This study was aimed to explore the regulation of the miRNA molecule has-miR-5196-3p on the NOD-like receptor protein 3 inflammasome (NLRP3) during the process of Brucella infecting THP1 cells.Use TargetScan,MiRDB and other bioinformatics prediction sites to predict the binding sites of has-miR-5196-3p and NLRP3-3'UTR.Its wild-type and mutant dual luciferase reporter vectors were construced,respectively,has-miR-5196-3p mimics and negative control were transfected into 293T cells,and the relative luciferase activity changes of each group were detected by the dual luciferase reporter system to verify the targeting relationship between has-miR-5196-3p and NLRP3-3'UTR.THP1 cells were infected with Brucella,the mRNA expressions of NLRP3,caspase1 and has-miR-5196-3p were detected by Real-time PCR,and the protein expressions of NLRP3 and caspase1 were detected by Western blotting..has-miR-5196-3p mimics,has-miR-5196-3p inhibitor and negative control were transfected into THP1 cells,Real-time PCR and Western blotting were used to detect the expression level of NLRP3 and cysteinyl aspartate specific protease (caspase1).The results showed that the wild-type and mutant dual luciferase reporter vector containing NLRP3-3'UTR was successfully constructed.has-miR-5196-3p mimics extremely significantly reduced the relative luciferase activity of pmirGLO-NLRP3-3'UTR-wt(P<0.01).After Brucella infecting THP1 cells,NLRP3 inflammasome was activated,while has-miR-5196-3p did not change significantly (P>0.05).has-mi-R-5196-3p mimics could significantly inhibit the expression of NLRP3 (P<0.05),while has-miR-5196-3p inhibitor was the opposite.It showed that the miRNA molecule has-miR-5196-3p could bind to NLRP3-3'UTR and inhibit its expression.During the process of Brucella infecting THP1 cells,has-miR-5196-3p targets and negatively regulated NLRP3 inflammasome expression and inflammation happened.

Tyrosinase (Tyr) gene plays a key role in the process of regulating animal pigment synthesis.In order to understand the relationship between tyr gene and body colour variation of rainbow trout,in this study,the protein sequence analysis of tyr-1 and tyr-2 genes in rainbow trout was performed,the expression of 19 different development stages and 13 different tissues was determined by Real-time quantitative PCR in wild-type rainbow trout (WT) and yellow mutant rainbow trout (YT).Protein sequence analysis results showed that Tyr-1 and Tyr-2 proteins were both hydrophilic proteins and the structure was mainly random coil and alpha helix;Real-time quantitative PCR results showed that tyr-1 gene was expressed in WT and YT embryos at all stages.In WT,the expression of tyr-1 gene was the highest in the fertilized-stage,followed by the multi-cell stage,which was significantly higher than other stages (P<0.05),In YT,the expression of tyr-1 gene expression was the highest at the 16-cell,which significantly higher than other periods (P<0.05);tyr-2 gene was expressed from the WT blastula stage and the YT gastrula stage,the expression level reached the highest in the heartbeating stage,which was significantly higher than other periods (P<0.05).The expression levels of tyr-1 and tyr-2 genes were the highest in WT at 5 days post hatch and YT at 3 months post hatch.The expression levels of tyr-1 and tyr-2 genes in WT were higher than those in YT at embryonic stage and generally lower than those in YT during post hatch.In adult fish tissues,tyr-1 gene was highly expressed in skin,eyes,kidney,liver and other tissues,the tyr-2 gene was highly expressed in skin and eyes,and it was rarely expressed in other tissues.The above results indicated that tyr-1 and tyr-2 genes had a certain correlation with the body color variation of rainbow trout,which provided a theoretical basis for in-depth elucidation of the body color variation mechanism and body color genetic improvement of rainbow trout.

The purpose of this experiment was to study the expression of RNA m6A modification related genes ALK B homologue 5 (ALKBH5),fat mass and obesity associated protein (FTO),methyltransferase like 3 (METTLl3),methyltransferase like 14 (METTL14) and Wilms' tumor 1-associated protein (WTAP) in chicken skeletal muscle development,and analyzed its correlation with the level of skeletal muscle m6A methylation.Firstly,Real-time quantitative PCR was used to detect the mRNA expression levels of m6A methylation related genes in 12 (E12),14 (E14),16 (E16),18 (E18) embryonic and 1-day-old leg and chest muscles of Jinmao Hua chicken.And the mRNA expression levels in chicken myoblasts at 50%,100% proliferation and 1,2,3,4,5 d differentiation phases.Subsequently,the level of m6A methylation modification in the E12 and 1-day-old leg muscles and chest muscle tissues of Jinmao Hua chicken were detected by m6A methylation reagent test kit,and the correlation between the methylation level and the expression level of genes related to m6A methylation was analyzed.The results showed that the mRNA expression levels of m6A demethylation genes ALKBH5 and FTO were significantly up-regulated during skeletal muscle development (P<0.05),that wass,low expression at E12 and E14,and gradually up-regulated at E16 and E18,reaching the highest level at 1-day-old.The mRNA expression levels of the m6A methylation write genes METL14,METTL3 and WTAP gradually increased at E12,E14 and E16,and decreased at E18,and then the expression level rose back to 1-day-old.In the process of cell proliferation,the expression of ALKBH5, FTO,METL14,METL3 and WTAP genes were all up-regulated,during the process of cell differentiation,the expression levels of ALKBH5 and FTO genes were significantly up-regulated (P<0.05),reaching the highest on the fifth day of differentiation.The mRNA expression levels of METTL14,METTL3 and WTAP genes showed a downward trend on the 1st,2nd,3rd,and 4th day after cell differentiation,but increased on the 5th day after differentiation.The methylation level detection results showed that the change trend of the m6A methylation level of leg muscles and chest muscles was the same,and both decreased significantly during embryonic development (P<0.05),and reached the lowest at 1-day-old.The results of correlation analysis showed that the methylation level of chicken skeletal muscle RNA m6A was significantly negatively correlated with the mRNA expression levels of m6A demethylation modifier genes ALKBH5 and FTO (P<0.05).Based on the above experimental results,that m6A methylation modification was related to chicken skeletal muscle development,and the demethylation genes ALKBH5 and FTO might affect chicken skeletal muscle development by regulating the level of RNA m6A methylation.This study provided theoretical basis for further research on the function and molecular mechanism of m6A methylation in regulating chicken skeletal muscle growth and development.

The purpose of this study was to explore the diversity and genetic evolution of Streptococcus uberis (S.uberis) from different regions in China.In this study,38 strains of S.uberis isolated from some regions of China were used as the research objects.Through the determination of their minimum inhibitory concentration (MIC) and the sequencing of the whole genome frame,the related information of drug resistance,drug resistance gene and virulence gene of 38 strains were obtained.The alleles,sequence type (ST) and clone complexes (CCs) of 7 housekeeping genes were analyzed by multilocus sequence typing technique (MLST),and their evolutionary tree was established.A total of 15 antibiotics were used in MIC test,it was found that 38 strains of S.uberis were resistant to 13 antibiotics except for cefotaxime and florfenicol,and the drug resistance in Jiangsu province was significantly stronger than that in Xinjiang province.After comparison of resistance and virulence genes,only 24 strains were found to carry drug resistance genes,and the remaining 14 strains did not carry drug resistance genes and belonged to Xinjiang isolates,which was consistent with the results of MIC test.Among the 15 virulence genes,except for cfu,hasA/B and lbp,the carrying rate of the remaining 12 virulence genes was as high as 100%.Based on the MLST's results,38 strains of S.uberis belonged to 17 ST types,of which 15 ST types were not reported,and only 1 strain of supan01 belonged to ST-5 CCs.The phylogenetic tree results showed that the 38 isolates largely unrelated,and their distribution was region-dependent.Moreover,the phylogenetic tree results suggested that the S.uberis strains prevalent in some parts of China were largely different from those prevalent in Western countries.This study contributed to the MLST database of S.uberis and provided a reference basis for understanding of the genetic and distribution characteristics of S.uberis in China.

The aim of this experiment was to clone the specificity protein 1 (SP1) gene,analyze its sequence,and investigate the effect of SP1 gene on the preadipocytes differentiation of Small-tailed Han sheep.The 1-month-old adipose tissue in the tail of Small-tailed Han sheep was selected for the isolation,culture and induction differentiation of preadipocytes.SP1 gene CDS was cloned by overlapping PCR.Bioinformatics softwares were used to analyze the SP1 gene CDS,and predict the structure,functional domain,subcellular localization and post-translational modification of SP1 protein.Overexpression and inhibition of SP1 gene in preadipocytes was performed by lentivirus infection.The overexpression and antisense vectors of SP1 gene and control plasmids packed with lentivirus were transfected into 293T cells,and the preadipocytes of Small-tailed Han sheep were infected with lentiviruses.Oil Red O staining was used to observe the ability of lipid accumulation in adipocytes.The mRNA expression of SP1 gene and adipogenic marker genes were detected by Real-time quantitative PCR.The results showed that the full-length of SP1 gene CDS was 2 340 bp,encoding 779 amino acids.Sequence similarity analysis showed that SP1 gene CDS of Small-tailed Han sheep had the highest similarity with Ovis aries and Capra hircas,followed by Bos taurus,Equus caballus,Sus scrofa,Homo sapiens,Mus musculus and Rattus norvegicus,and the lowest similarity with Gallus gallus,which was consistent with the result of phylogenetic analysis.The SP1 protein was located in the nucleus with 109 phosphorylation sites.Its secondary structure consisted of alpha helix,beta turn,radom coil and extended chain,and the proportion of them were 16.94%,8.60%,54.17% and 20.28%,respectively.Three zinc finger domains were found in both secondary and tertiary structures.The overexpression,interference and control plasmids were transfected into 293T cells,and the cells in each group presented strong green fluorescence,which indicated the vector was successfully transfected into 293T cells.After the lentiviruses infects the preadipocytes of the Small-tail Han sheep,the mRNA expression of SP1 gene in overexpression group was extremely significant higher than that in control group (P<0.01),while the expression of SP1 gene mRNA in inhibition group was extremely significant lower than that in control group (P<0.01).After SP1 gene was overexpressed,the mRNA expression of lipogenic marker genes were extremely significant up-regulated (P<0.01),and more lipid droplets were deposited.The results were opposite after SP1 gene was inhibited.Therefore,SP1 gene had a positive regulatory effect on the differentiation of preadipocyte in the caudal adipose tissue of Small-tailed Han sheep.

This experiment aimed to study the effect of vitamin E (VE) on H₂O₂-induced oxidative damage and tight junction-related gene expression in MAC-T cells.There were three groups in this study,control,H₂O₂ and H₂O₂+VE groups.MTT was used to detect cell survival rate,while colorimetric method was employed to detect the activity of superoxide dismutase (SOD) and catalase (CAT),the content of malondialdehyde (MDA) and reactive oxygen species (ROS) in cell culture medium.The expression levels of tight junction genes ZO-1,occludin and claudin-1 were detected by Real-time PCR.The results showed that compared with the control group,the survival rate of MAC-T cells in the H₂O₂ group decreased significantly (P<0.05),the content of ROS and MDA increased significantly (P<0.05),and the activity of SOD and CAT decreased significantly (P<0.05).Compared with the H₂O₂ group,the cell survival rate of the H₂O₂＋20 μmol/L VE group increased significantly (P<0.05),the content of ROS and MDA decreased significantly (P<0.05),and the activity of SOD and CAT increased significantly (P<0.05).In addition,compared with the control group,the H₂O₂ group extremely significantly inhibited the relative expression of tight junction genes (P<0.01).Compared with the H₂O₂ group,the relative expression of tight junction genes was extremely significantly increased after the addition of vitamin E (P<0.01).This study demonstrated that vitamin E could lessen the oxidative damage induced by H₂O₂ in MAC-T cells,and significantly alleviate the down-regulate tight junction genes expression in MAC-T cells induced by H₂O₂.

The objective of this study was to investigate the protective effect of superoxide dismutase mimetic (SODm) on intestinal epithelial cell (IPEC-J2) under H₂O₂-induced oxidative stress.Using MTT method to screen out the proper concentration of H₂O₂ applied in structuring oxidative stress model.IPEC-J2 were cultured with 0,0.05,0.5,5,50,500 and 2 000 U/mL SODm,respectively.The survival of each group was detected by MTT in 2,4,8 and 12 h to determine the proper range and duration of a SODm dose.Based on the model of oxidative stress and the proper range and duration of SODm,IPEC-J2 were randomly grouped as the control group,the model group with H₂O₂,the disposed group with SODm (SODm_0.5,SODm₅,SODm₅₀),the disposed group with SOD (SOD_0.5,SOD₅,SOD₅₀),then cell viability,ROS levels and SOD,GSH-Px,MDA,T-AOC levels in cells were measured in each group.The results were showed as follows:①1.0 mmol/mL H₂O₂ was the proper concentration in structuring oxidative stress model.②The survival ratios of the disposed group with SODm of 0.5~50 U/mL in 4,8 and 12 h was significantly higher than that of the control groups (P<0.05).The survival ratio was highest at 8 h and decreased at 12 h.③The IPEC-J2 survival rate of the SODm₅ and SOD₅ groups were significantly higher than that in other groups (P<0.05) except for the blank group,and the ROS contents in the SODm and SOD groups were significantly lower than that in the model group (P<0.05).The activities of SOD,GSH-Px and T-AOC were significantly decreased,and MDA content were significantly increased in model group compared with the control group (P<0.05).The treatments of SODm and SOD increased the activities of SOD,GSH-Px and T-AOC,and significantly decreased MDA content compared with the model group(P<0.05),The T-AOC level of SOD₅₀ group was higher than that of SODm₅₀ group (P<0.05).In conclusion,SODm had protective effect on IPEC-J2 cells under H₂O₂-induced oxidative stress.

To clarify the nutritional and immune status of Honghe Yellow cattle under the grazing condition,60 Honghe Yellow cattles comprising half males and half females were selected and divided into 6 groups according to sex and age (12,36 and 60 months old).Their sera were collected from jugular vein to determine biochemical and immune indicators.As a result,①There were significantly lower contents of total proteins (TP) and globulin (GLOB) within the same sex at the age of 12 months than those at the age of 60 months (P<0.05);The activity of alkaline phosphatase (ALP) and the content of glucose (GLU) were significantly higher in 12-month-old cattle than in 60-month-old cattle within the female (P<0.05),and no significant difference existed for the other serum biochemical indicators between ages.With the increase of age,the contents of TP and GLOB increased while the average activity of ALP decreased in the sera of both males and females.The contents of TP,albumin (ALB),GLOB and aspartate aminotransferase (AST) of females at the three ages were higher than those of males,but the activity of ALP and the content of GLU of males were higher than those of females.② For the same sex,the serum levels of interleukin (IL)-2,IL-8,IL-10,immunoglobulin(Ig)A,IgG,IgM,interferon-γ (IFN-γ),and tumor necrosis factor α (TNF-α) were not significantly different among the three ages (P>0.05),except for a significant difference in serum IL-8 between 12 and 36-month females (P<0.05).The contents of IL-2,IgA,IgG,IgM,IFN-γ and TNF-α in 12,36 and 60-month-old females were higher than those in males (P<0.05).In conclusion,with the increase of age,the serum TP and GLOB contents significantly increased while the ALP activity decreased,and basically,females had higher serum biochemical indexes and immune indexes than females in Honghe Yellow cattle.

This experiment was aimed to study the isolation,culture,multi-lineage differentiation potential of umbilical cord mesenchymal stem cells (UC-MSCs) from argali in vitro.UC-MSCs were isolated from umbilical cord of newborn male fetus after delivery in argali by tissue block culture and cultured in vitro.UC-MSCs were induced to differentiate into osteoblasts,chondroblasts and adipoblasts to detect their multi-lineage differentiation potential.The results showed that the UC-MSCs of argali had the unique biological characteristics of being cluster and fibrous by tissue block culture,and the growth curve was "S" shape.The average doubling time of the cells was 33.50 h,and it could differentiate into osteoblasts,chondroblasts and adipoblasts.The osteoblasts were stained with Alizarin red S showing typical orange-red calcium deposits,chondroblasts were stained with Alcian blue showing a blue cartilage matrix,and adipoblasts were stained with oil red O showing dark red lipid droplets.Real-time quantitative PCR detection results revealed that with the extension of inducting time,the relative expression of bone gamma-carboxyglutamate protein (BGLAP),osteopontin (OPN) and Runt-related transcription factor 2 (RUNX2) genes in the osteogenic differentiation cells increased extremely significantly (P<0.01).The expression of lumican (LUM) gene in the chondrogenic differentiation cells increased significantly,while the expression of biglycan (BGN) and sex determining region Y-box 9 (SOX9) genes increased extremely significantly (P<0.01).The relative expression of lipoprotein lipase(LPL) and peroxisome proliferator-activated receptor γ(PPAR-γ) genes in the adipogenic differentiation cells increased extremely significantly (P<0.01).The results indicated that UC-MSCs could be isolated from umbilical cord tissue attached to placenta and differentiated into osteoblasts,chondroblasts and adipoblasts.

The purpose of this experiment was to screen the appropriate level and proportion of rumen protected unsaturated fat (RPUF) and flaxseed in the diet of Holstein steers.Four Holstein steers (average body weight 580 kg±50 kg,2.5 years old) with rumen fistula were added with 2 sources of fat (RPUF and flaxseed) at 3 supplementation levels (4%,5% and 6%) interacted with 4 different ratios (100:0,85:15,70:30 and 55:45) per kg diet were used in 3×4 two-factorial design.Fat additional level/ratio in the twelve groups were 4/100,4/85,4/70,4/55,5/100,5/85,5/70,5/55,6/100,6/85,6/70 and 6/55,respectively.The effects of rumen protected unsaturated fat and whole flaxseed with different levels and ratio on rumen fermentation,nutrients degradability and purine derivatives were studied.The experiment was divided into 12 stages.The first 14 d of each stage was an adjustment period and the following 5 d was for sample collection.The results showed that:①Ruminal pH,concentrations of acetate,propionate and total volatile fatty acids were not affected by lipid supplement levels interacted with different ratios (P>0.05),ammonia nitrogen concentration and the ratio of acetate to propionate of 6% fat groups were significantly increased compared to 4% groups (P<0.05).②For the dry matter effective degradability,4% fat groups were significantly higher than 5% and 6% fat groups (P<0.05),4/85,4/70 and 4/55 groups were significantly higher than 5/55 (P<0.05).For the crude protein effective degradability,70:30 group was significantly higher than 100:0 group (P<0.05),5/70 group was significantly higher than 6/100 group (P<0.05).③The content of allantoin and purine derivatives in 4% fat groups were significantly higher than 5% and 6% fat groups (P<0.05).In conclusion,from the perspective of rumen fermentation,nutrients degradability and urine purine derivatives yield,the optimum of lipid addition level was 4%,and the optimum of ratio (RPUF:flaxseed) was 70:30.

The intestine is an important digestive,endocrine and immune organ of broilers,and intestinal health depends on the dynamic balance between nutrients,microbial flora and intestinal mucosa.Intestinal digestion and absorption are directly related to the growth of intestinal epithelial cells and intestinal morphology integrity.As an energy source,sodium butyrate can stimulate intestinal epithelial cell proliferation,and improve intestinal mucosal morphology in broiler and promote villus growth and intestinal tissue development.Through the intestinal free fatty acid receptors FFAR2 and FFAR3,sodium butyrate mediates the secretion of various hormones from intestinal endocrine cells,promotes gastrointestinal mucosa development,and stimulates the secretion of digestive enzymes from the stomach and pancreas,and promote nutrient digestion and absorption.Sodium butyrate is an essential regulator of intestinal homeostasis,which can stimulate mucin production,increase the thickness of mucus layer,reduce the permeability of colonic epithelium,and maintain intestinal integrity and mucosal barrier function.Sodium butyrate promotes the synthesis of host defense peptides (HPDs),inhibits intestinal harmful bacteria's proliferation,and reduces endotoxin's damage to intestinal epithelial cells.By inhibiting NF-κB kinase (IKK),sodium butyrate can down-regulate the pro-inflammatory pathway,inhibit the activation of NF-κB,prevent intestinal mucosal inflammation,promote digestion and absorption,and protect intestinal health.Therefore,sodium butyrate has intestinal protection and antibacterial effect,enhances intestinal integrity,promote digestion and absorption of nutrients and improves immunity and disease resistance.Under the background of the prohibition of antibiotics in the feed industry,it is very important to study and apply sodium butyrate as an antibiotic substitute.The authors focused on the mechanism of the effect of sodium butyrate on intestinal function,in order to provide a scientific basis for the replacement of antibiotics in broiler feed.

This study aimed to determine the effect of rumen fermentation products of acetic acid and propionic acid as well as acetic acid to propionic acid ratio on dry matter intake,milk yield,and milk composition in dairy cow.By reviewing the published literature,a model was constructed using meta-analysis method to perform regression analysis and correlation analysis.A total of 181 treatments have been sorted out from 51 related literatures.The articles described the effects of different feed ingredients,nutrients,and feed additives on the rumen fermentation indicators and production performance of lactating dairy cows at different physiological stages.The results of meta-analysis of 51 experiments showed that,compared to rumen fermentation production of acetic acid and propionic acid,acetic acid to propionic acid ratio was more closely related to milk yield (R²=-0.426,P<0.01),milk fat percentage (R²=0.359,P<0.01),milk fat yield (R²=-0.257,P<0.01),volatile fatty acid (R²=-0.226,P<0.05),and dry matter intake (R²=-0.485,P<0.01).There was a extremely significant unary linear regression between acetic acid to propionic acid ratio and dry matter intake,milk fat yield,milk production,and milk fat percentage (P<0.01).Dry matter intake,milk fat yield,milk production would decrease extremely significantly when the ratio of acetic acid to propionic acid increased and milk fat percentage would increase extremely significantly (P<0.01).When the ratio of acetic acid to propionic acid in the rumen increased by 1,the milk fat proportion increased by 0.26%,and the dry matter intake,and milk production of dairy cows decreased by 2.34 and 4.99 kg respectively.Through meta-analysis,it was found that acetic acid to propionic acid ratio could predict the proportion of milk fat in milk more scientifically,and accurately reflect the change of milk fat rate.This study could provide reference for the selection of diet and the regulation of milk quality.

In order to further explore the effective utilization of Oleum cinnamomi,based on the previous studies,the present study was conducted to investigate the effect of dietary addition of 300 mg/kg coated Oleum cinnamomi on microbial diversity and composition in the ileum of broiler chickens.Single factor experimental design was employed and a total of 132 one-day-old Cobb 500 broiler chickens were randomly assigned into control and experimental groups,respectively.There were 6 replicates in each group with 11 chickens (5 males and 6 females) in each replicate.The chickens in control group were fed the basal diet and the ones in experimental group were fed basal diet supplemented with 300 mg/kg of coated Oleum cinnamomi.The trail lasted for 21 d.The results showed that:Dietary addition of 300 mg/kg of coated Oleum cinnamomi did not affect growth performance (P>0.05).Compared with the control group,experimental group significantly increased Shannon and Sobs indices and significantly decreased the Simpson index (P<0.05).There were 133 shared OTUs in the ileal microflora of both control and experimental groups,4 unique OTUs in the control group,and 135 unique OTUs in the experimental group.The ANOSIM analysis showed that the inter-group difference of ileal microbiota composition was higher than that of intra-group (P<0.05).The relative abundance of Streptococcus spp.in the ileum of chickens in experimental group was lower than that in the control group (P<0.05).There was no significant difference in ileal abundance of other genus between two groups (P>0.05).In summary,dietary addition of 300 mg/kg of coated Oleum cinnamomi increased the microbial diversity,and reduced the relative abundance of Streptococcus spp.in the ileum of broiler chickens at 21 days of age.

The aim of this study was to explore the correlation between genetic polymorphism of erythropoietin (EPO), peroxisome proliferator-activated receptor α (PPARα) genes and hypoxia adaptability in yak.375 ears sample from 6 yak groups (Zhongdian yak,Maiwa yak,Sibu yak,Leiwuqi yak,Pali yak and Shenzha yak) and Sanjiang cattle at different altitudes were collected to extracted DNA and constructed DNA pool.The polymorphism of EPO and PPARα genes were detected by direct sequencing and PCR-RFLP.Finally,the association between the SNPs of the candidate genes and the high altitude hypoxia adaptation of yak were analyzed by SHEsis.The results showed that both EPO and PPARα genes had 3 SNPs sites,in EPO gene there were rs527G→A,rs1031A→T and rs1192T→C,while in PPARα gene the 3 SNPs sites were rs77363C→T,rs77471C→A and rs77534C→T.The fitness test indicated that the three SNPs sites of EPO gene all conformed to Hardy-Weinberg equilibrium;The rs77363C→T locus of PPARα gene was accorded with Hardy-Weinberg equilibrium in 6 yak groups (P>0.05).However,rs77471C→A and rs77534C→T of PPARα gene were deviated in Maiwa yak (P<0.05).The haplotype analysis showed that the distribution frequency of the ATC haplotype of EPO gene increased in high altitude yaks with the increase of altitude.Furthermore,the frequency of the TAC haplotype of PPARα gene was significantly higher than other haplotypes in 6 yak groups.Taken together,EPO and PPARα genes could be used as molecular markers for further exploring the mechanism of high altitude hypoxia adaptation of yak.

In order to explore the maternal genetic difference between Menyuan White yaks in Qinghai province and Tianzhu White yaks in Gansu province,the mtDNA D-loop sequences of 31 Menyuan White yaks were sequenced in this study.Combined with 235 D-loop sequences of Tianzhu White yaks reported in GenBank,266 White yaks D-loop sequences were analyzed.The results showed that 80 polymorphic sites were detected in the 638 bp D-loop sequences,including 5 single polymorphic sites and 75 parsimony informative sites after excluding 64 InDel sites.A total of 56 haplotypes were identified according to the nucleotide sequence variations.Totally,Menyuan White yaks had 7 specific haplotypes and Tianzhu White yaks owned 43 specific haplotypes,but only 2 haplotypes were shared by them.The haplotypes of Menyuan White yaks belonged to A,B and C haplogroups,while the haplotypes of Tianzhu White yaks falled within A,B,C,D,E and F haplogroups and contained 4 Bos taurus haplotypes,which indicated that there was genetic introgression of Bos taurus in Tianzhu White yak breed.The haplotype diversity and nucleotide diversity of Menyuan White yaks were 0.862 and 0.007,respectively,while those of Tianzhu White yaks were 0.946 and 0.014,respectively,which showed that the level of genetic diversity of Tianzhu White yaks was higher than that of Menyuan White yaks.The genetic differentiation index (Fst) between two populations was 0.114,which indicated that the genetic differentiation between them was moderate.The result of phylogenetic analysis showed that the 56 haplotypes could be divided into two distinct lineages (Ⅰ and Ⅱ),indicating that White yaks had two maternal origins.In conclusion,both Menyuan White yaks and Tianzhu White yaks had rich maternal genetic diversity,but the latter had higher level of genetic diversity than the former.There was a moderate differentiation between two populations and Tianzhu White yaks had a certain degree of Bos taurus genetic introgression.Each White yak breed (population) was composed of two maternal lineages,suggesting that White yaks had two maternal origins.Considering the moderate genetic differentiation between two populations and the specific haplotypes owned by them,we suggested that they should be considered as independent "genetic unit"for genetic resource protection and then corresponding conservation and breeding practice would be carried out.

In this study,polymorphisms of yak lactoferrin (LF) gene were investigated and their effects on some milk quality traits were estimated so as to enrich the molecular genetic data for yak.Variations in 5'UTR and intron 4 of LF gene were checked by PCR-SSCP in 3 yak populations (Gannan yak,Tianzhu White yak and Tibetan yak) and their effects on the milk quality traits were analyzed in Gannan yak.The results showed that:Variation of c.-568 G>A and c.499+56 C>A were found in 5'UTR and intron 4 of yak LF gene,respectively.Allele A₁,genotype A₁B₁ of 5'UTR and genotype A₂B₂ of intron 4 were the dominant allele and genotype in three yaks.The allele A₂ of intron 4 was dominant in Gannan yak and Tianzhu White yak,but allele B₂ was dominant in Tibetan yak.The polymorphism information content (PIC) in 5'UTR and intron 4 of LF gene in yaks ranged from 0.25 to 0.50,belonging to moderate polymorphism.The association analysis showed that the variations in LF gene affected the milk quality traits in different parities of Gannan yak.Individuals with genotypes A₁A₁ and A₁B₁ of 5'UTR in second parity yak had significantly higher non-fat solids rate than B₁B₁ genotype (P<0.05),the genotypes A₂B₂ and B₂B₂ of intron 4 in the third parity had significantly higher milk fat rate than A₂A₂ genotype (P<0.05),as well as yaks with B₂B₂ genotype in fourth parity had significant lower milk rate than A₂A₂ and A₂B₂ genotype (P<0.05).In conclusion,the polymorphisms were found in LF gene of Gannan yak,Tianzhu White yak and Tibetan yak,and these variations in 5'UTR and intron 4 that were association with content of non-fat solids and milk fat rate of Gannan yak,respectively,could be used as potential molecular genetic markers for milk quality traits.

The aims of current study were to show the process of genomic imputation and investigate the factors affecting accuracy of genomic imputation.The data of 50K panel imputed to 150K for dairy cattle was used to compare accuracy and concordance of imputation for two software (Beagle 5.1 and Minimac 3).Concordance was calculated by cross validation of individuals with imputed data and real data.The target population for imputation should be phased by Minimac 3 and the function of Beagle 5.1 including pre-phasing and imputation.The correlation of concordance between Minimac 3 and Beagle 5.1 were 0.98.For Beagle 5.1,the average of imputation accuracy (r²) and concordance were 0.9841 and 0.9914,respectively,and the correlation between imputation accuracy and concordance was 0.39.For Minimac 3,the average of imputation accuracy (r²) and concordance were 0.9782 and 0.9911,respectively,and the correlation between imputation accuracy (r²) and concordance was 0.36.Imputation accuracy (r²) was associated with minor allele frequency due to the formula for calculating accuracy from the software.With the increasing of minor allele frequency and heterozygosity for makers,the concordance of imputation was decreased.There was a steep decline when heterozygosity was higher than 0.6 (concordance of imputation was lower than 0.8).However, the accuracy of Beagle 5.1 software was better than that of Minimac 3 software under the same minor allele frequency and heterozygosity of imputed site. The accuracy of imputation (r²) was mainly affected by heterozygosity of SNPs and Beagle 5.1 had better performance on imputation accuracy than that of Minimac 3. Using concordance as the accuracy of imputation to select SNPs could avoid losing useful makers for further study.

Wnt family member 3a (Wnt3a) gene is one of the important members in the Wnt signaling pathway,and the Wnt signaling pathway plays important roles in the development of animal skin and feather follicles.In this study,Wnt3a was selected as a candidate gene to detect the mRNA expression pattern for different tissues and the genetic effect of single nucleotide polymorphisms sites (SNPs) on feather follicle density in chicken.Real-time quantitative PCR was used to detect the mRNA expression profiles among different tissues.PCR amplification and direct sequencing technologies were used to identify the SNPs of the Wnt3a gene and analyze the difference of feather follicle density among different SNP genotypes.Furthermore,the Wnt3a mRNA expression difference of skin tissue between different chicken breeds was detected by Real-time-quantitative PCR.The results showed that the mRNA expression of skin was significantly higher than that of other tissues (P<0.05),the following tissues were liver,testis,ovary and hypothalamus,and heart,chest muscle,pituitary gland and spleen were the lowest.This study identified 3 SNPs,they were g.2587569 G>A,g.2555812 T>C and g.2555377 T>C,located in exons 2 and 3,and intron 3,respectively.Chi-square test showed that the genotype distribution of these SNPs had deviated significantly from Hardy-Weinberg equilibrium.Population genetic parameters analysis showed that g.2555812 T>C was moderately polymorphic,the number of effective alleles was 1.7,which indicated that the degree of variation in the population was higher than other SNPs.The correlation analysis between SNPs and feather follicle density showed that CC genotype at g.2555377 T>C were significantly higher than that of TT and CT genotypes (P<0.05).The mRNA expression level of the Wnt3a gene in skin tissues was significantly different between varieties,which showed a significant difference in feather density (P<0.05).The results of the study would provide a reference for further analyzing the role of the Wnt3a gene in chicken skin and feather follicles development and screening of molecular markers that contribute to feather follicle density traits.

In order to further understand the genetic evolutionary relationship of Ovis ammon polii and domestic sheep,in this study,DNA of 20 Ovis ammon polii was extracted and mitochondrial DNA (mtDNA) cytochrome b (Cytb) gene was amplified by PCR.The mtDNA Cytb gene sequence in GenBank was downloaded,and the phylogenetic tree was constructed by Neighbor-Joining,so as to clarify the genetic diversity and evolution of wild sheep and domestic sheep.The results showed that the mtDNA Cytb gene of Ovis ammon polii was 1 140 bp,containing 58.3% A and T bases.There were 10 haplotypes,and the average haplotype diversity (Hd) was 0.884.There were 122 SNPs,accounting for 2.35% of the total nucleotide.Among them,there were 2 single polymorphic sites and 120 simple information sites,all of which were two-base mutations.The frequency of conversion was higher than that of inversion.The mean nucleotide diversity (pi) was 0.02090.The mean nucleotide difference (K) was 23.826.The results indicated that the mtDNA Cytb region of Ovis ammon polii was rich in haplotype and nucleotide genetic diversity.Phylogenetic analysis showed that wild sheep were divided into three distinct branches,among which Ovis ammon polii formed a single branch,and the genetic distance with Ovis ammon hodgsoni was the closest,and the genetic distance with Ovis ammon karelini, Ovis ammon darwini was the furthest,the domestic sheep breeds was closely related to Ovis musimon,but far from Ovis ammon polii.The results showed that Ovis ammon polii had a low contribution to the origin and evolution of domestic sheep,which supported the judgement that Ovis musimon might be the ancestor of domestic sheep.

To explore the polymorphism and gene function of LHCGR gene of 4 sheep breeds,the 2 loci (g.75747421A>C and g.75748200T>A) of LHCGR gene among Dorper sheep,Tan sheep,Small-tailed Han sheep and Tan×Han hybrid sheep were detected by Sequenom MassARRAYSNP.The secondary structure of mRNA and the physicochemical properties,structure and protein interaction between the 2 sites before and after the mutation were predicted and analyzed by bioinformatics software.The results showed that there were 3 genotypes of LHCGR gene g.75747421A>C in 4 breeds,which were CC,CA and AA. g.75748200T>A had 3 genotypes of AA,AT and TT in 4 breeds.χ² test indicated that g.75747421A>C and g.75748200T>A were in Hardy-Weinberg equilibrium in 4 sheep breeds (P>0.05).Bioinformatics analysis showed that the relative molecular weight of LHCGR gene was 73 599.70,the theoretical isoelectric point was 8.30,the fat coefficient was 98.90,and the total average hydrophilicity was 0.191,which was an unstable hydrophobic protein.The secondary structure of LHCGR gene mRNA and the secondary structure and tertiary structure of the encoded protein were all changed before and after mutation.The results of protein-protein interaction showed that the proteins interacting with LHCGR protein mainly included bone morphogenetic protein 15 (BMP15), chromogranin A (CGA), mevalonate kinase (MVK), cholesterol side chain cleavage enzyme (CYP11A1), guanine nucleotide binding protein α subunit (GNAS).This study successfully screened the LHCGR gene polymorphism loci of 4 sheep breeds,which could provide a certain theoretical basis for the marker-assisted selection and breeding of sheep fecundity.

To investigate the effect of cryopreservation on function of landrace sperm,fresh and frozen semen were selected as research materials.Sperm motility was detected by sperm analyzer.Sperm viability was detected by Trypan blue staining.The cleavage rate and blastocyst rate were measured by in vitro fertilization (IVF).The acrosome integrity rate,mitochondrial permeability transition pore (MPTP) activity,mitochondrial membrane potential (MMP),mitochondrial activity and reactive oxygen species (ROS),and integrity of sperm were measured using different kits.Expression levels of asthenospermia related genes SMCP, TEKT3,DNAH1 and TCTE3 were measured by Real-time quantitative PCR.The results showed that the motility,viability and acrosome integrity rate of frozen sperm were significantly lower than that of fresh sperm (P<0.05).The cleavage rate and blastocyst rate of frozen sperm decreased significantly compared with fresh sperm (P<0.05).The results of mitochondrial function analysis showed that the relative fluorescence units (RFU) value of MPTP,optical density (OD) value of mitochondrial activity and mitochondrial fluorescence ratio of MMP in frozen sperm were significantly lower than that of fresh sperm (P<0.05).The detection of ROS in sperm mitochondria showed that the RUF value of ROS in frozen sperm was significantly higher than that of fresh sperm (P<0.05).DNA integrity test results showed that the sperm tail dragging rate of frozen sperm increased significantly compared with fresh sperm (P<0.05).However,the genes expression of asthenospermia related proteins had no significant difference compared with fresh sperm (P>0.05).In conclusion,cryopreservation decreased the motility,viability,mitochondrial function and DNA integrity of boar sperm,resulting in the decrease of the fertilization ability of frozen sperm.

To explore the role of long noncoding RNA (lncRNA) at early stage of enterotoxigenic Escherichia coli (ETEC) infection,the expression profile of lncRNAs in the porcine intestinal epithelial cells (IPEC-J2) infected with ETEC was analyzed.ETEC F41 strain was used to infect IPEC-J2 cells.The cells were collected before infection and at early infection (4 h post infection),followed by high-throughput sequencing via the Illumina HiSeq Xten platform.A total of 9 975 lncRNAs were discovered in this study.Compared with cells before infection,100 differentially expressed lncRNAs were found at the early stage of ETEC infection.Among them,40 differentially expressed lncRNAs were up-regulated,and 60 differentially expressed lncRNAs were down-regulated.The target genes of differentially expressed lncRNAs were predicted using miRanda-3.3a and psRobot_v1.2 software,followed by GO and KEGG pathway enrichment analysis.The results showed that at the early stage of infection,the target genes of differentially expressed lncRNAs were significantly enriched in the GO functional items such as nuclear,nucleolus,metabolic process regulation and development process.The KEGG analysis showed that the target genes of differentially expressed lncRNAs at the early stage of infection were significantly enriched in the pathways including PI3K-Akt signaling pathway,regulation of actin cytoskeleton,focal adhesion and cell cycle.Real-time quantitative PCR was performed to validate the expression of 5 randomly selected differentially expressed lncRNAs,which showed that the expression differences before and after infection were consistent with the sequencing results.This study analyzed the lncRNA expression profile of IPEC-J2 cells infected with ETEC at the early stage,which provided a basis for exploring the functional mechanism of key lncRNAs during ETEC infection.

To understand the species of ticks from pet dogs and their carrying status of Brucella in Shihezi,Xinjiang,on the basis of morphological classification,the ticks from pet dogs were detected by molecular biology method based on mitochondrial gene 12S rRNA and cytochrome oxidase Ⅰ (COⅠ).DNAMAN software was used to compare and analyze sequence homology,and sequence analysis software Mega 7.0 was used to construct a phylogenetic tree of tick species by using the Neighbor Joining method to analyze the genetic evolutionary relationship of tick species.Based on the Brucella outer membrane protein Omp22 gene,ticks from pet dogs were tested for Brucella by PCR to determine the Brucella carrying status of ticks from pet dogs.The results showed that the morphology of 436 ticks from pet dogs were consistent with the molecular biology identification results of mitochondrial genes (12S rRNA and COⅠ),and they were all Rhipicephalus turanicus,one of the dominant species in Xinjiang.The phylogenetic tree of tick species constructed based on the 12S rRNA gene showed that the sequence of Rhipicephalus turanicus from pet dogs obtained in this experiment was clustered well with the sequence of the corresponding tick known in GenBank.The results of nested PCR amplification based on the Omp22 gene of Brucella showed that the positive rate of Brucella carried by Rhipicephalus turanicus from pet dogs was 4.82% (21/436),and the homology was 100%.BLAST comparison showed that Brucella carried by Rhipicephalus turanicus from pet dogs obtained in this experiment was related to the Xinjiang epidemic strain YC31 (GenBank accession No.:MK201679.1),QH5 (GenBank accession No.:MK201678.1),EM3 (GenBank accession No.:MK201677.1) and ML9 (GenBank accession No.:MK201676.1),and the homologies were all 100%.In this study,the species of ticks from pet dogs in Shihezi,Xinjiang,and their carrying Brucella were studied by morphology and molecular biology,and provided basic information for the species of parasitic ticks on pet dogs and the monitoring and control of tick-borne diseases.

In recent years,exotic insect-borne diseases,such as African swine fever,nodular dermatosis have infected China.However,the risk of some insect-borne pathogens,such as African horse plague,which have been popular abroad,being introduced into China is becoming higher and higher,seriously endangering the development of animal husbandry and public health safety in China.Especially since August 2018,the prevalence of African swine fever in China has brought important warning information to the pig industry,that is,to prevent the entry of vectors in pig farms and block the transmission of pathogens will become the focus of pig farms' biosafety.Mosquitoes,flies,midges and ticks are important vector factors for the transmission of epidemic diseases in pig farms and infected pigs.The types of vectors and the pathogens isolated from vectors,such as viruses,bacteria and parasites,are complex.The differences of biological characteristics of vectors have different effects on the transmission mode and transmission ability of pathogens.In view of this,the author summarized in detail the main types of insect vectors existing in pig farms and the important pathogens of swine diseases,including Porcine encephalitis B virus,Porcine reproductive and respiratory syndrome virus,African swine fever virus,Gatah virus,Swine enteric coronavirus,Escherichia coli,Streptococcus suis,and so on,and discussed their harmfulness and transmission risks,so as to reduce the economic losses caused by insect borne diseases in pig farms loss.

In order to investigate the prevalence,species of Eimeria and risk factors in rabbits in Gansu province,in this study,592 fecal samples of 5 different species of rabbits were collected from Pingliang,Jiuquan and Wuwei cities of Gansu province,and the genomic DNA of each sample was extracted.The Eimeria rabbit species-specific PCR method was used to amplify the Eimeria rabbit gene fragment based on the ribosomal first inner transcribed spacer (ITS1) primers.The results showed that overall prevalence of Eimeria in rabbits was 50%(296/592).Sequencing analysis showed that a total of 7 different Eimeria species in rabbits were identified,including E.magna (24.5%,145/592),E.intestinalis (4.1%,24/592),E.media (11.5%,68/592),E.perforans (19.8%,117/ 592),E.vejdovskyi (24.5%,145/592),E.exigua (4.9%,29/592) and E.coecicola (0.84%,5/592).The E.magna,E.vejdovskyi and E.perforans were the dominant species in rabbits.Moreover,multiple infections had been found,and up to quadruple infections.Statistical analysis showed that region (P<0.05),breed (P<0.05) and age (P<0.05) were the risk factors affecting the prevalence of Eimeria in rabbits,suggesting that we should take effective measures to prevent and control these three factors.The present study revealed the prevalence,Eimeria species and risk factor in rabbits infection in some areas of Gansu province.The results provided scientific basis information for the prevention and control of Eimeria in rabbits in Gansu province.

The aim of current study was to construct a safer and more effective new type of immune castration DNA vaccine.With the help of the self-cleavage function of the 2A peptide and introducting of a balanced lethal system instead the resistance gene screening process,the hypot halamic-pituitary-gonadal axis (HPG) upstream regulatory gene kissin 1 (KISS1) and hormone-releasing hormone (GnRH) being selected as the target were successfully transferred into the non-resistant screening plasmid pVAX-asd.Enzyme digestion verification and sequencing comparison verification on the insertion direction and sequence of the target gene were completely correct.When recombinant plasmid being transfected into HeLa cells,the results of amplification of the target gene after reverse transcription showed that the recombinant plasmid could be transcribed normally in eukaryotic cells,ensuring that the recombinant plasmid could be expressed normally after being introduced into the body,thereby causing a specific immune response.The successfully constructed plasmid was transformed into the attenuated Salmonella choleraesuis C500 to obtain a live vector vaccine that could be directly orally immunized.The results of enzyme digestion and sequencing showed that the co-expression recombinant plasmid was successfully introduced into the engineered bacteria.The live bacteria vaccine was continuously passaged in vitro for 50 times,and strains of 0,2,5,10,20,30,40,and 50 generations were selected for stability study.The growth curve test results showed that there was no significant change in its growth characteristics when the engineered bacteria were continuously passaged in vitro for 50 times,and it was consistent with the growth characteristics of attenuated Salmonella choleraesuis C500.There also was no significant changes were found when it carrying plasmids.At the same time,Salmonella marker genes (invA) and virulence genes (crp) was used to amplified with each generation of bacterial fluid,the results showed that the engineered bacteria still had the characteristics of Salmonella after multiple passages,and their attenuation characteristics had not changed.Direct enzyme digestion verification of bacterial liquid with various generations showed that multiple passages did not affect the stability of the plasmid,and the recombinant co-expression plasmid could maintain normal copy function in Salmonella C500.In summary,both the recombinant plasmid and the engineered bacterial vaccine had good stability,which could be directly applied to the study of animal immunocastration.

Orf is a zoonotic infectious disease,which is mainly caused by Orf virus (OrfV) infection.There is currently no specific medicine to treat it.Envelope protein is the main antigenic protein of OrfV.Its mechanism is mainly to create a series of conditions for the virus to invade host cells,while reducing the host's immunity and enhancing the virulence of the virus.When the virus invades the host,the host will produce an antiviral immune response to eliminate virus particles,including specific humoral immunity and non-specific cellular immune response,but mainly non-specific cellular immune response.The host will produce an antiviral immune response to the virus,and the virus will also form an immune evasion mechanism against the host's immune response.This mechanism is used to avoid the capture and removal of virus particles by the host immune cells,which is the proliferation of virus particles in the host,maturity and enhancement of virus virulence have created various conditions.This article summarized the research status of the interaction between OrfV and the host in recent years,expounded the biological function of the envelope protein of OrfV,the antiviral immune response of the host,the immune evasion mechanism of the virus in the host,and the mechanism of OrfV about the pathogenic effects of some other virulence factors,in order to provide a theoretical reference for the study of the pathogenic mechanism of the ovine aphtha virus and vaccine control.

The purpose of this experiment was to study the anti-inflammatory and analgesic effects of Pulu Ruyan San,and to provide the basis for the later clinical development.The anti-inflammatory effect was studied by xylene induced ear swelling,acetic acid induced peritoneal capillary permeability and carrageenan induced toe swelling in mice.The analgesic effect was studied by formalin and hot plate apparatus.In each experiment,mice were randomly divided into 5 groups, 10 mice in each group, half male and half female, which were negative control group, low, medium and high dose groups of Pulu Ruyan powder, and aspirin positive control group (aspirin group). The low, medium and high-dose groups of Pulu Ruyan powder and aspirin group were given 20, 30, 40 and 0.15 mg/g crude drugs by gavage once a day, respectively. The negative control group was given the same volume of normal saline by gavage for 7 days, and the follow-up was carried out according to different experiments.The results of mouse auricle swelling test showed that compared with the negative control group,each medication group could extremely significantly reduce the auricle swelling induced by xylene (P<0.01),but compared with aspirin group,there was no significant difference between Pulu Ruyan San middle and high-dose groups (P>0.05).The results of acetic acid induced peritoneal capillary permeability test showed that compared with the negative control group,each medication group had extremely significant inhibitory effect on acetic acid induced permeability increase (P<0.01).Compared with aspirin group,the difference between Pulu Ruyan San low and middle dose groups was very extremely significant (P<0.01),while the difference between high dose group was not significant (P>0.05).The results of carrageenan induced toe swelling test showed that compared with the negative control group,each medication group had different degrees of swelling inhibition (P<0.01).Compared with aspirin group,the difference was extremely significant in Pulu Ruyan San low dose group (P<0.01),but the difference was not significant in the middle and high-dose groups (P>0.05).The results of formalin induced pain test showed that compared with the negative control group,the times of licking feet in each treatment group were extremely significantly reduced in phase Ⅰ (P<0.01).In phase Ⅱ,the differences between each group were significant or extremely significant (P<0.05;P<0.01).The results of hot plate instrument pain test showed that compared with the negative control group,the pain threshold of each medication group was significantly prolonged at each time after administration (P<0.01).Compared with aspirin group,the effect of reducing pain threshold of Pulu Ruyan San high dose group was extremely significantly stronger than that of aspirin group (P<0.01),and the effects of Pulu Ruyan San low and middle dose groups were extremely significantly weaker than that of aspirin group (P<0.01).The results showed that Pulu Ruyan San had good anti-inflammatory and analgesic effects on mice.

To establish a rapid detection method for sarafloxacin (SAR) drug residues in animal-derived foods,SAR monoclonal antibody was prepared by immunizing mice with sarafloxacin hydrochloride molecular modification and coupling protein synthesis complete immunogen.The checkerboard method was used to determine the optimal coating concentration (SAR-1-OVA) and primary antibody dilution,and optimize the optimal reaction conditions to establish an indirect chemiluminescence enzyme-linked immunoassay (ic-CLEIA).The method was evaluated by sensitivity,precision,cross-reaction rate,and additive recovery test.The results showed that the complete immunogen coupling was successful after UV scanning,and the prepared SAR monoclonal antibody titer reached 1:4×10⁶.The optimal reaction conditions of ic-CLEIA were as follows:Coating concentration was 0.25 μg/mL,dilution of antibody was 1:750 000,coating at 4 ℃ overnight,5% skim milk sealed,competitive incubation for 1 h,dilution of enzyme labeled secondary antibody was 1:10 000,incubation for 1 h.The standard curve was linear,the linear range was 0.0625 to 10 ng/mL,R²=0.995,the sensitivity IC₅₀ was 1.45 ng/mL.The average intra- and inter-assay coefficients of variation were both <10%.Except the cross reaction rate with difloxacin was 98.08%,the cross reaction rates with other fluoroquinolones were <8%,and there was no cross reaction with other non fluoroquinolones.The minimum detection limit (LOD) of standard SAR was 0.32 ng/mL,and the LOD of SAR in chicken samples was 0.46 μg/kg.The recovery rate of standard addition was in the range of 88.3% to 106.7%,and the coefficient of variation was ≤12.2%.The above results showed that the method was fast and sensitive,was suitable for the large-scale detection of sarafloxacin residues in animal-derived foods,and this results provided a new method for the detection of sarafloxacin residues.

The purpose of this study was to evaluate the clinical efficacy of a novel pour-on agent of moxidectin (MOX) on Melophagus ovinus and gastrointestinal tract nematodes in sheep through the expelling test of MOX on sheep.150 Altay ewes were randomly divided into 6 groups,25 in each group,control group without medication,diazinon medical bath group,MOX injection group and MOX pour-on agent low (0.05 mL/(kg·BW)),medium (0.1 mL/(kg·BW)) and high (0.2 mL/(kg·BW)) groups.The experiment lasted for one week.On the first day of the experiment,the method and dosage of the above-mentioned grouping were used to expel insects once.The clinical manifestations,body temperature,pulse rate,respiration (TPR),blood physiological,biochemical and urine routine indexes were observed.The reduction rate and cure rate of Melophagus ovinus were determined.Fecal samples were taken to determine the reduction rate and negative conversion rate of egg number per gram (EPG) of feces.The results showed that compared with the control group without medication,there were no significant differences in TPR,blood physiological,biochemical and urine routine indexes among the groups on the 7th day after administration (P>0.05).The reduction rate of Melophagus ovinus was 43.7% in diazinon medical bath group,and 100% in MOX injection group and MOX low,medium and high dose groups.The cure rate of diazinon medical bath group was 13.0%,while that of MOX injection group and MOX low,medium and high dose groups were all 100%.Compared with the control group without medication,the reduction rate of gastrointestinal tract nematode eggs of diazinon medical bath group was 0,while that of MOX injection group and MOX low,medium and high dose groups were 91.6%,93.1%,94.9% and 97.8%,respectively.The negative conversion rate of diazinon medical bath group was 0,while that of MOX injection group and MOX low,medium,and high dose groups were 77.0%,69.2%,69.2% and 84.6%,respectively.The results showed that the novel pour-on agent of MOX had a significant effect on ectoparasites and gastrointestinal tract nematodes,which was better than the diazinon medical bath and equivalent to MOX injection.In practice,the optimal dose of MOX was 0.5 mL/(10 kg·BW)(0.25 mg/(kg·BW)).

To investigate the genetic variation of Porcine pseudorabies virus (PRV) in Jilin province,in this study,the virus isolation and identification were performed by cell passage,PCR and sequencing in clinical samples were collected from pigs with suspected porcine pseudorabies.The viruses titer was determined by TCID₅₀,and the pathogenicity of the isolated strains to rabbits was determined by the rabbit infection test.Futhermore,PCR amplification and sequencing of TK,gB,gC,gD and gE genes were performed to analyze their genetic variation.The results showed that 3 samples of clinical samples had cytopathy after passage by PK15 cells.PCR identification and sequencing analysis showed that 3 strains of PRV were isolated and identified and named as JL03,JL12 and JL15 strain,respectively.The titers of the 3 strains were 10^6.5,10^6.5 and 10^7.5 TCID₅₀/mL on PK15 cells.Six rabbits were inoculated with 3 isolated strains (10^3.5 TCID₅₀ each rabbit),all showed symptoms such as elevated body temperature,itching at the injection site and paralysis of limbs,and all died 3 to 4 days after virus inoculation.The results of the deduced amino acid sequence analysis of the TK,gB,gC,gD and gE genes of the 3 isolated viruses showed that the TK gene had no amino acid variation compared with the reference strains.Amino acid deletions,insertions or mutations occurred in some positions of gB,gC,gD and gE genes.Among them,an aspartic acid insertion in the 48^th and 496^th positions of the gE gene were consistent with the variation characteristics of epidemic strains reported in China.Nucleotide genetic phylogenetic tree analysis showed that the TK,gB,gC,gD and gE genes of the 3 isolates were highly homologous with those of the reference strains JS-2012 and ZJ01 isolated after 2012 in China,and belong to the same branch.They were far from foreign strains NIA3 and Becker, and located in different branches,and the genetic relationship with the early domestic virus strains SC and Ea was between them.In short,we successfully isolated and identified three PRV variants which had strong pathogenicity to rabbits.These results provided new data for the epidemiological study of PRV in Jilin province and lay the foundation for relevant research.

In order to investigate the virulence and drug resistance of Proteus mirabilis from swine in Guangxi province,98 samples of diseased swine were collected from farms with different scales in this study,and identified the isolated bacteria through isolation and culture,morphological observation,straining microscopy,16S rRNA sequencing.Drug susceptibility testing was performed by broth microdilution method.PCR was used to detect virulence genes,drug resistance genes,integrons and variable regions.The variable region amplification products were cloned into pMD^TM19-T for sequencing.The identification results showed that 22 strains of bacteria grew diffusely on the TSA medium,formed a center black marginal white single colony on the SS medium,and microscopic examination showed Gram-negative short bacillus and Proteus specific gene (TUF) positive.The results of 16S rRNA sequencing showed that 21 isolates had 99% homology with Proteus mirabilis,and one isolate had 99% homology with Proteus penneri.The drug sensitivity results showed that the drug resistance rate of 21 strains of Proteus mirabilis to tetracycline,doxycycline,ampicillin and sulfamethoxazole reached 100%,the drug resistance rate of cefalexin,cefuroxime,cefotaxime,imipenem and kanamycin was above 57.1%,and all the isolated strains were multiple drug resistant bacteria.PCR results showed that the virulence gene detection rates of atfA,hpmA,ireA,mrpA,pmfA,rsbA,ureC,zapA and ptA were all 100% and that of ucaA was 95.2%.The strains of ESBLs were bla_TEM type,bla_CTX type or bla_TEMtype and bla_CTX type,and the AmpC strains were bla_DHAtype.The proportion of strains carrying ESBLs or AmpC genes was 57.1%, 14.3%, and the proportion of strains carrying both ESBLs and AmpC genes was 14.3%.The detection rates of qnrA,qnrB,qnrS and aac(6')-Ⅰb-cr were 9.5%,0,4.8% and 80.1%,respectively.21 strains of isolated bacteria classⅠ integron (intⅠ1) positive rate was 61.9%,nine different gene cassette arrays were detected (aadA2-linF,estX,dfrA32-ereA-aadA2,drfA5,drfA12-orfF-aadA2,arr3-aac(6')-Ⅰb-cr5,aac(6')-Ⅰb-cr5-bla_OXA-1-catB3-aar3,drfA1-orfC and aadA2);classⅡ integron (intⅠ2) positive rate was 76.2%,only one gene cassette array (drfA1-sat1-aadA1) was detected.The results of this study showed that the virulence and drug resistance of swine-origin Proteus mirabilis were high in Guangxi,which provided data support for the prevention and control of Proteus mirabilis.

Antibacterial synergist is a kind of synthetic diaminopyrimidine drugs.When these drugs are used in combination with antibacterial agents,they will strengthen the activity of antibacterial agents through a specific mechanism.In recent years,the problem of bacterial resistance has become increasingly apparent due to the widespread use and even abuse of antibiotics.New preparations developed by adding antibacterial synergists can improve the utilization rate of antibiotics in animals,reduce drug dosage,enhance drug efficacy,reduce veterinary drug residues and reduce bacterial resistance.The preparation products prepared by the combination of TMP,DVD and ADP with various antibiotics were analyzed in recent years.It is found that the therapeutic effect of these preparations is generally better than those antibiotics alone.Besides,with the use of traditional Chinese medicine,the problem of bacterial resistance has been significantly improved,which opens a new prospect for the research direction of antibacterial synergist in the later study.With the research of veterinary drug preparation,it is no longer limited to simple dosage forms.Through the use of new technologies and new materials,the preparation developed can provide more accurate treatment for patients according to different drug delivery sites and routes,reduce the time for drugs to reach the target site,and increase the dosage of patients.This idea can provide support and reference for the follow-up research and development direction.In this paper,the research and development of the combination of antibiotics and antibacterial synergists are reviewed.

To investigate the effect of Sophora subprosrate polysaccharide (SSP) on the levels of inflammatory factors in 3D4/2 cells infected by Porcine circovirus virus Ⅱ type (PCV2),in this study,blank control group,PCV2 group,lipopolysaccharide (LPS) group and three concentrations of SSP group (100,200 and 400 μg/mL) were established,respectively.3D4/2 cells was infected by PCV2 in vitro, and then the cells were treated with different concentrations of SSP for 24 h.The secretions of monocyte chemotactic protein 1 (MCP-1) and interleukin-8 (IL-8) and the enzyme activities of cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) were detected by ELISA assays.The mRNA expression of inducible nitric oxide synthase (iNOS) and COX-2 genes were detected by Real-time quantitative PCR,and the protein expression of iNOS and COX-2 were detected by Western blotting.The results showed that the secretions of MCP-1,IL-8 and the activities of intracellular COX-1 and COX-2 were extremely significantly higher after PCV2 infection than those in the blank control group,the mRNA expression levels and protein expression of intracellular iNOS and COX-2 were also extremely significantly increased (P<0.01);After treatment with SSP at 100,200 and 400 μg/mL,the secretions of MCP-1,IL-8 and the activities of COX-1 and COX-2 in the cells were significantly or extremely significantly decreased (P<0.05,P<0.01),the effect was most significant in 400 μg/mL SSP group,and the mRNA expression of intracellular iNOS and COX-2 gene was also significantly or extremely significantly decreased(P<0.05,P<0.01).The increase of iNOS protein expression induced by PCV2 was significantly or extremely significantly inhibited by treatment with SSP at 100,200 and 400 μg/mL (P<0.05,P<0.01).The increase of COX-2 protein expression was significantly inhibited by treatment with SSP at 200 and 400 μg/mL,and the best effect was obtained at 400 μg/mL.The results showed that SSP could inhibit the increase of mRNA expression and protein expression of pro-inflammatory factors caused by PCV2 infection and played a certain role in alleviating inflammation.

The purpose of this experiment was to isolate Pseudorabies virus (PRV) from a sample tested as pseudorabies (PR) sent by a pig farm in Yunyan district,Guiyang city,Guizhou province.Methods such as cytotoxicity experiments,physical and chemical experiments,and transmission electron microscopy observations were used to isolate and identify the pathogen,and then to determine the virus value of the pathogen and then conduct animal experiments and comparative analysis of gD and gE genes sequences.The results showed that the cell inoculation experiment successfully isolated PRV that could cause Vero cells to produce typical lesions.The physicochemical experiment showed that the isolated virus was unstable in the environment of acid and heat,and sensitive to idoxuridine(IUDR) and chloroform.Transmission electron microscopy results showed that the mature virus particles with envelope in nucleus were approximatively round with a diameter of 150-160 nm,and the virus particles in nucleus were hollow or stuffed without envelope,and the capsids were lattice arrangement in mature viral inclusion bodies.Animal experiment showed that the strain had strong pathogenicity to rabbits,which could cause itching at the inoculation site.The results of nucleic acid identification showed that the open reading frame(ORF) of the amplified PRV gD gene was 1 209 bp,and the ORF of gE gene was 1 740 bp.The results of GenBank comparison showed that the gD gene of the isolated strain was 99.8% similarity with the reference strains of PRV JS2012 strain (accession No.:KP257591) and PRV HNB strain (accession No.:KM189914.3),and the similarity with vaccine strain Bartha-K61 (accession No.:JF797217.1) was 98.7%.The gE gene nucleotide sequence was 99.9% similar to PRV JS 2012 strain (accession No.:KP257591),PRV HNB strain (accession No.:KM189914.3) and PRV Qihe547 (accession No.:KU056477).According to the phylogenetic tree of gD and gE amino acid sequences,the isolated strain belonged to the same branch of the domestic mutant strains.These results indicated that the PRV variant strain was successfully isolated,which could provide references on PRV epidemiology,genetic evolution analysis and immune control of PR in Guizhou province.

The purpose of this study was to investigate the expression of ferritin 1 (Fer1) gene in different developmental stages,blood sucking stages and different organs of Dermacentor marginatus.Real-time quantitative PCR was used to analyze the expression profile of Fer1 gene in different developmental stages,blood vampire period and different organs of Dermacentor marginatus.The relative expression was calculated using 2^-△△Ct method using starved female ticks as a reference (1 fold-change,1 FC).The results showed that the female adults exhibited highest expression level of Fer1 gene at 72 h after blood feeding.In different development age,newly molted adult ticks (male and female),eggs,and larvae exhibited relatively low expression,while the relative expression level in nymph was higher.The relative expression level in larvea and nymphs under starvation state was higher than that in engorged state.In different tick organs,the expression level of Fer1 gene in midgut if semi-engorged tick was the highest.But there were no expressions detected in ovary and malpighian tube of semi-engorged female tick,and expressions of Fer1 were detected in salivary gland,midgut,ovary and malpighian tube in engorged female ticks,in which ovary and malpighian tube ranking ahead,while salivary gland was the last.The results showed that the relative expression level of Fer1 gene in the tick changed when feeding proceeded which was related to digestive enzymes in the tick,which laid a foundation for the study of biological characteristics and functional genes of this tick.This experiment provided basic data and reference for the study of key functional genes in the metabolic pathway of this tick species.

This experiment was conducted to explore transcriptome targets and pathways of Salvia miltiorrhiza polysaccharides in alleviating liver injury induced by flufenicol in broilers.Thirty one-day-old broilers were randomly divided into three groups:blank control group (LA group) were fed tap water,florfenicol model group (LB group) was given tap water containing 0.15 g/L florfenicol,and Salvia miltiorrhiza polysaccharides treatment group (LC group) was given tap water containing 0.15 g/L florfenicol and 5 g/L Salvia miltiorrhiza polysaccharides tap water.The drug was administered continuously for 5 days from the age of 1 day.On day 6,three chickens were randomly selected from each group and killed,and their liver tissues were aseptically extracted.After RNA extraction,purification and library construction of liver tissues,the Illumina HiSeq libraries were sequenced using the Illumina HiSeq platform.Then the original offline data was filtered and the high-quality sequences obtained after filtering were aligned to the reference genome of the species.The expression level of each gene was calculated according to the comparison results.3 groups chicken liver digital gene expression patterns (DGE) library were built respectively,the DGE library of differentially expressed genes were compared,looking for the key genes with most significant difference in gene expression,and GO function annotation,KEGG pathway were analyzed.The results showed that 1 989 (495 up-regulated,1 494 down-regulated),1 278 (344 up-regulated,934 down-regulated) and 380 genes (165 up-regulated,215 down-regulated) were screened out in the comparison groups of LA vs LB,LA vs LC and LB vs LC,respectively.The GO functional annotations of LA vs LB,LA vs LC and LB vs LC comparison groups were mainly classified into two branches:biological process and cell composition.The results of the KEGG pathway analysis showed that differentially expressed genes of LA vs LB were mainly enriched in drug metabolism-cytochrome P450,extracellular matrix receptor interaction,cytokine-cytokine receptor interaction,metabolism of xenobiotics by cytochrome P450,glutathione metabolism,peroxidase body growth activated receptor (PPAR) signaling pathway,glycine,serine and threonine metabolism,alanine,aspartate and glutamate metabolism pathways.The genes differentially expressed in LB vs LC were mainly concentrated in drug metabolism-cytochrome P450 and glycine,serine and threonine metabolism,PPAR signaling pathway.Salvia miltiorrhiza polysaccharides could be adjusted by the cytochrome P450 metabolic pathways related cytochrome P450 3A4 enzyme (CYP3A4),cytochrome P450 2C23b enzyme (CYP2C23b),glutathione S transferase (GSTA3),kind of glutathione S transferase alpha 2 (GSTAL2) mRNA expression and fatty acid binding protein 1 (FABP1) associated with PPAR signaling pathways and ACSBG2 coa ligase (long chain fatty acids) mRNA expression,thus inhibiting fluorobenzene nicol to produce harmful metabolites,promoting the normal metabolism of liver lipids and alleviating the liver injury caused by flufenicol.

In order to observe the effect of different doses of GS-441524 on naturally occurring feline infectious peritonitis(FIP),25 cats diagnosed with FIP in China Agricultural University Veterinary Teaching Hospital were randomly divided into high-dose group (5 mg/(kg·d))(12) and low-dose group (2.5 mg/(kg·d))(13).They were given GS-441524 everyday by subcutaneous injection and recorded daily weight,temperature,pain response of injection and weekly laboratory and imaging examination results.The treatment period was 4 weeks,and some cases were extended according to the actual condition.Finally,20 wet FIP cats and 3 dry FIP cats completed the test,2 cats quit the test.The whole medication was between 4 and 18 weeks.Of the 20 wet FIP cats,2 cats died within 1 week,1 cat's body cavity fluid increased,17 cats' body fluid decreased after 1 week of medication and then disappeared after 2 to 3 weeks of medication.Of the 3 dry FIP cats,2 cats had intestinal membrane lymph nodes that decreased after 1 to 2 weeks of medication,and 1 cat didn't change.The spirit and appetite of all cats improved within 3 to 5 days,weight began to increase within 1 to 2 weeks,5 cats with fever returned to normal after 2 to 3 days,14 anemia cats returned to normal after 1 to 10 weeks,16 cats showed an increase in the number of white blood cells and a decrease in the number of lymphocytes or an increase in the number of neutral granulocytes,and began to improve within 1 to 2 weeks.The ratio of albumin to globulin in all cat serums was less than 0.8,of which 9 cats had an increase in total protein,4 cats had a decrease in albumin,14 cats had an increase in globulin,and began to improve after 1 to 2 weeks.The effective and cure rate of 4 weeks' treatment in high-dose group were 81.8% and 60.0%,respectively.The effective and cure rate of 4 weeks' treatment in the low-dose group were 75.0% and 22.2%,respectively.In addition,statistical results showed significant or extremely significant differences in the indicators of cats in the high-dose group except for white blood cell count and albumin after 4 weeks of medication (P<0.05;P<0.01),while the cat in the low-dose group only had significant weight differences.In addition to hemoglobin,white blood cell count and albumin,the drug had significant differences in weight,red blood cells,red blood cell pressure,lymphocytes,neutral granulocytes,total protein and the ratio of albumin to globulin (P<0.05).Overall,GS-441524 was effective for naturally occurring FIP,and the optimum dosage was 5 mg/(kg·d).

To explore the molecular mechanisms of the antagonistic effect of selenium on aluminum-induced oxidative stress and inflammation in the spleen of mice,mice were divided into four groups as follows:Blank control group,aluminum group,selenium group,and selenium＋aluminum group.The spleen tissues of mice were taken 4 weeks after oral administration.Pathological changes of spleen were evaluated by HE staining and immunofluorescence staining of pathological tissue sections.Glutathione peroxidase (GPx),reduced glutathione (GSH),malondialdehyde (MDA) and nitric oxide (NO) levels were measured by respective commercial kits.IL-1β,IL-4,IL-6,IFN-γ,HO-1 and NF-κB mRNA expression levels were quantified by Real-time PCR,and p-p65,IL-1β and HO-1 proteins expression level were detected by Western blotting.The results showed that aluminum treatment could increase the local cell spacing of the spleen,decrease the number of lymphocytes,accompanied by an increase in neutrophils and red blood cell infiltration.Moreover,the activity of GPx and the content of NO and GSH were reduced,and the content of MDA was significantly increased.Likewise,the mRNA and proteins expression levels of inflammation-related such as IL-1β and NF-κB were increased,indicating oxidative damage and inflammatory reactions to the spleen.Selenium could alleviate the toxicity of aluminum,increase the activity of GPx and the content of other antioxidants,reduce the production of MDA and the expression of mRNA and protein of inflammation related genes.The results showed that aluminum could damage the spleen by inducing oxidative stress and inflammatory reaction in mice,and selenium could alleviate the spleen injury induced by aluminum.The mechanism might be related to selenium enhancing the antioxidant level of spleen and protecting spleen against tissue injury induced by oxidative stress and inflammatory reaction.

The aim of this study was to express Clostridium chauvoei toxin A (CctA) gene in prokaryotic system and establish indirect ELISA detection method using purified recombinant protein,and to verify the immune effect of gangraena emphysematosa inactivated vaccine.The sequence of CctA gene was cloned into prokaryotic expression vector pET-28a(+) according to the codon bias of Escherichia coli (E.coli).The prokaryotic expression plasmid pET28a-CctA was identified by double enzyme digestion and sequencing.The high expression of recombinant CctA inclusion body protein was induced by IPTG after recombinant plasmid was transferred into E.coli.The inclusion body protein was purified by Ni column after denaturation and its purification effect was tested by SDS-PAGE.Western blotting was used to detect the reactivity of recombinant CctA protein.Establishment of indirect ELISA was accompanied by the checkerboard titration method.The recombinant CctA protein was used as detected antigen,and the concentration of the coated antigen,the type and concentration of the blocking solution,the optimal dilution of the antibody and the reaction conditions were explored.After the guinea pigs were immunized with 3 batches of gangraena emphysematosa inactivated vaccine,the serum was collected.The immune effect of the vaccine was verified both by challenge and indirect ELISA.The results of double digestion of plasmid showed that the band about 853 bp was in line with expectation,and the sequencing results was correct.SDS-PAGE results indicated that 35 ku recombinant CctA protein was successfully expressed and purified.The results of Western blotting showed that the antiserum of guinea pigs against Clostridium chauvoei had good reactivity with recombinant CctA protein.The optimal conditions of indirect ELISA method were as follows.The coating concentration of antigen was 0.5 μg/mL overnight at 4 ℃,10% fetal bovine serum was selected for blocking solution and incubated at 37 ℃ for 2 h,the dilution of the secondary antibody was 1:8 000,the reaction was stopped after 10 min at room temperature and D_{450 nm} was determined.When P/N>4.6,the results of indirect ELISA fitted well with the guinea pig challenge test.In this study,CctA gene was successfully expressed and recombinant CctA protein was purified,and indirect ELISA was established with recombinant CctA protein as detected antigen,which was expected to be an alternative method to verify the immune effect of gangraena emphysematosa inactivated vaccine.