The aim of the study was to investigate the effect of knockout of interferon regulatory factor 3 (IRF3) on the replication of pseudorabies virus (PRV).The IRF3 knockout PK15 cell line was established using lentiviral-mediated CRISPR/Cas9 genome editing technology.The recombinant plasmid pIRF3-sgRNA was constructed and transfected into HEK293T/17 cells.Lentivirus was obtained and infected with PK15 cells.The polyclonal cell line was obtained by puromycin screening.After T7 digestion,PK15-IRF3^-/- monoclonal stable cell line was obtained by limiting dilution method.To verify whether the stable cell line of IRF3 gene was successfully constructed,Real-time PCR and Western blotting were used to detect the expression of PRV-related genes and proteins.Fluorescence microscopy and flow cytometry were used to observe the virus replication.The results showed that the fluorescence intensity of PK15-IRF3^-/- cells was significantly stronger than that of PK15 cells after infection of PRV-GFP in PK15-IRF3^-/- cell line.The PK15-IRF3^-/- virus titer was significantly stronger than that of PK15 cells after infection with PRV-QXX,and the gE protein level of PRV was significantly higher than that of PK15 cells.The same results were obtained by detecting changes of PRV TK gene in the two cells at the mRNA level.Further studies showed that IFN-β mRNA in PK15 cells increased significantly with time after infection with PRV-QXX (P<0.05),but there was no significant change in IFN-β mRNA in PK15-IRF3^-/- cells.The above results indicated that knocking out IRF3 significantly promoted the proliferation of PRV,and IRF3 played an important role in virus replication,providing new methods and strategies for the prevention and control of pseudorabies.

In order to identify the genetic characteristics of the SPDEF gene,the CDS of different pig breeds were amplified,sequenced,spliced,and the CDS of Saba pigs were analyzed by bioinformatics.The exon sequence of pigs were detected by direct sequencing after PCR,and sequence splicing were performed using SeqMan.The open reading frame (ORF) of SPDEF gene,and basic physicochemical properties,hydrophobicity,signal peptide,transmembrane region,subcellular localization,phosphorylation site,glycosylation site,secondary structure,tertiary structure,functional domain of SPDEF protein were predicted,and phylogenetic tree was constructed using bioinformatics software.The results showed that only 2 synonymous SNPs were detected in exon 1 (C13971T) and exon 2 (C16541T) of SPDEF gene in different pigs,respectively,indicating that the coding region of SPDEF gene was highly conserved among the tested herds.SPDEF gene had a complete ORF at length of 1 092 bp.The Ser content was the highest,which accounted for 10.7%.SPDEF was an unstable hydrophilic protein with an estimated half-life of 30 h in vitro.The SPDEF protein has no signal peptide and transmembrane structure,and had 39 potential phosphorylation sites and 1 glycosylation site.The protein located the cytoplasm in analysis of subcellular localization.In the secondary structure prediction,the amino acids involved in the random coil were the most,accounting for 55.10%,followed by the α-helix region,accounting for 28.37%.Functional domain predictions revealed the existence of an ETS functional domain and an SAM-PNT functional domain.Sequence homology analysis showed that pig has higher homology with Bos taurus,Ovis aries and Capra hircus.This study could provide references for further analysis of the function of SPDEF gene in Saba pigs.

This study was aimed to clone the CDS sequence of small muscle protein X-link (SMPX) gene,analyze its potential structure and function,and explore the expression levels of SMPX gene in different tissues of Bos grunniens.The CDS sequence of SMPX gene was amplified and cloned by RT-PCR,the amino acid sequence homology of SMPX gene was analyzed,and the phylogenetic tree was constructed.The physical and chemical properties,secondary structure and tertiary structure of SMPX protein were analyzed by bioinformatics softwares.The expression patterns of SMPX gene in right ventricle,gluteus maximus,lung and brain of Bos grunniens were detected by Real-time PCR.The results showed that the length of SMPX gene CDS and ORF sequences were 515 and 261 bp,respectively,which encoded 86 amino acids.The amino acid sequence homology of SMPX gene in Bos grunniens with Bos mutus,Bubalus bubalis,Canis lupus familiaris,Homo sapiens,Odocoileus virginianus texanus,Ovis aries,Pan troglodytes,Pantholops hodgsonii and Sus scrofa were 100%,97.7%,96.5%,96.5%,95.3%,95.3%,96.5%,94.2% and 91.9%,respectively,indicating that it was highly conserved in different species.Bioinformatics analysis represented that SMPX protein was unstable and hydrophilic.The secondary structure of SMPX mainly contained random coils and alpha helix.The SMPX protein had no signal peptide and transmembrane protein, and belonged to intramembrane protein.There were four phosphorylation sites in SMPX protein sequence.The results of subcellular localization showed that SMPX protein was located in the nucleus (52.2%),mitochondria (43.5%) and cytoplasm (4.3%).Real-time PCR results exhibited that the expression level of SMPX gene in right ventricle was significantly higher than gluteus maximus,lung and brain (P<0.05).This study might provide reference data for further study of physiological functions and regulatory mechanisms of SMPX gene in Bos grunniens.

A new multiplex PCR method was established to simultaneously detecting classical swine fever virus (CSFV),porcine reproductive and respiratory syndrome virus (PRRSV),pseudorabies virus (PRV),porcine parvovirus (PPV),porcine circovirus type 2 (PCV2), Fusobacterium necrophorum (Fn) and Haemophilus parasuis (Hps) seven kinds of common highly lethal epidemic pathogen in hoggery.Seven specific primers refer to each pathogen were designed,and one pair Cy-5 conjugated universal primers was designed.In this study,the universal primers were respectively fused in the 5'end of each specific primer to form seven pairs of specific chimeric primers.In optimized reaction condition,each chimeric primer was mixed with universal primer respectively and amplified cDNA/DNA of seven pathogens to verify the specificity of single PCR.This study further relied on GenomeLab Genetic Analysis System (GeXP),we mixed all the seven specific chimeric primers and universal primers,amplified cDNA/DNA of a single pathogen to verify multiplex PCR specificity.The genomes of other common pig disease pathogenes were taken as positive internal.Seven pairs of specific chimeric primers and universal primers were used to amplify cDNA/DNA of seven pathogens,then we mixed templates with positive samples to determine their anti-interference.The sensitivity of GeXP multiplex PCR assay was determined by gradient recombinant plasmid dilution.The results showed that GeXP multiplex and single detection both could detect the signal of specific target fragments,no other obvious interference signals were detected.GeXP multiplex anti-interference showed that mixed 3 pathogen genomes of other common pig diseases,the assay could also detect the seven pathogens simultaneously.GeXP multiplex sensitivity analysis showed that specific results of seven genes were detected,and the detection limit was 10³ copies/μL.The GeXP multiplex assay was established in this study for simultaneous detection of 7 kinds of common and highly lethal epidemic pathogens in pig farms,and it had the characteristics of high throughput,specificity and sensitivity,providing a new detection method for rapid diagnosis of cross-infection and mixed infection of swine epidemic diseases.

This study was aimed to clone the sequence of inhibin beta A (INHβA) in Kazakh sheep,and explore the sequence characteristics of INHβA gene and the structure and function of its encoded protein.One pair of specific primers was disigned based on the sequence of sheep INHβA gene (accession No.:NM_001009458.1) in GenBank,the sequence of INHβA gene in Kazakh sheep was amplified by RT-PCR.INHβA gene was inserted into pMD19-T vector for cloning and sequencing,its nucleotide sequence,amino acids sequence,protein transmembrane,protein modification site and secondary structure,tertiary structure model were predicted and analyzed by bioinformatics methods.The results showed that INHβA gene in Kazakh sheep was 1 278 bp in length and encoded 425 amino acids.The homology of INHβA gene in Kazakh sheep with Ovis aries,Bos taurus,Sus scrofa,Mus musculus,Homo sapiens,Rattus norvegicus,Felis catus,Rytolagus cuniculus and Equus caballus was 98.6%,97.7%,90.4%,87.9%,91.1%,88.2%,91.8%,89.8% and 91.8%,respectively,indicating that INHβA gene was highly conserved among different species.The molecular formula of INHβA protein was C₂₀₇₂H₃₃₂₅N₆₀₃O₆₂₈S₂₆,the molecular weight was 47.57 ku,the theoretical isoelectric point (pI) was 7.87,the instability coefficient was 66.16,the fat solubility index was 78.47,and the hydrophilic value was -0.507.The INHβA protein was an alkaline labile hydrophilic liposoluble protein containing a signal peptide,with no transmembrane structure.The secondary structure prediction showed that alpha helix,beta turn,random coil,and extended chain of INHβA protein accounted for 22.59%,4.47%,54.12% and 18.82%,respectively.The tertiary structure prediction showed that the spatial structure of the protein was mainly alpha helix,and contained β-barrel structures of 8 protein modification sites.This results provided a data reference for the later study of the function of INHβA protein and the effect of INHβA gene on improving the fecundity in Kazakh sheep.

To identify CDS sequence of tyrosinase-related protein 1 (TYRP1) gene in Black raccoon dog and perform bioinformatics analysis,the back skin tissue of Black raccoon dog was collected to extract RNA in this study,and RNA was reverse transcribed into cDNA.One pair of specific primers was designed according to the canine TYRP1 gene mRNA sequence (accession No.:NM_001194966.1) in GenBank.The TYRP1 gene CDS was amplified by RT-PCR,PCR products were inserted into the cloning vector,and verified by positive colony PCR and sequenced.The results showed that the CDS sequence of TYRP1 gene in Black raccoon dog was 1 614 bp in length and encoded 537 amino acids,the TYRP1 protein was an unstable soluble hydrophilic protein.The genetic distance was closer between Black raccoon dog and fox,indicating that TYRP1 gene was highly conserved in different species and evolution processes.The proportion of random coil in the secondary structures of TYRP1 protein was the highest (54.0%),followed by alpha helix,which was 29.8%.The tertiary structure of TYRP1 protein was mainly composed of a large number of random coil and alpha helix,which was consistent with the secondary structure.TYRP1 protein had oxidoreductase activity,which was involved in DNA repair and replication,protein synthesis and degradation,and interacted with various proteins such as TYR,DCT,TP53 and TRPC1.The results of this experiment provided a theoretical basis for further exploration of its function in mammalian hair color genetic mechanism of TYRP1 gene in Black raccoon dog.

Enhancers have traditionally been defined as genomic cis-regulatory sequences that regulate the transcription of one or more genes independently of the transcription direction and relative position to the target promoter.The activity of enhancers plays a key regulatory role in growth and development,gene expression diversity,evolution and human disease occurrence.Therefore,understanding the functional characteristics of enhancers will not only help us understand the space-time specificity of gene expression,but also help us finally reveal the key biological issues such as cells differentiate,species evolve,and non-coding region variation leads to disease.With the rapid development of high-throughput sequencing technology,the methods and tools for the prediction,identification and functional annotation of enhancers also show diversity,including sequence motif analysis,in vivo binding sites of transcription factors and cofactors,feature chromatin and histone modification,large-scale parallel enhancer analysis,and CRSPR related technologies.This article reviews six methods of enhancer identification,recognition and prediction based on high-throughput sequencing technology at the whole genome level using regulatory factor binding sites,chromatin accessibility,histone modification,enhancer-promoter interaction,eRNAs and in vitro luciferase,and expounds in detail how to target enhancer research using gene editing technology.Finally,the role of enhancer research and individual research enhancer at the whole genome level is prospected,and the methods of targeted prediction of enhancer are described in detail,and the advantages and limitations of each method are compared,providing a reference for further research in this field.

In this study,protective effect of camel urine on liver injury induced by CCl₄ in mice was investigated.Forty mice were randomly divided into five groups:Normal group,model group,low-dose camel urine group (170 mg/kg),medium dose camel urine group (340 mg/kg) and high dose camel urine group (500 mg/kg),respectively.The normal group and model group received the same amount of saline infusions once a day,while 3 camel urine groups received different amount of camel urine.All mice were given continuous administration for 14 d and injected with 20% CCl₄ and olive oil solution to establish an acute liver injury model except the control group at the end of the experiment.Mice have sacrificed after 12 h post-CCl₄ injection when blood and livers were collected,and the liver weight and the liver index were recorded and calculated.Then the related serum indicators and the pathological examination of liver tissue were detected.The results showed that compared with the model group,the level of serum ALT,AST,TBIL,ALP,GLU,TP in low,middle,high dose camel urine groups were decreased,while the serum ALB level was increased with high dose group had a better effect.According to the results of the pathological section,the liver histopathological damage of mice in 3 camel urine groups were all alleviate,and that in the high dose camel urine group was very similar to the normal group.In addition,it could be seen from the results that among the three of camel urine groups,the higher concentration had a better therapeutic effect,indicating that the effect of camel urine was dose-dependent.In summary,camel urine had a certain preventive effect on CCl₄-induced liver injury in mice,and the high dose was the better.

This study was aimed to discuss the stable and reliable method of isolation and primary culture of epithelial cells of sperm storage tubules (SSTs),and provide a cell model for studying the mechanism of chicken sperm storage.The uterovaginal junction of the chicken oviduct was used to culture the epithelial cells of sperm storage tubules with enzyme digestion and explant culture methods.The culture of SSTs cells in hens and the growth of the cells isolated with different cell culture methods was observed.The results showed that after the uterovaginal junction of hen was digested with collagenase or trypsin and then filtered with 100 mesh, the epithelial cells of SSTs could adhere to wall after 24 h,but the cells died after 48-72 h.After the uterovaginal junction of hen was digested with collagenase Ⅺ (0.01 g/mL) and trypsin (0.25%) and then filtered with 100 mesh,there was obvious proliferation at 24 to 48 h,the cells slowly proliferated after 72 h,and then began to die.The epithelial cells of SSTs could be obtained with tissue block culture for 7 days,and the cells could be transferred for 2 to 3 generations.Immunohistochemistry was used to test whether the protein of differentially expressed neurexophilin 1 (NXPH1) of chicken SSTs was located in the primary cells with tissue explant culture.The results showed that NXPH1 expressed in the nuclei and cytoplasm of the cultured cells,indicating that the primary cells with tissue explant culture could be used in subsequent experiments.In conclusion,the epithelial cells of sperm storage tubules obtained with explant culture could provide a cell model for studying the mechanism of sperm storage in hens.

In order to study the effects of Ampelopsis grossedentata extract on growth performance,blood biochemical indexes and antioxidant activities in pigs at different weight stages,ninety crossbred (Duroc×Landrace×Yorkshire) castrated boars with an average initial body weight of 30 kg were randomly divided into three groups,CON group (fed with basal diet),group A (fed with basal diet adding 0.03% plant essential oil complex)and group B (fed with basal diet adding 0.03% Ampelopsis grossedentata extract),respectively.Pigs were fed with different basic diets according to body weight stages (30-50,50-75,75-100 kg and 100 kg-out).Pigs were weighted and blood samples were collected before and after each change of basic diets to analysis the effects of 0.03% Ampelopsis grossedentata extract on the growth performance,blood biochemical parameters and antioxidant activity of different weight stages pigs.The results showed that:Compared with the CON group,adding 0.03% Ampelopsis grossedentata extract significantly increased ADFI and ADG of the 30-75 kg pigs (P<0.05).The content of blood lactic acid (LA) was significantly decreased at 30-50 kg body weight (P<0.05).At 50-75 kg,the insulin (INS) concentration in blood was significantly increased (P<0.05),but there was no significant difference in blood glucose (GLU) concentration (P>0.05).At 75-100 kg,GLU concentration was significantly elevated (P<0.05),meanwhile there was an increased trend for INS concentration (P>0.05),and the content of lactate dehydrogenase (LDH) was significantly decreased (P<0.05).There was an increased trend for serum growth hormone (GH) content at each weight stage (P>0.05).Serum T-SOD activity,MDA content,GSH-Px activity,and TAOC were not significantly different compared with the CON group at any weight stage (P>0.05).Compared with the plant essential oil complex group,addition 0.03% Ampelopsis grossedentata extract significantly increased the ADFI of the pigs at 30-100 kg body weight stage (P<0.05),and significantly reduced the content of LA of 30-50 kg pigs (P<0.05),and significantly increased the GSH-Px activity at the weight of 50 kg or more (P<0.05).In conclusion,adding 0.03% Ampelopsis grossedentata extract could promote the growth and metabolism of pigs at 30-75 kg,and improve the carbohydrate metabolism of pigs at 50-100 kg body weight to some extent,and the effect of adding Ampelopsis grossedentata extract was superior to the plant essential oil complex at the same amount.

In order to study the effect of alkali storage on the ruminal nutrients degradability of spent mushroom substrate,four kinds of alkalis such as CaO,Ca(OH)₂,CaO+3% urea and Ca(OH)₂+3% urea were used for storage in this experiment.Each treatment of alkali (CaO/Ca(OH)₂) had four levels (3%,4%,5% and 6% additive amount),that was named CO3,CO4,CO5 and CO6;CH3,CH4,CH5 and CH6;COU3,COU4,COU5 and COU6;CHU3,CHU4,CHU5 and CHU6,respectively,with 4 replicates per treatment.There were 2 blank groups,one group (CK group) was only spent mushroom substrate,the other one (CKU) was 3% urea in spent mushroom substrate.6 Chuanzhong Black goats with permanent rumen fistula were chosen for collecting ruminal fluids,and the dynamic degradation rates of DM,NDF,ADF,CP of spent mushroom substrate at 4,8,12,24,48,and 72 h were measured and analyzed,respectively.The results showed that the degradability and effective degradability(ED) of DM,CP,NDF and ADF were higher than those of control group after storage with different levels of alkali.Among them,the COU alkali storage treatment groups had the best effect.Compared to CKU group,the 72 h DM rumen degradability in the COU alkali storage treatment groups was increased by 13.01%,20.03%,31.65% and 32.33%(P<0.05),respectively.The degradability of NDF at 72 h was increased by 27.61%,45.78%,62.88% and 50.23% (P<0.05).And that of ADF was increased by 26.14%,45.75%,51.76% and 39.65% (P<0.05).The 72 h CP rumen degradability in the COU alkali storage treatment groups was increased by 12.33%,14.62%,20.55% and 19.07% (P<0.05),and COU5 treatment was optimal.

This study was aimed to test the optimization and characterization of high-yield cellulase produced by an endophytic Bacillus velezensis (B.velezensis) 157 from Eucommia bark,which provided a reference for exploring the relevant characteristics of B.velezensis 157 CMCase.Single-factor experiment was designed to optimize fermentation conditions such as pH,inoculation amount,temperature,fermentation time,carbon source and nitrogen source,and to explore the effects of temperature,pH,substrate specificity,metal ions and surfactants on the enzymatic properties of CMCase.The optimized conditions of CMCase production by B.velezensis 157 were as follows:Maize straw as carbon source (30 g/L),soybean meal as nitrogen source (30 g/L),3% inoculation,and pH 7.0 under the condition of 37℃ for 48 h.After the optimization,the CMCase activity of B.velezensis 157 was up to (5.14±0.18) U/mL.The CMCase properties analysis of B.velezensis 157 found that the optimal temperature of enzyme reaction was 60℃,it could still maintain above 80% cellulase activity when temperature was from 40 to 50℃;With the increase of temperature,the relative residual enzyme activity decreased,and it decreased to 50% when the temperature was higher than 55℃.The optimal pH of cellulase was 5.0,and it could still maintain over 90% cellulase activity in a pH range of 5.0 to 10.0;The pH had little effect on the relative residual enzyme activity with the extension of the action time.The cellulase from B.velezensis 157 belonged to typical endoglueanases.Na⁺,Mg²⁺,Ca²⁺ could increase the activity of B.velezensis 157 CMCase,Co²⁺,Hg²⁺,Fe²⁺,Cu²⁺ and SDS inhibited the activity of CMCase;The surfactant Tween-80,Tween-20 and Triton X-100 had no effect on CMCase activity.In this study,the enzyme production conditions and enzymatic properties of B.velezensis 157 CMCase were optimized,and it was concluded that B.velezensis 157 had a high yield of CMCase and tolerated a wide range of pH,which had certain application value in feed additive,detergent and paper industry.

This study was conducted to determine the effects of dietary Bacillus coagulans supplementation on plasma antioxidant indexes and intestinal mucosal growth of piglets.Thirty healthy weaned piglets (21-day-old) were randomly divided into three experimental groups (10 replicates per group):Control group,group Ⅰ and group Ⅱ.The control group was fed the basal diet,and the experimental groups Ⅰ and Ⅱ were fed the basal diet+2.0×10⁶ and 2.0×10⁷ CFU/g of Bacillus coagulans,respectively.On day 21 of the trail,blood samples were collected and then all the piglets were sacrificed and intestinal mucosa were collected for analysis.The results showed that:Compared with control group,the plasma superoxide dismutase (SOD) activity,the ratio of villus height to crypt depth in jejunum and ileum,the ileal TP content and the ratios of RNA to DNA of pigs in both groups Ⅰand Ⅱ were significantly increased (P<0.05); The plasma malondialdehyde (MDA) content of pigs in group Ⅱ was significantly decreased (P<0.05), while the TP content and the ratio of TP to DNA in jejunum of pigs in group Ⅰ increased significantly (P<0.05).In conclusion,under the conditions of this experiment,dietary supplementation with Bacillus coagulans could enhance the antioxidant capacity of weaned piglets,promote the proliferation and renewal of intestinal mucosal cells,and improve the intestinal mucosal morphology.

To systematically explore the microbial diversity and function in the rumen of Chinese Simmental cattle,this experiment used 16S rRNA gene high-throughput sequencing technology to detect and analyze the rumen fluid samples of Simmental cattle (about 20 months old,the average weight was 550 kg),and its function was predicted by PICRUSt.The results showed that a total of 66 712 high-quality sequences were obtained from the Illumina Miseq sequencing platform,and 1 797 operational classification units (OTU) were obtained by cluster analysis.The taxonomic identification was divided into 13 phylums,20 classes,22 orders,33 families and 59 genuses;Firmicutes and Bacteroidetes were dominant bacteria,accounting for 62.46% and 22.45%,respectively;Based on the genus composition,[Eubacterium]_coprostanoligenes_group (9.16%),Ruminococcus_2 (7.90%),norank_f__Bacteroidales_RF16_group(6.23%),norank_f__Ruminococcaceae(4.79%),Rikenellaceae_RC9_gut_group(4.72%),norank_f_Bacteroidales_BS11_gut_group(4.49%),Ruminococcus_1(4.43%),etc.The PICRUSt function of the 16S rRNA genome predicted that the rumen flora function was mainly concentrated in carbohydrate transport and metabolism,and might contain a large amount of cellulose and lignin degrading enzyme genes.In summary,the high-throughput sequencing technology based on 16S rRNA gene fully revealed the diversity of rumen bacteria in Simmental cattle,and predicted that it was rich in protein decomposition and lignocellulose degrading enzymes.The basis of microbial cognition provided a reference for further research on rumen microbial functional genes closely related to some important nutritional and physiological functions.

The aim of this study was to evaluate the effects of dietary superoxdie dismutase mimics (SODm) on growth performance,meat quality and activity of superoxdie dismutase(SOD) in broilers.Four hundred and thirty two 1-day-old healthy male AA broilers were randomly assigned to 6 groups with 6 replicates per group and 12 birds per replicate.The control group was fed corn-soybean meal basal diet,and 5 experiment diets were added 0.10%,0.15%,0.20%,0.25% and 0.30% SODm,respectively.The test lasted for 42 d.The results showed as follows:①The ADFI,ADG and F/G were not significantly affected by the different levels of SODm (P>0.05).②Compared with the control group,the L^* value of breast muscle in 0.20%,0.25% and 0.25% SODm groups were significantly decreased (P<0.05).Except for 0.10% SODm group,the shear force of breast muscle was significantly lower than that of control group (P<0.05),while the drip loss was significantly increased than that of control group (P<0.05).With SODm supplementation,the L^* value of leg muscle were found to decrease significantly compared with control group (P<0.05);The shear force of leg muscle in 0.10% and 0.15% SODm groups was significantly lower than that in control group (P<0.05).③At 21 days of age,compared with control group,the activity of SOD in serum was higher (P>0.05),but except for 0.30% SODm group which was significantly decreased (P<0.05).The activity of SOD in serum at 42 days of age was discovered significantly increased with SODm supplementation (P<0.05).④Compared with control group,the activity of SOD in liver at 21 days of age was significantly increased with dietary SODm supplementation (P<0.05).SODm did not significantly affect the SOD activity of breast muscle and heart at 21 days of age (P>0.05).The activity of SOD in liver and heart in 0.15% and 0.25% SODm groups at 42 days of age were significantly higher than that of control group (P<0.05),while the supplement of 0.25% and 0.30% SODm had an outstanding effect on the activity of SOD of breast and thigh muscle (P<0.05).It was concluded that the supplement of SODm had no negative effects on the growth performance,and 0.15% to 0.25% dietary SODm could improve SOD activity and the tenderness,L^* value and shear value of muscle.

Mulberry leaf is an ideal protein resources as it contains protein,vitamins (vitamin A and vitamin C),trace elements (Fe,Mn,Cu and Zn) and flavonoids active ingredients,which are the nutrients that animals need to sustain their lives.Researches have showed that 15% mulberry leaf may affect the growth of the finishing pigs.Moderate amount of mulberry leaf (6%) can improve the meat quality by delaying the decline of pH and increasing antioxidant capacity.At the same time,mulberry leaf also can influence the amino acids and fatty acids composition (increase the content of unsaturated fatty acid) of muscle to improve the flavor of meat.In poultry production,adding mulberry leaf can reduce the pH of chicken meat,and improve the quality of meat by changing the fatty acid composition of muscle.In addition,the active ingredients of mulberry leaf have been shown to improve the antioxidant ability of mice,decrease blood sugar,blood fat and atherosclerosis and so on.In this paper,the nutritional value of mulberry leaves and its application in pigs,broilers and laying hens were reviewed to provide references for its further application as protein resource in livestock production.

In order to optimize the reproductive efficiency of sows and explore the effects of backfat thickness during pregnancy on the sow's farrowing duration and reproductive performance,the data from 2 969 Large White sows and 1 787 Landrace sows,including backfat thickness,farrowing duration,total litter size,alive litter size and litter weight of the three stages of sow gestation (30,80 and 107 d of gestation) in January 2017 to October 2017 were collected.The results showed that the farrowing duration of Large White sows was significantly lower than that of Landrance sows (P<0.05).The total litter size,alive litter size and birth weight of Large White sows were significantly higher than that of Landrance sows(P<0.05).At 30 days of gestation,with the backfat thickness of 18-20 mm in Large White sows,the total litter size,alive litter size and the litter weight were the highest;For the backfat thickness of 18-20 mm in Landrace sows,the total litter size and alive litter size was the biggest.At the 80 days of gestation,when the backfat thickness was more than 20 mm in Large White sows,the total litter size,alive litter size and litter weight were the biggest,and the sows had the shortest farrowing duration;For the backfat thickness of 16-18 mm in Landrace sows,the total litter size,alive litter size and the litter weight were the biggest,and the farrowing duration was shorter.At 107 days of gestation,the backfat thickness of 14-16 mm in Large White sows,the total litter size,alive litter size and the litter weight were the biggest,and farrowing duration was not significantly different from the other groups (P>0.05);While for the back fat thickness of ≥ 20 mm in Landrace sows,the total litter size and alive litter size were the biggest,and the litter weight was larger.From 30 days to 107 days of pregnancy,for Large White sows with backfat thickness reduced by 1-2 mm,the total number of litters and alive litter size were the biggest,but the litter weight was smaller,and farrowing duration was longer.For Landrace sows with backfat thickness reduced by more than 2 mm,the total number of litters and alive litter size were the biggest,the litter weight was larger,and the farrowing duration was shorter.The research had shown that optimize the backfat thickness of sows could effectively improve reproductive performance of sow and welfare level of sow during perinatal period.The pig farm could establish database of backfat thickness according to the nutritional level and sow breeds,adjusting the backfat thickness in different stages of pregnancy by feeding accurately and controlling the reasonable change of backfat during pregnancy.

The aim of this study was to elucidate the association between tachykinin receptor 3 (Tacr3) gene polymorphism (g.21484478A>C,g.21560640C>T and g.21560688T>C loci) and litter size in sheep,providing new genetic markers for high prolific molecular breeding of sheep.Year-round estrous sheep breeds (Small-tail Han sheep,Hu sheep and Cele Black sheep) and seasonal estrous sheep breeds (Tan sheep,Sunite sheep and Prairie Tibetan sheep) were selected and Sequenom MassARRAY^® SNP assay was applied to detect the genotypes of g.21484478A > C,g.21560640C > T and g.21560688T > C loci of Tacr3 gene,then the association was analyzed between Tacr3 gene polymorphism and litter size in Small-tail Han sheep.The results showed that three genotypes were found at g.21484478A>C (AA,CA and CC),g.21560640C>T (TT,TC and CC) and at g.21560688T>C (TT,CT and CC) loci in both year-round estrous and seasonal estrous sheep,both genotype frequency and gene frequency of three loci of Tacr3 gene were extremely significantly different between year-round estrous and seasonal estrous sheep (P<0.01).Population genetic analysis showed that the g.21484478A>C locus was at moderate polymorphism (0.25 < PIC < 0.5) in Tan sheep and Cele Black sheep,it was at low polymorphism (PIC<0.25) in Small-tail Han sheep,Sunite sheep and Hu sheep;The g.21560640C>T locus was at moderate polymorphism in 6 sheep breeds;The g.21560688T>C locus was at low polymorphism in Small-tail Han sheep,Sunite sheep,Hu sheep and Prairie Tibetan sheep.The χ² test indicated that the g.21560640C>T locus of 6 sheep breeds was in Hardy-Weinberg equilibrium (P>0.05);The g.21484478A>C locus of Prairie Tibetan sheep was not in Hardy-Weinberg equilibrium (P<0.05);The g.21560688T>C locus of Tan sheep and Cele Black sheep was not in Hardy-Weinberg equilibrium (P<0.05).The association analysis indicated that the polymorphism of g.21484478A > C,g.21560640C > T and g.21560688T > C had no correlation with the litter size of the first,second or third parity in Small-tail Han sheep (P>0.05).In conclusion,the three polymorphic loci of Tacr3 gene might not be suitable for breeding of Small-tailed Han sheep.

In order to investigate the relationship between CAPN1,H-FABP genes and the growth and intramuscular fat contentcontent (IMF) of Yellow-feathered broilers under the free-range feeding system,130 3-week-old health Yellow-feathered broiler hens with similar weight were selected as test material and randomly divided into two groups with cage rearing (control group) and free-range group (test group).A total of 360 samples of chest muscles,leg muscles and abdominal fat of Yellow-feathered broiler hens were collected,the CAPN1 and H-FABP genes mRNA expression were analyzed by Real-time quantitative PCR,and the IMF content was determined by the method of Soxhlet extraction.The results showed that the CAPN1 and H-FABP mRNAs of Yellow-feathered broilers were expressed in different degrees in the chest muscles,leg muscles and abdominal fat.With the increase of age,the overall expression trend of CAPN1 in free-range group was increase-decrease-increase-decrease,and the expression level of CAPN1 mRNA in chest and abdominal fat were significantly higher in cage rearing than that in free-range during 3 to 6 w (P<0.05).The overall expression trend of H-FABP decreased with the increasing age,and the expression trends in the chest and leg muscles were the same and they were significantly higher in free-range than the cage rearing (P<0.05).The expression of H-FABP in abdominal fat was higher in cage rearing group (P>0.05).Compared to free-range chickens,cage rearing chickens grew faster,and the rate of abdominal fat and IMF content were higher also.The test results could provide some reference for the CAPN1 and H-FABP genes molecular breeding of Yellow-feathered chickens.

In order to elucidate whether PP1γ2 is present or not in tree shrews'spermatozoa and its form in epididymal sperm,and further explore the regulation of PP1γ2 on sperm maturation and motility in tree shrews,the present form and phosphorylation levels of PP1γ2 in caput and cauda epididymal spermatozoa under various conditions have been analyzed by Western blotting.The effects of db-cAMP,IBMX or Ca²⁺ on the phosphorylation levels of PP1γ2 in tree shrew sperm were investigated.The effects of okadaic acid (OA) and calyculin A (CA) on the phosphorylation level of PP1γ2 and motility in caput and cauda epididymal spermatozoa were further studied with tree shrews as experimental animals in the present study.The results showed that PP1γ2 was present in caput and cauda epididymal spermatozoa of tree shrew,and the phosphorylation level of PP1γ2 was far higher in cauda epididymal spermatozoa than that of caput spermatozoa.db-cAMP,IBMX or Ca²⁺ did not change the phosphorylation level of PP1γ2,OA or CA could significantly increase not only the phosphorylation level of PP1γ2 in caput and cauda epididymal spermatozoa but also sperm motility (P<0.05),especially for caput epididymal spermatozoa.The optimal concentrations of OA and CA were 1 μmol/L and 10 nmol/L,respectively,and the optimal incubation time were 15 and 20 min,respectively.This study indicated that PP1γ2 played an important role in regulating sperm maturation and motility in tree shrews through phosphorylation and dephosphorylation.

Skeletal muscle development is a complex process involving the formation and proliferation of muscle cells,the formation of muscle ducts and muscle fibers,and the final maturation process.The growth and development of skeletal muscle directly affects the production performance in sheep.With the development of sequencing technology,more and more people are trying to elucidate the systematic regulatory network of skeletal muscle development from the genetic level,and explore the molecular markers related to the important functional traits such as meat quality and meat production performance.Cell enhancement factor 2 (MEF2) is a conserved and functional transcription factor that controls the expression of muscle genes and plays a role in regulating muscle generation,neural development and differentiation.Myogenic differentiation (MyoD) gene family regulates the differentiation of myocytes and the maturation of skeletal muscle system.In order to further understand the function of sheep skeletal muscle and its growth and development regulatory genes,starting from the structural features of skeletal muscle,this paper focuses on the structure and function of MEF2 and MyoD genes family and the regulation of non-coding gene on gene expression in skeletal muscle.

The objective of this study was to determine the genetic diversity and evolution of Danzhou chicken.The complete mitochondrial DNA (mtDNA) D-loop regions of 36 Danzhou chickens were amplified,sequenced and analyzed.The sequencing reads were compared with the complete mtDNA D-loop sequence of several relative strains of chicken annotated in GenBank,and analyzed by bioinformatics methods.The genetic diversity and its evolutionary relationship in Danzhou chicken were analyzed.The results showed that the lengths of PCR products at the D-loop region were 1 210 bp,with 59.9% being A+T and 40.1% as C+G.The variable regions were 167-1 215 bp,and the high variable regions were mainly 167-367 bp.A total of 20 variable sites that defined 6 haplotypes were identified.The average haplotype diversity (Hd) and average number of nucleotide difference (k) were 0.571 and 6.449,respectively,the nucleotide diversity (Pi) was 0.00537,and the Tajima's D value of neutrality test was 1.61643.6 haplotypes could be grouped to 3 haplogroups (A,B and C) as determined by phylogenic analysis,with B clade,as the most abundant population.It concluded that the genetic diversity and haplotype diversity of Danzhou chicken were relatively low.Phylogenetic tree showed that the genetic composition of Danzhou chicken came from 3 maternal ancestors,Gallus gallus spadiceus,Gallus gallus bankiva and Gallus gallus jabouillei were potential ancestors.There was few influence of exotic lineage detected,which indicated that Danzhou chicken was a relatively conserved breed.

To understand the molecular prevalence of feline panleukopenia virus (FPV), feline astrovirus (FAstV) and feline enteric coronavirus (FEcoV) in Chengdu, this experiment collected 315 domestic cats and 34 community stray cats anal swabs containing 228 clinically infected cat samples and 121 clinical healthy cat samples. Viral nucleic acids in all samples were detected by PCR. At the same time, pathogen epidemiology analysis was carried out according to factors such as animal age, living state and immune status, and genetic evolution trees were constructed based on the sequences of viral gene fragments. The results showed that the detection rates of FPV, FAstV and FECoV were 38.4% (134/349), 23.2% (81/349) and 19.5% (68/349), respectively. In the case of mixed infection, FPV/FAstV (9.5%, 33/349), FAstV/FECoV (4.6%, 16/349) and FPV/FECoV (4.3%, 15/349) were the main infections. Based on the analysis of partial gene sequences, the homology of the FPV VP2 gene sequence between samples was 95.1% to 100%. The sick cat strains were divided into two branches, one of which was closely related to the Portuguese strain (GenBank accession No.:KT240136) and the Canadian strain (GenBank accession No.:MF069445), and the other was closely related to the Chinese CPV strain (GenBank accession No.:KY937664).The sequence homology of FAstV ORF1b gene between samples was 90.9% to 99.7%. One of the two branches of the sick cat strain was closely related to the Portuguese strain (GenBank accession No.:KF374704) and the American strain (GenBank accession No.:KM017743). The other branch was closely related to the Hong Kong strain of China (GenBank accession No.:KF499111).The N gene sequence homology of FECoV between samples was 89.3% to 100%, all the FECoV strains sequences were in a branch showing high homology to American (GenBank accession No.:FJ938059),Italy(GenBank accession No.:GU017092/GU017107) and Chinese strain (GenBank accession No.:KT852997).The survey results showed that the situation of cats carrying viral diarrhea in Chengdu was more common, some cats had multiple pathogen mixed infections, and the viral gene sequence had significant variation, increasing the risk of pathogen epidemics and the incidence of poisoned cats. It was necessary to actively carry out clinical prevention and control.

This study was aimed to explore the sequence characteristics of OmpK17 gene of Klebsiella pneumonia (K.pneumonia) and its immunogenicity.Aaccording to the OmpK17 gene sequence (accession No.:U52843.1) of K.pneumonia in GenBank,one pair of specific primers was designed,and the sequence of OmpK17 gene CDS of K.pneumonia Yuzhong (YZ) strain was amplified using PCR,the nucleotide and deduced amino acid sequences of OmpK17 gene were analyzed using bioinformatics softwares.The prokaryotic expression vector pET-OmpK17 was constructed and transformed into E.coli BL21(DE3),then the recombinant protein was expressed by IPTG induction.The recombinant protein was purified and the anti-sera against His-OmpK17 was prepared through immunizing rabbit.The immunogenicity of recombinant protein was analyzed by ELISA and Western blotting.The results showed that the sequence of OmpK17 gene of K.pneumonia YZ strain was highly conserved,its CDS sequence was 513 bp in length and encoded 170 amino acids,and the homology of nucleotide sequences of OmpK17 gene was above 99.6% between YZ strain and other K.pneumonia in GenBank.The molecular weight of OmpK17 protein was 18.5 ku,the hydrophilicity was strong,which had 1 signal peptide,1 transmembrane region and 6 potential antigenic determinant regions.Moreover,OmpK17 was a barrel-shaped protein,which suggested that it was a porin protein.In addition,the SDS-PAGE results indicated that the recombinant protein was successfully expressed in E.coli BL21 (DE3),and the expression product was mainly present in the form of inclusion body.The ELISA and Western blotting results showed that the OmpK17 of K.pneumonia had good immunogenicity.This study cloned the sequence of OmpK17 gene of K.pneumonia and expressed its encoded protein which had good immunogenicity.It laid the foundation for the study of the biological characteristics of OmpK17 gene and the establishment of K.pneumoniae detection method,and provided a theoretical basis for the development of genetic engineering vaccine of K.pneumoniae.

In order to investigate and determine the pathogen from calf which showed pneumonia symptoms occurred in cattle farms in Xinjiang,one strain of Mycoplasma bovis was isolated from seven pneumonic lung tissue samples of calves using liquid and solid Mycoplasma medium.The isolated strain was characterized by the colonial morphologic observation and biochemistry test.Specific primers of Mycoplasma bovis and universal 16S rRNA primers of Mycoplasma were employed to amplify and the amplicons were sequenced as well.DNAStar software was used to compare the sequence between the isolate and the other strains deposited in GenBank.A phylogenetic tree of different strains based on 16S rRNA sequences was constructed by Mega 6.0 software with the Neighbor-Joining method.The results showed that colonies of isolated strain grew on solid medium were typical fried-eggs,the central depression of the colony was deep into the medium and thin and transparent periphery,and Dienes staining was dark blue.The isolated strain could not ferment glucose,not decompose urea,and not hydrolyze arginine.Hemadsorption test and hemolysis test were negative,while film and spot test and triphenyl tetrazolium chloride reduction reaction test were positive.PCR products of Mycoplasma bovis showed the specific band of 1 911 bp.Sequence analysis indicated that 16S rRNA of the isolated strain showed 99.8% homology with that of Mycoplasma bovis strain PG45,and 99.3% to 99.7% homology with that of local strains of Mycoplasma bovis (Mb NM2012,Mb HB0801,Mb Hubei-1,Mb Ningxia-1,Mb CQ-W70 and Mb 08M strains).The phylogenetic tree showed that the 16S rRNA gene of the isolated strain was closely relative with Mb Ningxia-1 and Mb 08M strains,which were in the same branch.The results confirmed that the pathogenic bacteria causing cattle death was Mycoplasma bovis,which provided theoretical basis and scientific basis for the prevention and treatment of Mycoplasma bovis in Xinjiang.

To investigate the epidemiological and genetic variation of porcine epidemic diarrhea virus (PEDV) in Jiangxi province,a total of 182 clinical porcine intestinal and fecal samples were collected from pig farms in some areas of Jiangxi province in 2017 and detected for PEDV by RT-PCR.Two pairs of primers were designed and the S gene from 37 PEDV positive samples were amplified,cloned and sequenced.Genetic evolution analysis of S gene was performed using bioinformatics software and referenced PEDV strains registered in GenBank.The results showed that the length of S gene genome of 37 Jiangxi PEDV strains was 4 158/4 161 bp,encoding 1 385/1 386 amino acids.All PEDV strains belong to Group 1 which had a close relationship with the United States strains,and differed genetically from European strains (Br1/87) and the vaccine strains (CV777).36 of 37 PEDV Jiangxi strains were G1-1 and 1 strain was G1-2.The nucleotides homology of S gene among 37 PEDV strains were 96.9% to 100.0%,and that of amino acids homology was 96.1% to 100.0%.The nucleotides and amino acids homology of S gene between PEDV strains and referenced PEDV strains were 92.7% to 100.0% and 91.5% to 100.0%,respectively.Compared with the vaccine strains (CV777),S gene of PEDV Jiangxi strains existed bases deletions,insertions and mutation.This study confirmed the prevalence and variation of PEDV in Jiangxi province from the perspective of molecular epidemiology,and provided references for guiding the scientific prevention and control of PEDV in Jiangxi province.

To understand the prevalence and molecular biology of porcine circovirus type 3 (PCV3) in Jilin province,a total of 484 serum samples in Jilin province from 2015 to 2017 were detected by PCR in this study.The ORF2 gene sequence of PCV3 positive samples was sequenced,the molecular characterization of ORF2 gene sequences was analyzed using bioinformatics softwares of DNAStar and Mega 6.06.The results showed that the total infection rates of PCV3 were 28.1% (136/484) at serum samples and 65.8% (25/38) at pig farms,and showed increased infection rates from 2015 to 2017.The homology alignment analysis showed that the nucleotide and amino acid homology of the 4 ORF2 gene sequences in this study shared 98.3% to 98.9% and 97.7% to 99.5% homologies,respectively,and also shared 97.7% to 99.7% and 96.7% to 100% homologies in the nucleotide and amino acid with reference ORF2 gene sequences in GenBank.The results of phylogenetic showed that there were two major branches of PCV3 strains,PCV3a and PCV3b.The PCV3 strains isolated in Jilin province were distributed on the two major branches,PCV3a (1 isolate) and PCV3b (3 isolates).The difference between the amino acid at sites 24 (A,V) and 27 (R,K) of the Cap protein might be generated during the evolutionary of PCV3 strains.These results showed that PCV3 had a high infection rates in pig herds and pig farms in Jilin province.The molecular characterization of PCV3 showed that PCV3 strains were highly conserved in natural evolution.This study provided an important reference for the molecular characteristics of PCV3.

To explore the effects of Ru Huangxiao oral liquid on yin-nourishing, strengthening righteousness and eliminating evil spirits, this study observed the clinical symptoms,diet,water consumption, body weight, thymus index, spleen index, contents of total triiodothyronine (T₃), total thyroxine (T₄) and thyroid stimulating hormone (TSH) in rats with yin deficiency model and the effect of mouse macrophage phagocytosis and inhibition on Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae and Streptococcus uberis. The results showed that the rats of yin deficiency model had normal mental state and were easy to grasp after the administration of Ru Huangxiao oral liquid, but the gloss of the coat was still poor, there was hair removal and the stool was normal. Compared with model group, the body weight (P>0.05), thymus index (P>0.05), spleen index (P>0.05), TSH content (P<0.05) of the experimental group increased, the food intake (P<0.05), T₃ (P<0.05) and T₄ content (P>0.05) decreased. However, the difference was not significantly different from the negative control group (P>0.05).Compared with model group, the water consumption of the rats in the experimental group significantly decreased (P<0.05), but it was still significantly higher than the negative control group (P<0.05). The phagocytic index of the high, middle and low dose groups and the negative control group were significantly different (P<0.05). The percentage of phagocytosis was significantly higher in the high-dose and middle-dose groups compared with the negative control group (P<0.05). The minimum inhibitory concentration (MIC) of Ru Huangxiao oral liquid to Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae and Streptococcus parauberis were 12.50, 1.56, 0.20 and 0.39 mg/mL, respectively. In summary, the Ru Huangxiao oral liquid could improve the clinical symptoms of yin deficiency in rats and enhance the phagocytic function of macrophages. At the same time, it could inhibit the main pathogens of cow mastitis, and had the effects of nourishing yin, strengthening righteousness and eliminating evil spirits, it could be used for the treatment of dairy cow mastitis.

This study was aimed to understand the degree and mechanism of cross resistance of Escherichia coli isolated from some pig farms in Neijiang city to antimicrobials and disinfectants.The minimal inhibitory concentration (MIC) values for enrofloxacin,oxytetracycline,benzalkonium bromide and glutaraldehyde of 66 strains of Escherichia coli isolated from some pig farms in Neijiang city were determined using broth microdilution method and selected cross resistance strains that were resistant to antimicrobials and disinfectants.Whether the cross resistance strains had qacE△1,acrA,acrB and tolC genes was detected by PCR technology and agarose gel electrophoresis.And the mutation of acrA,acrB and tolC genes of these strains were determined.All the 66 strains were resistant to enrofloxacin and oxytetracycline,and three strains (named as No.5,No.6 and No.7) were resistant to benzalkonium bromide,but no strains were resistant to glutaraldehyde.It showed that there were three strains were cross resistance strains.All the three cross resistance strains had acrA,acrB and tolC genes,but only No.6 had qacE△1 gene.All the genes acrA,acrB and tolC of the three strains occurred mutation,and the amino acid that transcribed by these genes also occurred mutation.The highest and lowest base mutation rate were 2.10% (No.7's tolC gene) and 0.97% (No.7's acrA gene).The highest and lowest amino acid mutation rate were 2.39% (No.7's acrB gene) and 0.22% (tolC gene of No.5,No.6 and No.7).And some mutation occurred at the same base site or the same amino acid site.All the 66 strains isolated from some pig farms in Neijiang city were resistant to enrofloxacin and oxytetracycline,and there were cross resistance strains that were resistant to antimicrobials and disinfectants,and the cross resistance mechanism of these strains might related to the efflux pumb AcrAB-TolC.

In order to understand the cause of diarrhea death in a rabbit field in Lhasa,a strain of bacterium was isolated from the intestinal contents of a rabbit,and was isolated and identified by Gram staining,biochemical identification,16S rRNA PCR amplification and sequence alignment,animal regression test and drug sensitivity test.The results showed that the isolated strain was a single or pair of short rod-shaped Gram-negative bacteria,and the homology of the isolate with Proteus mirabilis in GenBank were 73.5% to 99.8%,the homologous analysis of the 16S rRNA sequence of the isolates and 11 reference strains was carried out by Mega 7.0 software,and the phylogenetic tree was constructed.The isolated strain was identified as Proteus mirabilis,named as T2018.In the animal regression test,there were 3 cases of diarrhea,but none of them died.The results of the necropsy showed that the jejunum and ileum were thin and transparent,with translucent jelly inside;Colon and cecal mucosa were congested,there were bleeding spots on the serosa.Susceptibility tests showed that the isolated strains were only highly sensitive to the five antibacterial agents of amoxicillin/clavulanic acid,gentamicin and ofloxacin,ciprofloxacin and norfloxacin,intermediate to lacillin,and resistant to 24 other antibacterials.The above results indicated that the diarrhea death of rabbits was caused by Proteus mirabilis.

In order to elucidate the mechanism of zearalenone (ZEA) on male reproductive toxicity and investigate the pathway of sertoli cell (SC) apoptosis induced by ZEA,the primary SC was used as the experiment material,exposed to different concentration of ZEA(0,5,10,20 μmol/L) for 24 h.qRT-PCR was used to detect the level of gene transcription of Bax and Bcl-2;Western blotting method was used to detect the expression of proteins related to mitochondria-dependent apoptotic pathway.The changes of apoptosis rate and apoptosis-related protein were detected by 20 μmol/L ZEA combined with the inhibitor of Caspase-8 (Z-IETD-FMK).The results showed that compared with the control group,the level of gene transcription of Bcl-2 was decreased significantly in the group treated with 20 μmol/L of ZEA (P<0.05),while the level of gene transcription of Bax increased significantly (P<0.05).The ratio of Bax/Bcl-2 and the expression of Cyto-CytC,Cleaved caspase-3 and Cleaved caspase-9 were extremely significantly increased while the expression of Mito-CytC was decreased extremely significantly in the group treated with 10,20 μmol/L of ZEA (P<0.01).Compared with ZEA group,the apoptosis rate induced by ZEA decreased extremely significantly after being treated with ZEA and Z-IETD-FMK (P<0.01).The ratio of tBid/Bid and Bax/Bcl-2,Cyto-CytC,Cleaved Caspase-3 and Cleaved Caspase-9 were decreased significantly or extremely significantly (P<0.05;P<0.01),while the Mito-CytC protein expression was increased extremely significantly (P<0.01).The results showed that ZEA could induce the apoptosis of SC via the mitochondria-dependent apoptotic pathway.

The aim of this study was to investigate the effects of low level glucose on mRNA expression of tumor necrosis factor-α (TNF-α),lysozyme (LYZ),induced nitric oxide synthase (iNOS),interleukin-6 (IL-6),lactoferrin (LF) and interleukin-8 (CXCL8) in bovine mammary epithelial cells.After enzymatic digestion,the bovine mammary epithelial cells were isolated and purified,and then cultured in complete medium containing 0.25,1.00 and 4.50 mg/mL glucose respectively.The expressions of related genes in cells were detected by Real-time fluorescence quantitative PCR.The results showed that there were no significant differences in the relative mRNA expression of the above genes among three glucose groups (P>0.05).The expression levels of TNF-α and LYZ in each concentration group were very low.The expression levels of iNOS were in the middle.However,the expression of IL-6,LF and CXCL8 in different concentration groups was relatively high.It suggested that the decrease of glucose concentration did not directly affect the basic expressions of TNF-α,LYZ,iNOS,IL-6,LF and CXCL8 mRNA in mammary epithelial cells.While without infection,mammary epithelial cells could express a certain amount of iNOS,IL-6,LF and CXCL8,which could be used as a reserve for innate immune defense in order to quickly respond to the invasion of pathogens.

A multiplex PCR combined with DNA microarray method was developed for detection of 5 swine reproductive diseases virus in this study.Five pairs of specific primers and probes were designed according to the published genomic sequences of swine fever virus (CSFV),porcine parvovirus (PPV),porcine reproductive and respiratory syndrome virus (PRRSV),Japanese encephalitis virus (JEV) and porcine circovirus type 2 (PCV2).The corresponding oligonucleotide chips were prepared,16 clinical samples and the standard strains of five reproductive viruses were tested.Specific gene fragments with fluorescent label were amplified by multiplex PCR and hybridized with microarray that immobilized with specific probes.The results showed that the multiplex PCR combined with DNA microarray to detect viral diseases was highly specific and stable,and the sensitivity was up to 10² copies/μL.The test results of 16 clinical samples showed a positive rate of 87.5% (14/16).These data indicated that the sensitive and specific detection method could be applied in these 5 viruses detection for clinical diagnosis and pathogenic epidemiologic investigation.

In order to evaluate the therapeutic effect of total flavonoids from Spatholobus suberectus Dunn (TFSD) on Escherichia coli (E.coli) sepsis,sepsis animal models were established in mice by intraperitoneally injected with E.coli,mice were administrated with various dose of TFSD after 1 h,the clinical manifestations of mice were observed and scored after 12 h.Then the mice were dissected,the liver,thymus and spleen were observed and weighted for the calculation of their organ indexes,pathological sections of livers were prepared for detail analysis of pathological change.Blood routine indicators were measured to analyze the levels of aspartate aminotransferase (AST),alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in serum.The levels of inflammatory cytokines such as interleukin-1β (IL-1β),IL-2,IL-6,IL-10 and tumor necrosis factor α (TNF-α) in serum were determined.The results showed that the injection of E.coli resulted in irregular hair coat,poor spirit,inflamed eyes,increase of secretion from eyes,tachypnea,even convulsion or shock.Atrophy was observed from liver,thymus and spleen tissues,corresponding with reduced organ index.Pathological sections indicated disorder of hepatic cord,swell of hepatic sinus,fuzziness of cell edges,and infiltration of inflammatory cells.The concentrations of leukocytes (WBC),lymphocyte (LYM),hemoglobin (HGB),red blood cell (RBC) and platelet (PLT) were significantly altered by the injection of E.coli (P<0.05).The levels of ALT,AST,LDH and inflammatory cytokines were significantly increased (P<0.05).However,the treatment of TFSD and amikacin was able to alleviate the clinical manifestation and inhibit the change of organ indexes,which tended to be similar to the control group.The results indicated that TFSD was able to alleviate E.coli induced injury by improving the immune response and inhibit inflammatory reaction in mice.

To diagnose the pathogen of a diseased racoon dog in a Jilin farm,in this experiment,bacterial culture,staining microscopy,biochemical identification,molecular biology identification and blood smear microscopy were used to isolate and identify the pathogens of the collected lungs,liver,spleen,kidney,intestines and blood.And the isolated pathogens were tested for pathogenicity and susceptibility test.The results showed that a dominant strain was isolated,which was presented as a rod-shaped Gram-negative bacilli with obtuse ends at the optical microscope.The results of biochemical tests showed that it could decompose glucose-lactose,mannitol,maltose and sucrose to produce acid gas,which was positive for M-R test,sputum test and starch hydrolysis test.The 16S rRNA sequence had 99% homology with the known E.coli sequence registered in the NCBI database.The animal lethality test results showed that the strain was pathogenic to mice.The results of drug susceptibility test showed that the isolate was most sensitive to the quinolone drugs norfloxacin and ciprofloxacin,and had different degrees of resistance to other drugs.The anti-coagulation smears of sick racoon dog proved to be infected by Eperythrozoon,and the infection rate of red blood cells reached more than 98%.In summary,the pathogenic strain of the disease infection was E.coli and the E.coli was pathogenic,at the same time,the sick raccoon dog suffered from Eperythrozoon and led to its growth retardation.

The purpose of this experiment was to study the in vitro bacteriostasis of different kinds of traditional Chinese herbal medicines combined with enrofloxacin hydrochloride against drug-resistant strains of Escherichia coli (E.coli).The minimum inhibitory concentration (MIC) of enrofloxacin hydrochloride (a commonly used drug) and different Chinese herbal medicines against E.coli resistant strains and their combined use was determinated by micro-broth method.The results showed that the antimicrobial effects of Cacumen Biotae,Rosa Laevigata and Schisandra Chinensis Fructus were better in water extracts of single traditional Chinese herbal medicine,and the MIC value were 3.91,3.91 and 7.81 mg/mL,respectively;The antimicrobial effects of Cacumen Biotae and Portulaca Oleracea were better in ethanol extracts of single traditional Chinese herbal medicine,and the MIC value were 7.81 and 3.91 mg/mL,respectively.When enrofloxacin hydrochloride was combined with water extracts of Schisandra Chinensis and Rosa Laevigata,the MIC value of drug-resistant strains of E.coli reduced to 0.0078 and 0.0039 mg/mL,respectively.When enrofloxacin hydrochloride was combined with ethanol extracts of Portulaca Oleracea,Radix Paeoniae Rubra,Cacumen Biotae and Rosa Laevigata,the MIC value of drug-resistant strains significantly decreased.When the enrofloxacin hydrochloride was combined with water extract of Houttuynia Herba,Radix Paeoniae Rubra and Ailanthi Cortex,and alcohol extract of Portulaca Herba,Atractylodis Rhizoma,Berberidis Radix,Paeoniae Radix Rubra,Allii Macrostemi Bulbus,Allii Macrostemonis Bulbus,Platycladi Cacumen,Rosa Laevigatae and Ixeris Denticulata,it showed adding or synergistic bacteriostatic effect on the E.coli resistant strains,the FIC values were 0.75,0.62,0.62,0.27,0.75,0.75,0.31,0.62,0.50,0.53,0.28 and 0.62,respectively.It was preliminarily believed that there were traditional Chinese herbal medicines that could effectively reduce the drug resistance of E.coli among the different kinds of traditional Chinese herbal medicines,there were also traditional Chinese herbal medicines combined with enrofloxacin hydrochloride,which had synergistic or additive effects.It was possible to find effective drug combinations for the prevention and treatment of E.coli disease to against multi-drug-resistant bacteria.