femur,tibia,and duodenal mucosa were collected.Growth performance,bone mineralization and mRNA expressions of sodium phosphate transporter (NaPi-Ⅱb),nuclear vitamin D receptor (nVDR) and membrane vitamin D receptor (mVDR) were analyzed.The results showed that:Compared with the basic group,addition 5 μg/kg 1,25-(OH)₂-D₃ did not significantly affect the body weight gain,feed intake,feed and gain ration,and the weight,length,diameter,ash weight,ash content,calcium content,and phosphorus content of femur and tibia,and mRNA expression of NaPi-Ⅱb,nVDR and mVDR in the duodenum of broilers from 1 to 14 days of age (P > 0.05).Adding 10 μg/kg 1,25-(OH)₂-D₃ significantly reduced body weight gain,feed intake and feed conversion,the quality of femur and tibia,and the mRNA expression of NaPi-Ⅱb,nVDR,and mVDR in the duodenum of broilers (P < 0.05).In conclusion,adding 5 μg/kg 1,25-(OH)₂-D₃ to normal diets did not affect the growth,bone mineralization or the mRNA expression of NaPi-Ⅱb,nVDR and mVDR in duodenum of broilers.However,the addition of 10 μg/kg 1,25-(OH)₂-D₃ reduced the growth performance,skeletal mineralization and inhibits duodenal NaPi-Ⅱb,nVDR and mVDR gene expression at the transcriptional level.

The shortage of protein feed resources has always been one of the bottlenecks restricting the healthy and stable development of Chinese feed industry.Single-cell proteins have great potential in solving the problem of shortage of food and protein in the world.After years of research,single-cell proteins have made great progress in strain selection,production techniques,and fermentation raw materials.However,the cost of single-cell protein feeds remains high,and has not been widely used in the feed industry.Increasing the utilization efficiency and growth rate of non-protein nitrogen (NPN) production strains for production of single cell proteins is the key to reducing the production cost of single cell proteins.In this test,strains with strong NPN use ability were screened out from multiple strains of yeast and subjected to UV irradiation to obtain mutant strains with high yeast protein yield.Through the study of 14 kinds of yeasts using different carbon and nitrogen sources,the most suitable carbon and nitrogen sources were determined.According to the ranking of the growth potential,yeast protein production and NPN utilization ability of 14 strains of yeast,yeast M(Saccharomyces cerevisiae strain YI59) and N2 (Saccharomyces cerevisiae isolate AA2) with better overall performance were screened out.Yeast M and N2 were used as starting yeast for UV irradiation mutagenesis.Using glucose,ammonium sulfate as carbon and nitrogen source,preliminary screening and rescreening were performed according to the plate colony size and the liquid fermentation cell protein production.Three strains of MU23,MU3 and MU5 with the largest colonies were finally obtained.After liquid fermentation,compared to the original yeast M,the protein contents of MU3 and MU5 were increased by 12.93% and 11.82%,respectively (P < 0.05),while the yeast protein production had no significant difference (P > 0.05);The yeast protein content of strain MU23 had no significant difference,and it's yeast protein production increased by 13.04%,reached to 0.26 g/L (P > 0.05).In conclusion,the use of ultraviolet irradiation mutagenesis could effectively improve the utilization ability of yeast NPN and the production of yeast protein.

This study was aimed to investigate the effect of dietary supplementation with graded levels of flaxseed oil on production performance,egg quality,yolk fatty acid composition,and sensory profile of eggs.Two hundred and eighty-eight HyLine Grey laying hens at the age of 40-week-old were randomly assigned to 4 dietary treatments with 8 replicates per group,9 chickens per replicate.The diets were isonitrogenous and isoenergetic,containing 0,0.5%,1.0% or 3.0% flaxseed oil,respectively.The trial lasted for 4 weeks with 1 week of prefeeding.The results showed as follow:①Laying performance and egg quality were not significantly influenced by dietary flaxseed oil supplementation (P > 0.05);②Feeding flaxseed oil-included diets resulted in a linear and quadratic increase of ALA,EPA,DHA and total n-3 PUFA contents in yolk (P < 0.05);③The results of sensory experiment showed that egg yolk aroma and flavor scores significantly decreased and the intensity of fishy aroma and flavor significantly increased in response to 3.0% dietary flaxseed oil supplementation (P < 0.05).Correspondingly,the acceptance scores was significantly decreased (P < 0.05).And there was a linear and quadratic change of above indexes with the increase of dietary flaxseed oil supplementation (P < 0.05).④The yolk n-3 PUFA content was significantly related to fishy aroma and flavor (P < 0.05),while acceptance scores of egg yolks decreased as the n-3 PUFA concentration increased (R²=0.801).In conclusion,adding flaxseed oil to diet could increase the total n-3 PUFA content of egg yolk,but there would be bad influence on the flavor of egg cause by high dosage,leading to the decrease of overall acceptability.The eggs were accepted when yolk n-3 PUFA concentration was less than 21.26 mg/g of yolk and acceptable scores greater than 5.

This trial was conducted to investigate the effect of dietary Picria fel-tarrae Lour extracts on serum biochemical indexes,anti-oxidant indexes and some immune indexes of piglets to provide theoretical basis and production guidance for rational utilization of Picria fel-terrae Lour extracts in the production of piglets.A total of 250 healthy cross-breed pigs (Duroc×Landrace×Yorkshire) at 28-35 days were selected and randomly divided into 5 groups (groups A,B,C,D and E) with 5 replicates per treatment and 10 piglets per replicate.The piglets in groups A,B and C were fed basal diet supplemented with high (3.5 g/kg),middle (1.75 g/kg)and low doses (0.875 g/kg) Picria fel-tarrae Lour extracts,respectively,that in group D fed basal diet with 4.0 g/kg Qibusan,while that in group E was fed basal diet.The pre-feeding period was 7 d and the trial period was 30 d.Blood samples were obtained from the anterior vena cava by randomly selecting 10 piglets from each group on the 14th day after administration and the 14th day after drug withdrawal to analyze the biochemical,antioxidant and immune indexes.The results showed as follows:①There was no significant difference in blood biochemical indexes among experimental groups,indicating Picria fel-terrae Lour extracts had no adverse effects on glucose metabolism,lipid metabolism and digestive system in piglets.②From the antioxidant indexes,after administration for 14 d,NO content in group B was extremely significantly higher than that of other groups (P < 0.01),and the contents of T-AOC in group A was extremely significantly higher than that of other groups (P < 0.01),and that in groups B and D were extremely significantly or significantly higher than groups C and E (P < 0.01;P < 0.05).At 14th day after drug withdrawal,the contents of GSH in group E was significantly higher than that of groups B,C and D (P < 0.05).There was no significant difference in other indexes among all groups (P > 0.05).③From the immune indexes,after administration for 14 d,the level of IgG in group B was extremely significantly higher than that in other groups (P < 0.01) and the content of IL-6 in group A was significantly higher than that in groups B,D and E (P < 0.05).At 14th day after drug withdrawal,there was no significant difference in immune indexes among all groups (P > 0.05).In conclusion,the dietary supplementation of 0.875 to 3.5 g/kg Picria fel-tarrae Lour extracts had a good effect on enhancing the antioxidant function and improving the immune level of piglets with no adverse effects on piglets health.

The study was aimed to explore the effects of different molasses levels on in vitro ruminal fermentation characteristics and nutrient degradation of whole "Zhang Hybrid Millet" silage.Three Holstein dairy cows with permanent rumen fistula and similar body condition were chosen and the rumen fluid was collected.0(control group),2%,4% and 6% molasses were added to whole "Zhang Hybrid Millet" silage for 60 d fermentation at indoors temperature.After that the quality of whole "Zhang Hybrid Millet" silage were valued.The results showed that:① The DM of 4% molasses group was significantly higher than that of 2% and 6% molasses groups (P < 0.05),which was not significantly different with control group (P > 0.05).There was no significant difference of CP content among the groups (P > 0.05);The content of NDF of 4% molasses group was significantly lower than that of control group and 2% molasses group (P < 0.05),while the ADF was significantly lower than other group (P < 0.05).② After adding 4% molasses,DMD was significantly higher than that in control group and 2% molasses group (P < 0.05).The CPD and NDFD of 4% molasses group was significantly higher than that of control group and 6% molasses group (P < 0.05),while the ADFD was significantly higher than that in control group (P < 0.05).③Compared with the control group,ME,NE_L and GP_{72 h} of molasses adding groups were increased (P > 0.05).And the C value of 4% molasses group was significantly lower than that of control group (P < 0.05) when the gas production volume reached the theoretical maximum yield of 1/2.④There was no significant difference of pH,NH₃-N,MCP,TVFA,NGR,acetic acid,propionic acid,butyric acid,valeric acid and branched-chain fatty acid between molasses adding groups and control group (P > 0.05),while the fermentation parameters of 4% molasses group were better.Under the condition of this study, the optimum level of molasses was 4%.

This experiment was aimed to study the effect of fructooligosaccharides on the structure and diversity of rumen bacterial community in dairy cows by 16S rDNA sequencing technology.Four lactating dairy cows with similar lactation stages and the same parity were randomly divided into two groups using two-phase cross design method.The cows in control group were fed with basal diet and that in test group were fed with the basal diet with 60 g/(d·head) fructooligosaccharides.The experiment was designed through 2×2 cross-over test with 21 d in each stage (14 d for pre-experiment and 7 d for test) and 14 d transitional period of cross test.The rumen fluid samples were collected at 0 (before feeding),2,4,6,9 and 12 h after feeding by cattle catheter.Each phase was collected continuously for 3 days,and the structure and diversity of rumen bacterial community were measured by 16S rDNA sequencing technology.The results of Alpha diversity indexes (Simpson,Shannon),richness indexes (Chao,Ace) and Beta diversity profile showed that the addition of fruitooligosaccharide decreased the bacterial diversity in the rumen of dairy cows.And the results of 16S rDNA sequencing showed that according to the analysis at phylum level,Bacteroidetes,Firmicutes and Proteobacteria accounted for more than 95% of the total bacterial population in cow rumen of two groups.The addition of fructooligosaccharides significantly decreased the abundance of Bacteria_NA (P < 0.05),and the abundance of Synergistetes showed an increasing trend (P=0.075).According to the analysis at genus level,fructooligosaccharides significantly increased the abundance of the Pseudomonas (P < 0.05),extremely significantly increased the abundance of Bacteroides (P < 0.01),while the abundance of Dehalobacterium significantly decreased (P < 0.05).Compared with the control group,the abundance of Ruminococcus,Butyrivibrio,Succiniclasticum and Lachnospiraceae_NA in test group was increased by 80.0%,7.5%,127.9% and 20.0%,respectively,but these differences were not significant (P > 0.05).Under the test conditions,the addition of fructooligosaccharides had a certain effect on the diversity and structure of rumen bacterial flora in dairy cows,which significantly increased the abundance of Pseudomonas which unfermented sugar,extremely significantly increased the abundance of Bacteroides which used carbohydrates as a source of fermentation,had an accelerating effect on the abundance of rumen fiber degrading bacteria.

This study was aimed to understand the population genetic variation and association relationship between the mutation sites of tyrosinase related protein 1(TYRP1) gene and different coat color phenotype in Chinese indigenous sheep populations.The directly sequencing and PCR-RFLP method were performed to identify single nucleotide polymorphisms (SNP) and genotype of TYRP1 gene,and then constructed haplotype,linkage disequilibrium analysis and genetic variation analysis were performed out by Beagle,PLINK and POPGENE softwares.The results showed that a total of 13 SNPs were identified in TYRP1 gene.10 SNPs of TYRP1 gene exons were chosen and used for genotyping in 10 sheep breeds.The results demonstrated that 10 sheep breeds presented variation at most mutation sites,except for some mutation sites had not polymorphisms in Large-tail Han sheep,China Merino sheep and Minxian Black-fur sheep.These results indicated that Chinese indigenous sheep populations had higher genetic diversity.The haplotypes analysis results indicated that a total of 42 haplotypes were detected in all sheep individuals,0000000000 (245/918) and 0100000001 (91/918) dominant haplotypes were detected in all sheep populations,except 0101100000 (93/918) was not detected in China Merino sheep,0001000001 (69/918) was not detected in China Merino sheep and Minxian Black-fur sheep,which were detected in other sheep populations.Linkage analysis results demonstrated that two block were detected in 10 SNPs of TYRP1 gene from all the sheep individuals.The genetic variation investigation indicated that Chinese sheep breeds had maintained a high level of within-population genetic differentiation,and the distinct genetic differentiation patterns and genetic relationships among the Chinese sheep breeds displayed a high consistency with the traditional classification.This study provided the theoretical basis for the further application of TYRP1 gene in coat color genetic traits of sheep.

This study was aimed to investigate the single nucleotide polymorphisms(SNP) of DGAT2 gene in buffalo,and explore the population genetic characteristics of the polymorphism in Murrah buffalo.A total of 57 Murrah buffaloes from Guangxi buffalo research institute were selected as materials,the exon 2,introns 2 and 3 sequences of DGAT2 gene were amplified by PCR,SNP was detected using conventional sequencing,gene frequency and genotype frequency,polymorphism information content (PIC),effective number of alleles (Ne) and detection of genetic heterozygosity (He) were analyzed by genetic diversity analysis software (POPGENE) and SPSS softwares.The results showed that 9 SNPs loci were found of DGAT2 gene exon 2,introns 2 and 3 in Murrah buffalo (IVS2.54 G > A,IVS2.158 A > G,EVS2.191 A > G,EVS2.228 A > G,IVS3.311 C > T,IVS3.444 A > G,IVS3.451 A > C,IVS3.466 C > T and IVS3.521 C > T),EVS2.191 A > G caused the amino acid to be changed from isoleucine to valine,and there was a certain degree of linkage inheritance between the mutation sites but close to the chain equilibrium state.There was more difference between two allele frequency of 6 SNPs loci (IVS2.158 A > G,EVS2.191 A > G,IVS3.311 C > T,IVS3.451 A > C,IVS3.466 C > T and IVS3.521 C > T).It was suggested that the higher allele frequency individuals might be more suitable for survival.9 SNPs loci in DGAT2 gene in the buffalo species were high polymorphism,the heterozygosity were 0.1744 to 0.4975,which illustrated the buffalo populations DGAT2 gene genetic polymorphism was rich,and had a great breeding value and strain improvement potential.

In order to study the effect of backfat loss of lactating sows on reproductive performance in Large White pig,1 178 Large White pigs were selected,the total number of born (TNB),number of born alive (NBA),number of healthy born (NHB),weight of weaning litter (WWL),weaning estrus interval (WEI) and backfat loss of lactating sows (BLL) were recorded during 6 parities.The sows were divided into 6 groups based on BLL as < 0,0-1,1-2,3-4,5-6 and > 6 mm.WWL was used as a covariate to examine the significant differences between groups of TNB,NBA,NHB and WEI using the least squares test.The results showed that the average TNB,NBA,NHB,WWL,WEI and BLL for 6 parities sows were 13.67 heads,11.51 heads,10.32 heads,65.90 kg,4.83 d and 2.89 mm,respectively.There was significant difference in litter size traits in different groups of BLL during 6 parities (P < 0.05).Based on the results of all 6 parities,there was no significant difference among groups 4,5 and 6 of BLL (P > 0.05),but it were significantly higher than those in groups 1,2,and 3 (P < 0.05).The TNB in group 4 was the highest,reaching 13.54 heads,1.90,2.29 and 1.63 heads more than those in groups 1,2 and 3 (P < 0.05),respectively.Although there were sometimes significant differences of NBA and NHB in each group and each parity,there was no significant difference among 6 groups in 6 parities (P > 0.05).From the standpoint of WEI,no significant differences were observed in groups (P > 0.05).The results indicated that in the production of Large White pigs,higher TNB could be achieved by controlling the BLL in the range of 3 to 4 mm.

In order to explore the biological characteristics of lactoferrin (LF) gene in dairy goat,and analyze the variations of LF of milk in Saanen dairy goats,CDS region of LF gene in Saanen dairy goat was cloned,the sequence,physicochemical property,conservative structure domain and homology comparison of LF gene were analyzed by bioinformatics softwares,milk samples from individual were obtained and determined using ELISA kit.The results showed that the length of LF gene in Saanen dairy goat was 2 127 bp,which coded 708 amino acids.The formula of LF protein was C₃₄₀₅H₅₃₆₅N₉₄₉O₁₀₂₄S₄₃,which was an unstable and hydrophilic protein.The secondary structure of LF mainly contained α-helix,extended chain and random coil,similar with that in transferrin.The molecular weight of LF was 77.359 ku.The protein sequence analysis result showed that the high similarity were found in Bovidae (> 92%),and the rest were quite different,LF gene of Saanen dairy goat exhibited the highest homology with goat,they shared 99% amino acid identity and were in the same branch of an evolutionary tree.ELISA results showed that the maximal concentrations were observed in the colostral samples,the common milk had the lower and stable concentrations.The results obtained the sequence and molecular structure of LF gene,and revealed the change of LF content in milk of Saanen dairy goats,which provided a theoretical basis for LF functional researches and product development.

In order to provide histological basis for molecular regulation mechanism of the growth of wool and cashmere,and provide reference for application of hair follicle traits in cashmere goat breeding,hair follicle structure and the correlation between characteristic parameters and cashmere production traits in Shanbei White cashmere goat were studied.Skin samples were collected from Shanbei White cashmere goats,and made into paraffin sections.The morphological characteristics of skin hair follicle were observed by microphotograph system and a number of parameters were measured.The correlation between characteristic parameters and cashmere production traits was analyzed with SPSS 17.0 software.The results showed that the hair follicles of Shanbei White cashmere goat were distributed in hair follicle groups and the main type of hair follicle group was three-type,which accounted for 85.80%.The cashmere yield had an extremely significant positive correlation with body weight,cashmere length,S/P value,SF-bulb width (P < 0.01),had a significant positive correlation with SF density (P < 0.05),and had a significant negative correlation with epidermis thickness (P < 0.05).The cashmere length had an extremely significant positive correlation with cashmere yield and SF-bulb width (P < 0.01),had a significant positive correlation with body weight,S/P value,SF diameter (P < 0.05),and had a significant negative correlation with epidermis thickness (P < 0.05).The cashmere fineness had a significant negative correlation with S/P value (P < 0.05).It was concluded that the skin hair follicle characteristics of Shanbei White cashmere goat,such as S/P value influenced the cashmere fineness,and body weight,cashmere length,S/P value,SF density (cashmere density) significantly influenced the cashmere yield (P < 0.05).It suggested that the S/P value could be used as a very important trait in Shanbei White cashmere goat breeding.High S/P value not only could improve the cashmere yield,but also could improve the cashmere length and reduce the cashmere fineness,thus solve the contradiction of improving the cashmere yield and quality at the same time.

In order to further study the major epitopes and antigen properties of African swine fever virus (ASFV) p54-1 protein,specific primers were designed according to p54 gene sequence retrieved from GenBank (accession No.:NC_001659.2),the target sequence of encoding p54-1 protein was amplified by PCR.Then it was ligated into pGEX6p-1 vector and constructed prokaryotic expression plasmid (pGEX6p-1-p54-1).The plasmid was transfected into E.coli BL21,and the expression of recombinant protein was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The fusion protein cleaved the GST-tag using thrombin,and then purified by anion exchange column.The protein was identified by ELISA and emulsify with adjuvant,the prepared immunogen was inoculated into mouse to prepare of p54-1 protein specific polyclonal antibody.The immune-activity and titers of the prepared polyclonal antibody were determined by ELISA and Western blotting.The results showed that the expressed recombinant protein p54-1 existed in a soluble form.The p54-1 protein cleaved GST-tag could react with the positive serum of ASFV,but no with negative serum,PRRSV and PCV3.The prepared polyclonal antibody titer was 1:128 000.Western blotting result demonstrated that the prepared polyclonal antibody could recognize the p54 protein.In conclusion,the high purified expressed protein of ASFV p54-1 had been successfully prepared and p54-1 specific polyclonal antibody showed wonderful immunocompetence and specificity,providing foundation for the development of sandwich ELISA detection method of ASF.

To investigate the genetic variation and molecular epidemiology of porcine reproductive and respiratory syndrome virus (PRRSV) in Jiangxi province in the recent years,a total of 453 tissue samples were collected from porcine reproductive and respiratory syndrome (PRRS) suspected cases in pig farms in different regions of Jiangxi province during 2016 to 2017,and then examined by RT-PCR.Of the 453 clinical samples,321 were identified positive for PRRSV,with a positive rate of 70.86%.The positive rates were varying from 19.15% to 84.85% in different regions.14 positive samples were sequenced,and the nucleotide homologies of ORF5 gene were among 83% to 100% for the strains identified in this study,and 59.9% to 98.5% for reference strains of PRRSV.14 sequenced strains all belonged to North America type (genotype Ⅱ) PRRSV in this study,4 strains belonged to the highly pathogenic PRRSV (HP-PRRSV,subtype Ⅰ),3 strains belonged to the classic PRRSV (subtype Ⅱ),3 strains belonged to NADC30-like PRRSV (subtype Ⅲ),and 4 strains belonged to the new subtype of PRRSV (subtype Ⅳ).Sequence analysis on deduced amino acid of GP5 coded by ORF5 gene indicated that the significant variations were observed in three epitopes and two important antigenic regions in PRRSVs belonged to the four subtypes (subtypesⅠ,Ⅱ,Ⅲ and Ⅳ).Subtype specific variations in subtypes Ⅲ and Ⅳ were also observed in amino acid of GP5,which might lead to the variation of antigenecity of GP5 protein.The results indicated that there was a new situation of PRRSV strains circulating in Jiangxi province during 2016 to 2017,with multi-subtypes of strains existed and co-infected in pigs.And the most popular subtype was HP-PRRSV,while there were high rates of newly emerged NADC30-like strains and new subtype strains;It was necessary to continuously monitor the epidemiology of PRRSV,which would be benefit for the development of new diagnostic methods,drugs and vaccines for the prevention and control of PRRS.

In order to study the genotype,pathogenicity and nucleotide difference between Newcastle disease virus (NDV) isolates and the traditional NDV vaccine strain (La Sota),a suspected NDV virus was isolated from a chicken farm.The isolate was preliminarily determined by HA and HI tests.A pair of primers was designed based on the partial fragment of NDV F gene published at GenBank(accession No.:JF950510.1).F gene was amplified by RT-PCR method,and cloned and sequenced,the sequencing result was compared with the F gene sequences published at GenBank,and the evolutionary tree was constracted to analyze the genotypes.The pathogenicity of the virus was determined by the mean death time (MDT) of chicken embryos,intracerebral pathogenicity index (ICPI) in one-day-old chickens and intravenous pathogenicity index (IVPI) in six-week-old chickens,respectively.Based on the NDV genome sequence published in GenBank(accession No.:JF950510.1),nine pairs of primers were designed to amplify the genome sequence of the isolate strain,and its structure was analyzed.The results showed that the length of F gene was about 500 bp,a NDV strain of genotype Ⅶ was isolated.The MDT,ICPI and IVPI were 52.8 h,1.675 and 2.46,respectively.The whole genome sequence analysis results showed that the full genome length of the isolated strain was 15 192 bp,and had 6 nucleotides more than the strain of La Sota,and the homology between the two strains was 82.8%.Therefore,a virulent NDV strain of genotype Ⅶ was isolated,and contained a large difference in the nucleotide sequence compared with the La Sota strain.

The aim of this study was to establish a direct fluorescent antibody assay for the isolation and identification of porcine epidemic diarrhea virus (PEDV).The PEDV S1 gene neutralized antigen epitope region (COE) was successfully fused by genetic engineering technology.Immunoglobulin G (IgG) from rabbit serum was purified by chromatographic column.We used agitation method to combine the antibody with fluorescein isothiocyanate (FITC).The protein antigen and antibody titer were identified by Western blotting and indirect ELISA,respectively.The specificity of labeled fluorescent antibody and the best working concentration were determined by direct immunofluorescence and gradient dilution method,respectively.In this study,the length of S1 gene fragment in the epitope region was 423 bp,the expression protein was named as pGEX-6P/COE fusion protein,and the molecular mass was about 40 ku.Western blotting result showed that the fusion protein had good antigenicity and the titer of serum antibody reached 1:25 600 at 35 d after immunization.According to the experiment results,optimal working concentration of the direct FITC antibody was diluted from 1:32 to 1:64.This method was specific,having no cross-reactivity with other common virus,such as TGEV,PoRV,PPV and CSFV,etc.The results showed that the prepared direct immunofluorescent antibody could be used for rapid detection of PEDV on the cell platform.It could provide the related detection data intuitively,which was of great guiding significance for the selection of the virus separation process.

In order to study the antibacterial mechanism of Lactobacillus fermentum isolated from natural fermented koumiss on pathogenic Escherichia coli O₈ (E.coli O₈),the strains with best bacteriostasis isolated from koumiss was screened out in this study,and then 16S rDNA sequence was identified by BLAST of NCBI website.After fermentation,the cell-free fermentation supernatant (CFS) was prepared.The properties and content of antibacterial substances in CFS were preliminarily determined by acid excretion,hydrogen peroxide excretion and different protease treatments.The best inhibitory concentration of CFS on the growth curve of pathogenic E.coli O₈ within 24 h was determined using the Oxford cup method and two fold dilution method.The influence of CFS on cell membrane and cell wall permeability of pathogenic E.coli O₈ was determined by kit method.The results showed that there were 22 strains which had antibacterial effects on pathogenic E.coli O₈ isolated from koumiss,and the best strain was Lactobacillus fermentum after 16S rDNA sequence and phylogenetic tree analysis.Protein was the main bacteriostatic substance of CFS,and its content was 399.5 μg/mL.The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) were 25.0 and 49.9 μg/mL,respectively.CFS could rapidly increase the AKP content of pathogenic E.coli O₈ within 1 h and then increase slowly.Moreover,it could obviously increase the protein content in the culture of the pathogen.In conclusion,the protein of Lactobacillus fermentum was the antibacterial substance on pathogenic E.coli O₈.And with the increase of protein concentration,the bacteriostasis ability was stronger.CFS could release AKP and intracellular proteins by destroying or altering the permeability of cell membrane and cell wall.Thus,it could inhibit the growth of pathogenic E.coli O₈ in a short time.

In the study,the network pharmacology method was used to reveal the mechanism of Baitouweng decoction in treating swine diarrhea with the active ingredient and target,gene function and signal pathway.All the chemical compounds and targets of Baitouweng decoction were searched in the pharmacological analysis database of traditional Chinese medicine (TCM).Then the compound-target network,protein-protein interaction (PPI) network and target-pathway network were constructed by STRING,DAVID,NCBI databases and Cytoscape software to study the mechanism of Baitouweng decoction in treating swine diarrhea.The results showed that 11 active compounds of Baitouweng decoction were screened through the oral availability and drug-likeness,and the results of compound-target network showed that there were 63 corresponding targets corresponding to 11 compounds.The Baitouweng decoction-swine diarrhea PPI network diagram contained 45 targets,in which the key targets were ESR1,CREBBP,MAPK1,AR and so on,TOP2B,ESR1,ESR2,NR3C1,AR and NCOA2 were directly related to the targets of swine diarrhea.There were 2 KEGG pathways and 5 gene ontology (GO) entries of which the number of biological process,molecular function and cellular component was 3,1 and 1,respectively.Baitouweng decoction could regulate the expression of TOP2B,ESR1,ESR2,NR3C1,AR and NCOA2 and other regulated genes by aureusidin,palmatine,corylifol C,fumarine,peltatin,capsularin and 8-hydroxypinoresinol.The phospholipase C activation G protein-coupled receptor signaling pathway,adenylate cyclase-activating adrenergic receptor signaling pathway,DNA template transcription,steroid binding,and cell membrane components were used to treat swine diarrhea through neuroactive ligand-receptor interaction signaling pathways.

To understand the epidemic situation and drug resistance of pathogenic Escherichia coli (E.coli) in diarrhea piglets from large-scale swine farms in Guizhou province,agglutination test,PCR and drug sensitive agar diffusion methods were used to perform serotyping and virulence gene classification and drug resistance analysis of 78 pathogenic E.coli isolates in this experiment.The results showed that the main serotypes of 78 pathogenic E.coli were O138 and O87,accounting for 60.8% of the established strains;The virulence genes were detected in 62 strains of pathogenic E.coli,the detection rate was 79.5%,and could be divided into 8 virulence gene types,belonging to enteropathogenic E.coli (EPEC),enterotoxigenic E.coli (ETEC) and enterohemorrhagic E.coli (EAEC),respectively.The virulence genes eaeA,elt and escV had higher detection rate,which were 38.5%,28.2% and 21.8%,respectively.The isolated pathogenic E.coli was highly resistant to β-lactam drugs,and was multi-drug resistant,with more than 8 drug-resistant species.The results showed that the pathogenicity of E.coli in piglets with diarrhea in large-scale pig farms in Guizhou province was high,and the genotypes were complex.The pathogenicity of E.coli was severe.This experiment could provide basic data and theoretical basis for prevention and control of piglets diarrhea in large-scale pig farms.

In order to observe the antagonistic effect of selenizing garlic polysaccharide on immunosuppressed chicken induced by cyclophosphamide,336 11-day-old roman chickens were randomly divided into seven groups:Blank control group (BC),vaccine group (VC),cyclophosphamide group(Cy),sGPS3 group,sGPS5 group,sGPS6 group and drug control group (APS),with 6 duplications per group and 8 chickens per duplication.Each chicken except that in BC and VC groups were intramuscularly injected with 8 mg/mL cyclophosphamide 0.5 mL,while each chicken in BC and VC groups were injected with the same amount of saline,once a day for three successive days.At the 14 d of age,The chickens except BC group were vaccinated with ND vaccine,repeated vaccination at 28 days old.At the first vaccination,the chickens in three sGPSs groups were injected with 0.5 mL of 1 mg/mL sGPS₃,sGPS₅ and sGPS₆,respectively.The chickens in APS group were intramuscularly injected with 0.5 mL Astragalus polysaccharide,while that in Cy,VC and BC groups were injected with the same amount of saline,once a day for three successive days.Six chickens were selected randomly from each group for collecting wing venous blood on 7,14,21 and 28 days after the first vaccination,and the serum ND-HI antibody titer were determined by β-micro method,IFN-γ and IL-2 contents were determined by ELISA.The body weighed,thymus,spleen,bursa of Fabricius were collected after culling,and the immune organ indexes were calculated.The result showed that the serum ND-HI antibody titer,IFN-γ and IL-2 contents and bursa of Fabricius index of each administration groups were higher than that of the Cy group (P < 0.05);the serum ND-HI antibody titer and IFN-γ contents at 21 d,IL-2 content at 21 and 28 d in sGPS₆ group were significantly higher than that of other groups (P < 0.05);At 21 and 28 d after first vaccination,the body weight and bursa of Fabricius index in sGPS₆ group were significantly higher than that of other three administration groups (P < 0.05).At 28 d after first vaccination,the thymus and spleen indexes in sGPS₆ group were significantly higher than that of other three administration groups (P < 0.05).In conlusion,the selenizing garlic polysaccharide had the antagonistic effect on immunosuppressed chicken induced by cyclophosphamide,and sGPS₆ had the better effect which could be used as candidate drug for novel immunosuppressive antagonists.

The aim of this study was to establish a sensitive,rapid and specific method for the detection of chloramphenicol (CAP).Based on immunoaffinity chromatography and enzyme-linked immunosorbent assay,a novel immunoaffinity chromatography test column (ICTC) for the rapid detection of CAP in shrimp was developed.The detection layer and control layer of ICTC column were prepared by sol-gel method,and the conditions of detection layer,control layer and CAP-HRP dilution factor were optimized.The performance parameters such as sensitivity and specificity of the ICTC column were determined,and the pretreatment method and matrix interference effect of the shrimp sample were optimized.This study determined that the ICTC column conditions were as follow:The detection layer,anti-CAP sol-gel:blank sol-gel was 10:1 000 (V/V);The control layer,anti-HRP sol-gel:blank sol-gel was 13:1 000 (V/V);CAP-HRP dilution was 1:10⁵.The detection result was determined by the detection layer decolorization method.The detection time was 10 min,and the detection limit of CAP in PBS buffer was 0.5 μg/L.The method had no visible cross-reaction with thiabendazole,florfenicol,florfenicol similar drugs or metabolites.The detection limit of this method in shrimp samples was 0.75 μg/kg.The ICTC column detection method established in this study was fast,sensitive and easy to use,and provided a new technical methods for rapid detection of CAP residues in shrimp on site.

This study was conducted to explore the role of Smad9 on follicular development in mice and provide new ideas for the study of follicular growth and its regulation mechanism.Six-week-old female Kunming mice were randomly divided into 3 groups and injected intraperitoneally with physiological saline,bone morphogenetic protein-4 (BMP4) and Smad9 inhibitor (LDN-193189),respectively,which were used as the control group,BMP4 group and LDN group.After 48 hours,the ovaries were collected to make paraffin sections.Follicles were observed after HE staining and the follicles at different stages of development were counted.Western blotting and Real-time PCR were used to detect Smad9 protein and mRNA levels.At the same time,the concentrations of estradiol (E₂),progesterone (P₄),luteinizing hormone (LH),follicle stimulating hormone (FSH) and aromatase in the serum of each group of mice were measured by ELISA,and the expression of genes such as CYP19a1,LHR,PRLR and FSHR were detected to further explore possible mechanisms of Smad9.The results showed that compared with control group,the Smad9 protein and mRNA levels were higher in BMP4 group and lower in LDN group;The number of antral follicles in BMP4 group was significantly increased (P < 0.05),while LDN group was significantly reduced (P < 0.05).In addition,ELISA results showed that the levels of serum E₂,FSH and aromatase increased,while the levels of P₄ and LH decreased in BMP4 group;The levels of E₂ and aromatase decreased in LDN group.The results of Real-time PCR showed that the mRNA levels of FSHR and CYP19a1 genes increased,while the mRNA levels of LHR and PRLR genes decreased in BMP4 group;The mRNA level of PRLR gene increased,while the mRNA level of CYP19a1 gene decreased in LDN group.From the above experimental results,it could be concluded that BMP4-induced Smad9 promoted antral follicles development,and it might work via affecting the production of E₂,PRL and aromatase.

To determine the antibacterial effect of 20 Miao national herbs such as Isatis tinctoria,gallnut and Astragalus and their prescriptions against Staphylococcus aureus (S.aureus),Salmonella and Escherichia coli (E.coli),the active ingredients of Miao herbal medicines were extracted using water decoction,ultrasonic water extraction and ultrasonic alcohol extraction,respectively.Double dilution method was used to assess the minimal inhibitory concentration (MIC) for S.aureus,Salmonella and E.coli.The results showed that antibacterial effect of Miao national single herbs in three different extraction methods was ultrasonic alcohol extraction method > ultrasonic water extraction method > water decoction method.The MIC of the ethanol extracts of gallnut and Agrimonia on S.aureus,Salmonella and E.coli were 0.18,0.71 and 1.43 mg/mL (gallnut) and 1.56,3.13 and 3.13 mg/mL (Agrimonia),respectively.Furthermore,the MIC of Kadsura,plum,thorn pear,Lonicera,Rhizoma Dryopteris Crassirhizomae and Eucommia were higher than gallnut and Agrimonia,but lower than other 12 kinds of Miao national single herbs.The MIC of these 6 kinds of Miao national single herbs extracts extracted in ultrasonic alcohol against S.aureus,Salmonella and E.coli were 3.69 to 50.00,3.13 to 50.00 and 3.31 to 66.25 mg/mL,respectively.The effect of prescriptions of Miao national herbs showed that ultrasonic water extraction method > ultrasonic alcohol extraction method > water decoction method.The MIC of prescription 5 ultrasonic water extract was the lowest for S.aureus,Salmonella and E.coli,which were 0.34,2.75 and 2.75 mg/mL,respectively.The MIC of the ultrasonic water extract of prescription 1 against S.aureus,Salmonella and E.coli was lower than the prescriptions 2,3 and 4,which were 2.88,5.75 and 5.75 mg/mL,respectively.The results showed that gallnut,Agrimonia and prescription 5 had strong antibacterial activity against 3 common pathogens,Kadsura,plum,thorn pear,Lonicera,Rhizoma Dryopteris Crassirhizomae,Eucommia and prescription 1 had certain antibacterial effects,and the antibacterial effects of other Miao drugs were relatively weak.

Astragali Radix is a Chinese herbal medicine which is used for both medicine and food,Astragalus polysaccharides is the most important natural active ingredients which has functions of improving immunity and antioxidant ability and intestinal microecology,and has the capability of anti-microbial and anti-virus without drug resistance,of which immune regulation is the most prominent.Numerous studies have demonstrated that Astragalus polysaccharides have significant immunomodulatory effect in vivo and in vitro.The authors introduced the regulatory effect of Astragalus polysaccharide on immune related genes,its role in organ immunity,cellular immunity,cytokines and intracellular messenger substances,cardiovascular system immunity,antibacterial immunity and antiviral immunity,and summarized its application used as immune adjuvant in CSF,PCVD,FMD,PRRS and MPS in the latest research progress.At present,most researches are limited to the structure of Astragalus polysaccharide,which is mainly used as immune adjuvant and animal feed additive,and its application range is narrow which lacks deep research and development.In the future,we should strengthen the separation and purification of low molecular weight Astragalus polysaccharide,explore its structure characteristics and immune regulation of the structure-activity relationship to improve its curative effect,promote its clinical application.

This study was aimed to illustrate the metabolic pathway of mequindox (MEQ) in aquatic animal.Ultra-high performance liquid chromatography tandem time-of-flight mass spectrum (UPLC-Q/TOF-MS) and metabolism analysis software Metabolynx^XS were adopted in this research,and zebrafish was adopted as aquatic model animal for MEQ metabolism investigation.After dipping with MEQ,zebrafish tissues were collected and MEQ metabolites were extracted with ethyl acetate and acetonitrile in turn.Then samples were filter through 0.22 μm filter membrane for further UPLC-Q/TOF-MS analysis.The number of metabolites could be confirmed according to the newly increased chromatographic peak by comparing the experimental group and the control.The chemical structure of metabolites could be confirmed according to the accurate mass spectrum by comparing the metabolites and the MEQ standard.The results showed that the major metabolic pathway of MEQ in zebrafish was N→O group reduction.Only three metabolites were identified,including N1-deoxymequindox (1-DMEQ),N4-deoxymequindox (4-DMEQ) and N1,N4-bisdeoxymequindox (1,4-BDMEQ).The depletion rule of MEQ and major metabolites in zebrafish was investigated.It was found that the concentration of MEQ reduced after administration,and it was less than the half of the highest concentration after 4 h.As to the three metabolites,the concentration first increased and then decreased.The highest concentration of 1-DMEQ and 4-DMEQ was at 2 h after administration,and then it was less than the half of the highest concentration after 8 h.For another metabolite 1,4-BDMEQ,the highest concentration was at 4 h after administration,and it was less than the half of the highest concentration after 12 h.This study would contribute to further control of aquatic animal derived food safety.In addition,the results would be benefit for the metabolism and pharmacokinetic investigation of MEQ in other aquatic food animal.

In order to successfully prepare florfenicol nanoemulsion and investigate its bacteriostatic effect,the prescription screening,physical properties investigation and bacteriostatic activity test were conducted.The oil phase,emulsifier and coemulsifier were optimized by determining the solubility of florfenicol in oil phases,emulsifiers and coemulsifier,respectively.The optimum formula was designed based on Kv of emulsifier-coemulsifier (Smix) and Smix-oil phase volume ratio which was selected using pseudo-ternary phase diagrm.Florfenicol nanoemulsion was distinguished by staining method,and the configuration of florfenicol nanoemulsion were measured by TEM,the particle size and Zeta potential were measured by laser particle size analyzer.The stability was investigated by a centrifugation and a long-term test.The antibacterial effect of florfenicol nanoemulsion on Staphylococcus aureus,Escherichia coli,Microbacterium and Bacillus subtilis was studied by a bacteriostatic activity test.The results showed that olive oil was the optimized oil phase,EL-40 was the optimized emulsifier,glycerin was the optimized coemulsifier,the optimized ratio of Kv was 2.0,and the volume ratio of Smix-oil was 8:2.The optimum formula of florfenicol nanoemulsion was the composition of fluorobenicol (120 mg),N,N-dimethylformamide(0.1 mL),olive oil (2 mL),EL-40(5.7 mL),propanol(2.3 mL),and distilled water(6 mL).The uniform size and no adhesion droplet shape of florfenicol nanoemulsion was O/W,with the average diameter of 28 nm and Zeta potential of -0.454 mV,maintained to be stable up to six months.The florfenicol nanoemulsion performanced a better antibacterial effect than the same concentration of fluorphenicol solution for four bacterias.The results demonstrated that the preparation method of florfenicol nanoemulsion was simple,feasible,and showed high stability and good bacteriostasis.The study proved that the florfenicol nanoemulsion had potential application value in the animal husbandry and aquaculture.

The test was aimed to study the expression of nuclear protein 1 (Nupr1) mRNA in mouse uterus during early pregnancy.The method of in situ hybridization was used to investigate Nupr1 mRNA expression in animal models that included early pregnancy,pseudopregnancy,delayed implantation and activation,artificial decidualization and hormonal treatments.The relative expression level of Nupr1 mRNA was detected in early pregnancy and pseudopregnancy using Real-time PCR.During mouse early pregnancy,the signal of Nupr1 mRNA was detected in luminal epithelium and glandular epithelium during the 1st to 4th day and in the decidua area during the 5th to 8th day.Nupr1 mRNA was mainly expressed in the luminal epithelium and glandular epithelium of mose uterus on the 1st to 5th day of pseudopregnancy.The signal was detected in luminal epithelium and glandular epithelium of the mouse uterus in the delayed implantation,which was similar to the results of early pregnancy on the 4th day.The signal was detected in decidua in the model of delayed activation,which was similar to the results of early pregnancy on the 5th day.The expression of Nupr1 mRNA in the model of artificial decidualization was detected in decidua area.In the control of artificial decidualization the slight signal appeared in luminal epithelium and glandular epithelium of the mouse uterus.After treated with oestrogen (E₂) the signal appeared in luminal epithelium and glandular epithelium of the mouse uterus,and the signal was enhanced.After treated with both of E₂ and progesterone (P₄), the expression of the signal was not changed significantly.Real-time PCR result showed that the relative expression on the 2nd day was higher than other days in early pregnancy and pseudopregnancy.The results indicated that the expression of Nupr1 mRNA in mouse uterus was related to the process of mouse early pregnancy.The expression of signal in luminal epithelium and glandular epithelium of the mouse uterus might be regulated by hormones.Nupr1 mRNA expression in uterine stroma was associated with decidualization and active blastocysts.

The goats in a farm in Western Jiangxi showed neurological symptoms such as circling movements,unilateral blindness and dyspnea,nearly 20 goats died.In order to identify the pathogens and establish effective prevention and treatment measures,the pathological necropsy was performed on three dead sheep that were sent for inspection.The viscera tissues such as liver,spleen and brain tissues were collected for the purification and cultivation of pathogenic bacteria.And the LB medium was used to make a secondary enrichment isolation selectively.Gram staining,biochemical tests,and 16S rRNA gene sequencing analysis were carried out.The animal experiments in mice and rabbits were conducted in order to identify the pathogenicity of pathogenic bacteria.To identify the sensitivity of pathogenic bacteria to antibacterial drugs,cefazolin,ceftriaxone sodium,penicillin,vancomycin and other 30 antibacterial agents were used in bacteriostasis experiment in vitro of pathogenic bacteria.The results were as follows:Two strains of Gram-positive bacteria were isolated from the brain tissue of two sheep,both of which were identified as Listeria monocytogenes with strong virulence.The homology between two strains was 100.0%,and that of two strains and other referenced Listeria were higher than 99.0%.The two isolates were highly sensitive to drugs of cefazolin,piperacillin,carbenicillin,tetracyclines,doxycycline,aboren,erythromycin,vancomycin and chloroamphenicol.However,they were resistant to the 1st and 2nd generation cephalosporin antibiotics,oxacillin and PCST.This farm should adopt comprehensive prevention and control measures according to drug sensitivity test results to better control the disease in order to keep the safety of goats and meat products.

A method of the determination for 8 triazine pesticides in milk and dairy products from total diet was developed by isotope dilution-high performance liquid chromatography-linear ion trap mass spectrometry (HPLC-LIT-MS).Milk and dairy products from total diet were collected from 144 locations in China.Samples were ultrasonically extracted with acetonitrile,and then passed through gel permeation chromatography (GPC) to clear-up.The LC separation was performed on a CAPCELL PAK CR 1:20 column using acetonitrile and 10 mmol/L formic acid/0.1% formic aqueous solution as mobile phase.The ionization of the analytes was performed by electrospray mode.Selective reaction monitoring (SRM) was used for the monitoring of MS² transitions for each compound.The one-to-one internal standards were used for quantation.The average recoveries were in the range of 83.9% to 103.3%.The relative standard deviations were 2.5% to 9.1%.The limits of detection (LODs) were 0.003 to 0.300 μg/kg.In conclusion,the method was suitable for dietary exposure assessment of 8 triazine pesticides in milk and dairy products from total diet.

The purpose of this experiment was to compare the amino acid content,nutritional value of milk protein and regional difference in the raw milk of Inner Mongolia,so as to provide a theoretical basis for the quality evaluation of raw milk.70 milk samples were selected from milk storage tank with the similar season in Hulunbuir,Xilinguole Meng,Wulanchabu,Chifeng,Hohhot,Xing'an Meng and Tongliao,to measure the content of 17 amino acids in the raw milk of Inner Mongolia,and evaluate the nutritional value of milk protein according to the latest amino acid score recommended by FAO (2011),meanwhile compare the regional difference.The results showed that the total amount of 17 amino acids in Inner Mongolia raw milk was 3.117%,the content of glutamic acid was the highest (0.593%),followed by proline,leucine and lysine,glycine (0.060%) and cystine (0.061%) were the lowest.The proportion of essential amino acids (EAA) and amino acid score in raw milk of Inner Mongolia were higher than that of FAO/WHO ideal protein standard and ideal protein pattern.The content of amino acids in milk of different regions of Inner Mongolia were quite different.Compared with the amino acids content in the milk of Chifeng,the content of EAA,non essential amino acids (NEAA) and total amino acids (TAA) in Xilinguole Meng milk were significantly higher (P < 0.05),the content of valine,lysine,histidine,glycine and alanine were higher in Hulunbuir (P < 0.05).The content of leucine,lysine,histidine,alanine,aspartic acid and tyrosine in Hohhot were relatively higher (P < 0.05),the differences in other regions were not significantly (P > 0.05).It was concluded that the protein of raw milk from Inner Mongolia had high quality,and the amino acid nutritional value of Xilinguole Meng,Hulun Buir and Hohhot were better.