This study was aimed to explore the physicochemical properties and structural characteristics of porcine semen 6-phosphofructokinase (PFKL) gene,elucidate the physiological role and regulatory mechanism of glycolysis in spermatogenesis.PFKL gene was amplified by RT-PCR,the obtained gene fragment was inserted into pUC57 vector for cloning and sequencing,and its amino acids sequence,transmembrane structure,modification site,secondary structure,tertiary structure,antigen site and functional annotation were predicted and analyzed by bioinformatics methods.The results showed that the total length of PFKL gene was 2 349 bp,encoding 782 amino acids,the molecular weight of protein was 83.819 ku,the isoelectric point was 6.96,and the molecular formula was C₃₆₇₄H₅₈₉₇N₁₀₅₁O₁₁₀₉S₄₀.The homology of porcine PFKL gene was more than 80.0% compared with the reference sequences of Mus musculus,Iumenta anatis,Ovis aries,Bos taurus,Pongo abelii and Homo sapiens.The protein structure had no transmembrane structure and signal peptide region,and contained 2 N-glycosylation modification sites,14 O-glycosylation sites and 35 phosphorylation sites.The structure of PFKL protein was mainly composed of random coil and alpha helix,and its homology with muscle phosphofructokinase and platelet phosphofructokinase was 68.24% and 70.84%,respectively.At the same time,the 27 antigenic sites were different in strength,flexibility,uniform distribution and high surface plasticity,and there were binding sites of PKC and PKA specific protein kinase,which were hydrophilic proteins with more potential B cell epitopes.The results of GO functional annotation analysis showed that PFKL gene had carbohydrate kinase activity and phosphofructokinase activity,and could participate in hexoses metabolism and monosaccharides metabolism.In conclusion,this reaults laid a molecular biological foundation for improving sperm quality and the incidence of sperm-egg binding.

The aim of this study was to clone bovine interferon regulatory factor 9 (BoIRF9) gene,analyze its biological information,and express its antigenic dominant region.In this study,total RNA was extracted from EBK cells infected with vesicular stomatitis virus (VSV).Using the first chain of retroviral DNA as template,the specific primers were designed to amplify BoIRF9 gene according to bovine IRF9 gene sequence in GenBank.The molecular characteristics,advanced structure and molecular evolution of the cloned BoIRF9 gene were analyzed.According to the hydrophilicity,antigen index and surface accessibility of BoIRF9,a pair of specific primers were designed to clone the dominant antigen region fragment of BoIRF9 and connected it to pET30a (+) vector to construct the recombinant expression plasmid pET30a-IRF9-Part.The correct plasmid was transformed into E.coli Rosetta competent cells,and the dominant region protein was induced by IPTG.The protein was identified by SDS-PAGE and Western blotting.The results showed that the full-length of BoIRF9 gene was 1 684 bp,the length of BoIRF9 gene CDS was 1 320 bp,incoding 439 amino acids.The amino acids sequence similarity of BoIRF9 was 80.0% to 96.2% with Homo sapiens,Equus caballus,Sus scrofa and Capra hircus.Phylogenetic tree analysis showed that Holstein dairy cows had the closest relationship with Capra hircus,followed by Sus scrofa,Equus caballus,Loxodonta africana and Homo sapiens.The molecular formula of BoIRF9 protein was C₂₁₃₉H₃₃₄₂N₅₉₄O₆₅₈S₁₈,the molecular weight was 48.5 ku,and the theoretical isoelectric point (pI) was 5.71.There was no transmembrane structure and no signal peptide in BoIRF9 protein,and there was a potential N-glycosylation site and multiple phosphorylation sites.The secondary structure of BoIRF9 protein contained 115 alpha helix,22 beta turn,68 extend chain and 234 random coil,respectively.There were two conserved domains of BoIRF9 protein,a 648 bp gene fragment was cloned from the 12 to 221 amino acids containing the first conserved domain,and the recombinant protein with a molecular weight of 27 ku was expressed.This results could provide a reference for the further study of interferon regulatory factors.

This study was aimed to clone acetyl-CoA synthetase 2(ACSS2) gene in goats,perform bioinformatics analysis,and detect its mRNA expression level in mammary gland during different lactation stages in goats.Mammary gland tissue RNA in goats was used as a template,and the complete CDS region of ACSS2 gene in goats was amplified and cloned.The sequencing result was analyzed by bioinformatics,and the expression of ACSS2 gene in mammary gland tissues during different lactation stages in goats were analyzed.The results showed that the total CDS region sequence of ACSS2 gene in goats was 2 106 bp,which encoded 701 amino acids.The homology of ACSS2 gene in goats with Bos taurus,Equus caballus,Homo sapiens,Canis lupus familiaris,Sus scrofa,Rattus norvegicus and Gallus gallus were 97.8%,92.0%,91.3%,91.3%,91.1%,88.1% and 73.3%,respectively.The physicochemical properties of ACSS2 protein showed that its molecular weight was 78.72 ku,and its theoretical isoelectric point was 6.03,which belonged to an acidic protein.No transmembrane structure and signal peptide was found in ACSS2 protein,but it contained a N-terminal domain of acetyl-CoA synthetase.Subcellular localization analysis indicated that ACSS2 protein was located in endoplasmic reticulum (44.4%),mitochondria (33.3%),cytoplasm (11.1%) and nucleus (11.1%).ACSS2 protein structure was mainly consisted of alpha helix (29.10%),extended chain (21.54%),beta turn(9.84%) and random coil (39.52%).Real-time PCR results revealed that the highest and the lowest expression of ACSS2 gene in goats were in the middle lactation and dry lactation periods,respectively.These results provided references for further study of the function and transcriptional regulation mechanism of ACSS2 gene in lipid metabolism of goats.

The study was aimed to reveal the regulation relationship between myostation (MSTN) and matrix metalloproteinase (MMPs),and explore the effects of MSTN on the expression of MMP-2/7/9 in PK15 cells.The backfat tissues of MSTN single allele knockout pig was analyzed by transcriptome sequencing.MSTN knockout PK15 cells line of MSTN single allele knockout PK3108 cells and MSTN double allele knockout L18 cells were prepared,the mRNA and protein expression of MSTN and MMP-2/7/9 in PK15,PK3108 and L18 cells were performed by Real-time PCR and Western blotting,respectively.The results showed that compared with the wild type pigs,the levels of transcription factor C/EBPδ and MMP-2/7 mRNA were extremely significantly reduced (P < 0.01),and the main components of extracellular matrix laminin (LN) and fibronectin (FN) were extremely significantly increased in MSTN single allele knockout pigs (P < 0.01).The revived PK3108 and L18 cells showed green fluorescence.Real-time PCR results showed that the mRNA expression of MSTN and MMP2/7/9 in PK3108 and L18 cells were extremely significantly reduced than that in PK15 cells (P < 0.01).Western blotting results indicated that the protein expression of MSTN and MMP2/7/9 in PK3108 and L18 cells were significantly reduced than that in PK15 cells.The results revealed that the expression of MMP-2/7/9 were significantly decreased by MSTN deletion in PK15 cells,and the expression trend of MMP-2/7/9 protein were consistent with the trend of MSTN protein expression.

To explore the biological characteristics and genome characteristics of Escherichia coli phages,in this study,a Shiga toxin-producing multi-drug resistant Escherichia coli strain GXEC010 was isolated from the sewage of a swine farm in Guangxi,and a lytic phage strain was isolated from the sewage,and through the transmission electron microscope,determination of best multiplicity of infection in the plural,one-step growth curve drawing,thermal sensitivity and pH stability evaluation,sterilization experiment and whole genome sequencing methods to analyze the biological characteristics and genome characteristics of the phage.The results showed that the phage which could efficiently lyse Escherichia coli GXEC010 was isolated and purified,and named as vB_EcoM_BP10.The optimal multiplicity of infection this bacterium was 1.The results of one-step growth curve showed that the incubation period of infected host bacteria was 5 min,the outbreak period was 65 min,and the outbreak quantity was 51.The tolerable temperature range was 30 to 60 ℃,and it could be maintained stable in the pH of 5 to 8.The results of bacteriophage bactericidal experiment showed that the bacteriophage vB_EcoM_BP10 had a good bactericidal effect when MOI=1.Completed genome sequencing results showed that the total fragment length of phage vB_EcoM_BP10 was 52 288 bp,GC content was 44.16%,and included 71 ORF.The GO functional notes indicated that phage vB_EcoM_BP10 could participate in small molecule metabolism,cell nitrogen compound metabolism and other biological processes,and had molecular functions such as nucleotide transferase activity,ion binding and DNA binding.This bacterium was closely related to Escherichia phage ST32 and Enterobacteria phage phiEcoM-GJ1.In conclusion,the phage vB_EcoM_BP10 in this study had clear host receptor specificity,good thermal stability and acid-base stability,which could lay a theoretical foundation for the effective prevention and control of Shiga toxin-producing multidrug-resistant Escherichia coli.

To know the distribution and structural characteristics of clustered regular interleaved short palindromic repeats (CRISPR) and distribution of cas genes in Escherichia coli O157∶H7,complete genome sequence,CRISPR position,CRISPR flanking sequence and cas gene clusters sequence of 92 strains Escherichia coli O157∶H7 were obtained through GenBank database and CRISPRdb database,then multiple sequence alignment,promoter prediction and RNA secondary structure prediction were used to obtain the CRISPR system.The results showed that the genomes of Escherichia coli O157∶H7 had three CRISPR loci,(CRISPR1,CRISPR2 and CRISPR3),and the gene sequences at each CRISPR locus were highly consistent.The repeats of CRISPR1 and CRISPR2 could form the stem ring structure,and the bases were easily changed on the ring.There was a sequence X which was 451 bp in CRISPR2,and it divided CRISPR2 into two parts (CRISPR2a and CRISPR2b).The ratio of base A to T of the segment X in CRISPR2 was 74% and the sequence was found in 91 strains of O157∶H7 Escherichia coli,in which at least 1 promoter and 9 transcription factor binding sites could be predicted.The ratio of base A to T of suspected leader which located in CRISPR2 downstream was not less than 69%,the sequence was 340 bp and the sequence was available in 92 strains of bacteria,which could predict at least 1 promoter and 3 transcription factor binding sites.In these 20 strains of Escherichia coli,15 strains had complete cas gene clusters,5 strains lacked cas3 gene.The CRISPR structure and cas gene cluster of Escherichia coli O157∶H7 were relatively stable,the CRISPR sequence was highly conservative.The CRISPR2 structure was different from other types.The sequence X found in this study was widely distributed and conserved in Escherichia coli O157∶H7,which could be used as a potential molecular target to identify Escherichia coli O157∶H7.

To study the expression of β2m gene in swine kidney PK-15 cell line,total RNA was extracted and β2m was amplified by RT-PCR.The recovered gene product was cloned into pMD18-T vector to get some recombinant plasmids.After being screened by EcoR Ⅰ and Hind Ⅲ double enzyme digestion,the positive clones were sequenced in a biological company.The sequence of the β2m from PK-15 was edited and analyzed by GENETYX version 9.0,then,it was compared with other swine by DNAMAN version 5.2.2.The phylogenetic tree of the β2m from PK-15 with other animal β2m was constructed by NJ method of Mega 5.0.The secondary structure and tertiary structure of β2m from PK-15 were also predicted and analyzed by SWISS-MODEL and PDBsum.Proteins extracted from PK-15 cells were used to detect the expression of β2m by Western blotting.The results showed that β2m gene was amplified successfully by RT-PCR.The fragment was about 360 bp.After sequencing,it showed that the real length of β2m gene was 364 bp coded 118 amino acids including a mature peptide with 98 amino acids on the carboxyl terminal and a signal peptide with 20 amino acids on the amino terminal.Sequences analyzing confirmed that there was no mutation of β2m gene in PK-15 cells.The phylogenetic tree showed that PK-15-β2m was the closest to bovine β2m,followed by sheep,horses,etc.The results of multiple sequence comparison showed that the mature peptide of PK-15-β2m along with other bovine,ovine and equine β2m was constituted by 98 amino acids,while 99 amino acids in other animal β2m.The secondary structure prediction showed that the mature peptide of PK-15-β2m was mainly composed of β-sheet,β-turn and γ-turn,without α-helix.The tertiary structure prediction showed that the mature peptide of PK-15-β2m was constituted by 7 β-sheet.The protein samples isolated from PK-15 cells were detected by Western blotting and it was proved that β2m was successfully expressed in the PK-15 cells.This study confirmed the stable expression of β2m in nucleic acid and protein level in PK-15 cell line,which laid a foundation for the further study of the antigen-presenting function of PK-15 cells.

In order to screen the reference genes that suitable for bovine skeletal muscle satellite cells (MSCs) differentiation process and not affected by MSTN gene expression,bovine MSCs from wild group (WT),MSTN interfered group (si-MSTN) and control group (NC-MSTN) were used as sample.HMBS,B2M,GAPDH,TUBB,SDHA,18S rRNA,ACTB,RPL4,PPIA,HPRT1 and YWHAZ were selected as candidate internal reference genes.The relative expression of candidate internal reference genes was determined by Real-time quantitative PCR and Ct value analysis.Firstly,the expression stability of each candidate internal reference gene in wild group (WT) bovine MSCs of proliferative (GM) and the differentiated third day after differention (DM3) cells was evaluated by three independent evaluation softwares:geNorm,NormFinder and BestKeeper,and the first five candidate reference genes with the most stable expression were screened out.Furthermore,the expression level and stability of these five internal reference genes in si-MSTN group and NC-MSTN group of GM and DM3 were analyzed by similar methods.The analysis of geNorm and NormFinder showed that the expression of HMBS,B2M,TUBB,GAPDH and ACTB genes were more stable before and after differentiation of bovine MSCs.The BestKeeper showed that the correlation coefficient (R) of ACTB and TUBB were ranked first,GAPDH,HMBS and B2M were not very high,but GAPDH had the lowest SD value,so these five candidate genes were selected for subsequent experiments.In siRNA-MSTN group and NC-MSTN group of GM and DM3,the results of geNorm and NormFinder showed that the expressions of GAPDH,TUBB and B2M in the five candidate reference genes were more stable than other.BestKeeper showed that the correlation coefficient of TUBB was not the highest,but its SD value was the lowest.Based on the above analysis,TUBB was selected as the most suitable internal reference gene in GM and DM3 of bovine MSCs siRNA-MSTN and NC-MSTN groups.The result of this study would provide reference for future research on gene expression analysis of bovine MSCs in different growth process and after MSTN expression was regulated.

With the discovery of the double helix structure of DNA,a preliminary study of genome has been carried out,and the central principle has become the core of life science.It has provided a beneficial start for the rapid development of molecular biology and detection technology in recent years.With this,animal gene sequencing research has become a new hot topic in biology.Although a great deal of gene data can be obtained by transcriptome sequencing,the further exploration of data needs further research,and these in-depth mining needs bioinformatics analysis tools,thus establishing a multi-group science,through its continuous improvement,making modern biological science form a complete system.It enables the study of biogenetics to be a layer-by-layer analysis that integrates information and data to describe life activities.Therefore,functional genome research has became an important direction of animal and plant genome research,especially for transcriptome research.This review summarized the background of transcriptome,the complexity of transcriptome research,the development history of DNA sequencing technology and the methods of transcriptome research.It briefly described the development of transcriptome and gave a simple and systematic introduction to the content of transcriptome research,so as to better understand transcriptome sequencing.It laid a foundation for a deeper understanding of transcriptome sequencing.

This study was conducted to screen the different concentrations and culture time of tunicamycin (TM) in porcine immortalized hepatic stellate cell,in order to model endoplasmic reticulum stress (ERS) successfully and explore the changes of ubiquitination under ERS.Porcine hepatic stellate cell was treated with TM with concentrations of 0,1,2,5,10 and 15 μg/mL,respectively,and cultured for 2,4,8,16,24 and 36 h.TM concentration and culture time were initially screened by detection of cell inhibition rate,and finally selected by detection of cell cycle and ERS marker genes and proteins expression,in order to model ERS successfully.In addition,the expression of ubiquitin related genes in porcine liver stellate cells under ERS were detected.The results showed that 5 μg/mL TM culture for 8 or 24 h might model ERS via detection of cell inhibition rate.ERS marker gene expressions were all extremely significantly upregulated by 5 μg /mL TM,cultured for 8 or 24 h (P < 0.01).In terms of ERS marker protein expression,only ATF4 increased significantly (P < 0.05),and only G2/M phase of cell proportion in cell cycle significantly decreased at 8 h of culture (P < 0.05).At 24 h of culture,ERS marker proteins were significantly increased (P < 0.05),and cell cycle arrest occurred in G1 phase,leading to extremely significant declines in cell proportion in S phase and G2/M phase (P < 0.01).The result indicated that ERS model was successfully constructed treated by 5 μg/mL TM for 24 h.Ubiquitin related genes UBA2 and UBE2E expressions were extremely significantly increased (P < 0.01).In conclusion,the ERS model was successfully established and ubiquitylation mechanism was initiated by cultured with 5 μg/mL TM for 24 h in porcine hepatic stellate cell.

This study was aimed to construct a eukaryotic expression vector for porcine Orexin 2 receptor (pOX2R) and explore its pharmacological function when treated with traditional Chinese medicine.The pcDNA3.1(+)-myc/pOX2R and PGL4.29[luc2p/CRE/Hygro] were transiently transfected into HEK293T cells.The intracellular cAMP level of the positive cell lines under the action of Orexin A and Orexin B and antagonist TCS was measured by luciferase reporter gene assay.6 positive cells and 5 negative cells were obtained by stably transfecting target plasmid and reporter plasmid into CHO-K1 cells,G418 and hygromycin B were used for resistance screening.36 kinds of extracts from 18 kinds of traditional Chinese medicine were screened,HEK293T cells transfected with pcDNA3.1(+)-myc/pOX2R and PGL4.29[luc2p/CRE/Hygro] were used to analyze the dose effect on the positive extracts.The results showed that Orexin A and Orexin B could increase the activity of firefly luciferase,and TCS could antagonize the activation of receptor.A stable and reliable agonist screening model was constructed based on the expression level of firefly luciferase by optimizing drug stimulation time and DMSO dosage.Licorice extracted by water,lotus seed and Chinese yam extracted by alcohol could increase the expression of firefly luciferase.The extract could stimulate the expression of intracellular luciferase in a certain concentration range,which was dose-effect relationship.These results would provide reference for screening compounds and related drugs that could improve pig production efficiency.

The purpose of this experiment was to study the effect of different calcium(Ca) levels on energy metabolism of Yunnan semi-fine wool sheep during lactation.50 Yunnan semi-fine wool sheep were randomly divided into 5 groups according to their weights 15 days before lambing.Each group was fed with 10 ewes.The calcium levels of each group were 0.35%,0.42%,0.61%,0.75% and 0.96% respectively.The normal trial period was 90 days.The results showed that:① DE and ME:0.42%,0.75% and 0.96% Ca level groups were significantly higher than 0.35% and 0.61% Ca level groups (P < 0.05;P < 0.01).② Net energy of lactation(NEL):There were no significant difference in different Ca levels of diets in the early lactation period (P > 0.05);In the middle period,0.42% and 0.96% Ca level groups were significantly higher than 0.35% Ca level group (P < 0.05).In the later period,0.35% Ca level group was the lowest,and there were no significant difference among the other four Ca level groups (P > 0.05).③ DE/GE:0.35% and 0.96% Ca levels in the early lactation group were significantly higher than 0.42% Ca levels (P < 0.05).0.35% Ca levels in the middle lactation group were significantly higher than 0.42% and 0.75% Ca levels (P < 0.05).There was no significant difference between different Ca levels in the later lactation group (P > 0.05).④ ME/GE:There was no significant difference in different Ca levels in the early and late lactation (P > 0.05).In the middle stage,0.35% Ca level group was significantly higher than 0.42%,0.61% and 0.75% Ca level groups (P < 0.05).⑤ ME/DE:0.75% Ca level group in the early lactation period was significantly higher than 0.61% and 0.96% Ca level groups (P < 0.05).0.35% Ca level group in the middle period was significantly higher than 0.96% Ca level group (P < 0.05).There were no significant difference under different Ca level diets in the later period (P > 0.05).In conclusion,no matter in the early stage,middle stage or later stage of lactation,the intake of digestive energy and metabolizable energy of Yunnan semi-fine wool sheep were the best in the 0.42% Ca level group,the highest in the 0.42% Ca level group in the middle stage of lactation,the highest in the 0.35% Ca level group in the early and middle stage of lactation,and the highest in the 0.35% Ca level group in the middle stage of lactation.Therefore,when the dietary Ca level was between 0.35% and 0.42%,it was beneficial to the effective energy intake and energy utilization efficiency of Yunnan semi-fine wool ewes during lactation.

This experiment mainly studied the growth curve,artificial gastroenteric fluid,pH and pig bile salt tolerance of Bacillus amyloliquefaciens DHN04,and its bacteriostatic characteristics and antibiotic sensitivity.The growth curve of the bacteria was measured by growth rate method,the tolerance,acid resistance and bile salt resistance of artificial gastrointestinal fluid were measured by viable count method,the antibacterial activity was measured by Oxford cup method,and the antibiotic sensitivity was measured by drug sensitive test piece.The results showed that Bacillus amyloliquefaciens DHN04 entered the logarithmic growth phase after a slow growth period of 4-6 h,and the stable growth period of the bacteria was 12-24 h.After 3 h in artificial gastric juice and artificial intestinal fluid,the survival rates were 55.55% and 53.33%,respectively.The survival rate of the bacteria was 14.14% at pH 2.0.With the decrease of acidity,the survival rate increased gradually,and the survival rate reached 93.85% at pH 7.0.With the increase of bile salt concentration,the survival rate of Bacillus amyloliquefaciens decreased gradually.The same concentration of bile salt,the survival rate of 24 h was lower than that of 3 h.Bacillus amyloliquefaciens DHN04 had a certain inhibitory effect on Escherichia coli,Salmonella,Staphylococcus aureus and Tetracocci.It was resistant to amoxicillin.In summary,the characteristics of the Bacillus amyloliquefaciens DHN04 were found to be well tolerated by artificial gastrointestinal,bile salts and pH,and had certain inhibitory effects on Escherichia coli,Salmonella and Staphylococcus aureus,and could be activated quickly,which could be used as a potential probiotic strain for livestock and poultry production.

In the past few decades,due to the abuse of feed antibiotics in animal husbandry,the normal intestinal flora of livestock and poultry has been destroyed and caused a series of problems such as intestinal bacterial resistance and environmental pollution,seriously damaged livestock and poultry intestinal health.At the same time,intensive farming has become the development direction of animal husbandry in recent years,and the scale of breeding is getting bigger and bigger.How to effectively improve the immunity of livestock and poultry has become the focus of research.A healthy intestinal tract is the basis for ensuring the healthy growth of livestock and poultry.Maintaining a healthy intestinal flora can effectively improve the immunity and disease resistance of the animal body.Mannan oligosaccharides is a new green feed additive.As a kind of functional oligosaccharide,it has the functions of improving intestinal health and improving immune response of livestock and poultry.In pig production,the addition of mannan oligosaccharides can improve the growth performance and immunity of weaned piglets.In poultry production,the addition of mannan oligosaccharides can improve the intestinal flora of poultry and improve production performance and meat quality.The article mainly introduces the structure and physicochemical properties of mannan oligosaccharides,improves intestinal health and immune regulation mechanism and its application in weaned piglets,finishing pigs,laying hens and broiler chickens.The paper summarizes the regulation mechanism and immune pathway of mannan oligosaccharides on intestinal health and immunity of livestock and poultry,and aims to provide theoretical basis for the in-depth study of manno oligosaccharides in intestinal health and immune regulation and its application in livestock and poultry breeding.

For effectively utilizing ginseng processing by-products resources and clarifying the effects of which on chicken carcass qualities and metabolism,864 healthy newborn Jilin Luhua chicken,male and weighted at (40.88±2.43) g were chosen and randomly divided into 9 treatments,8 repetitions in each treatment and 12 chicken in each repetition.These treatments were control,ginseng stem and leaf (GSL) treatments (0.6%,1.2%,1.8% and 2.4%) and ginseng residual parts (rhizome and root hair,GRP) treatments (0.2%,0.4%,0.6% and 0.8%).The experiment lasted 150 d and the effects of ginseng processing by-products on slaughter performance,carcass quality and blood biochemistry indices were comprehensively evaluated.The results showed that:①There was no significant difference in slaughter performance among all the treatments.②Water content of leg muscle in 1.8% GSL group was significantly higher than control and 0.6% GSL groups (P<0.05),fat contents of leg muscle in 0.6% and 1.8% GSL groups were significantly lower than control group (P < 0.05);Water content of chest muscle in 0.8% GRP group was significantly higher than 0.4% GRP group (P < 0.05),and 0.4% and 0.8% GRP increased protein contents of chest muscle compared with control and 0.6% GRP groups (P < 0.05);Fat contents of chest and leg muscle in 0.4% GRP group were higher than 0.8% GRP group (P < 0.05).③In 2.4% GSL treatment,the plasma LDL-C level was significantly higher than 0.6% GSL group(P < 0.05),creatinine (CRE) was significantly higher than other GSL treatments (P < 0.05),and blood urea nitrogen (BUN) was higher than 1.2% and 1.8% GSL treatments (P < 0.05).In 0.8% GRP group,plasma total protein (TP) was significantly higher than control and 0.6% GRP groups (P<0.05),and globulin (GLB) level was also higher than 0.6% GRP group (P < 0.05).Synthesizing the above results,the suitable additive ranges of GSL and GRP in Jilin Luhua chicken feed were 0.6% to 1.8% and 0.4% to 0.8%,respectively.

This study was conducted to investigate the changes of nutritional composition,the cottonseed peptides contents and the molecular weight distribution of cottonseed peptides of cottonseed meal fermented by Bacillus subtilis-1,Saccharomyces cerevisiae and their complex bacteria.The study consisted of four groups:Control group (unfermented cottonseed meal),group Ⅰ (cottonseed meal fermented by Bacillus subtilis-1),group Ⅱ (cottonseed meal fermented by Saccharomyces cerevisiae),group Ⅲ (cottonseed meal fermented by Bacillus subtilis-1 and Saccharomyces cerevisiae).The dry matter (DM),crude protein (CP),crude ash (Ash),neutral detergent fiber (NDF),acid detergent fiber (ADF),ether extract (EE),Ca and P,as well as cottonseed peptides contents and molecular weight distribution of cottonseed peptides were measured.The results showed that:①The contents of DM,CP and Ash of group Ⅰ were extremely significantly higher than other three groups (P < 0.01).Compared with other groups,the EE content of control group were extremely significantly increased (P < 0.01).The ADF content of group Ⅲ was the lowest among all the groups (P < 0.01).The contents of Ca and P were increased in three fermented groups,and group Ⅲ had the highest contents of Ca and P (P < 0.01).② There were no significant difference in free amino acids and total amino acid among all the groups (P > 0.05).Compared with control group,the total free amino acid in groupsⅠ,Ⅱ and Ⅲ were increased by 134.02%,75.59% and 75.44%,respectively.③ Compared with control group,the content of acid soluble protein and cottonseed peptide in three fermented groups were increased extremely significantly (P < 0.01),and in which the group Ⅰ was the highest.In addition,the peak area percentages below 1 000 u in fermented cottonseed meal groups were 89.41%,77.61% and 84.58%,respectively.In conclusion,fermented by Bacillus subtilis,Saccharomyces cerevisiae and their compound could improve the nutrition value of cottonseed meal.The results could provide a theoretical basis to the development and utilization for cottonseed meal.

To evaluate the possibility of substituting in-feed antibiotics with different combinations of substitute,240 crossbred piglets (Duroc×Landrace×Yorkshire) weaned at (28±3) days of age were selected and randomly assigned into 5 groups,the control group (basal diet),antibiotic group (basal diet＋75 mg/kg CTC+40 mg/kg bacitracin zinc+100 mg/kg olaquindox),treatment group 1 (basal diet＋2 000 mg/kg fermented Chinese herb),treatment group 2 (basal diet＋500 mg/kg clostridium butyricum+1 000 mg/kg tributyrin) and treatment group 3 (basal diet＋450 mg/kg Macleaya cordata extract+800 mg/kg betaine anhydrous).The trial had 5 replicates per group and 12 piglets per replicate,and lasted for 32 d.At the 14th and 32nd days,one pig was randomly selected from each replication,and the plasma samples were collected from the anterior vena cava.The growth performance,diarrhea rate,and plasma antioxidant indexes were measured.The results showed that:① From 1 to 14 d,compared with the control group,ADFI and ADG of weaned piglets in treatment group 3 were significantly increased (P < 0.05),and F/G was significantly decreased (P < 0.05).From 15 to 32 d,compared with the control and antibiotic groups,treatment group 3 could extremely significantly decreased F/G of weaned piglets (P < 0.01).From 1 to 32 d,compared with the antibiotic group,F/G in treatment group 3 was significantly decreased (P < 0.05).② From 1 to 14 d,treatment group 3 could extremely significantly decreased diarrhea index compared with the control group (P < 0.01).From 15 to 32 d and 1 to 32 d,diarrhea indexes of weaned piglets in treatment group 2 and 3 were extremely significantly lower than the control group (P < 0.01).The diarrhea index had no significant difference between the antibiotic group and treatment group 3 (P > 0.05),but they both decreased in some degree compared with the control group (P > 0.05).③ Compared with the control and antibiotic groups,in treatment group 3,at the 14th day,T-SOD and GSH increased significantly or extremely significantly (P < 0.05;P < 0.01),while MDA and H₂O₂ decreased significantly or extremely significantly (P < 0.05;P < 0.01).At the 32th day,GSH increased extremely significantly (P < 0.01),while MDA decreased significantly or extremely significantly (P < 0.05;P < 0.01).The results suggested that supplementation of Macleaya cordata extract (450 mg/kg) and betaine anhydrous (800 mg/kg) could improve the antioxidant capacity,decreased F/G and diarrhea rate.Its effect was superior to other combinations,and it could be used as the substitute for antibiotics in feed.

This study was conducted to elucidate the genetic diversity of mitochondrial DNA (mtDNA) D-loop region in Qingyuan partridge chicken group 1,Qingyuan partridge chicken group 2,Yangshan chicken and Qingyuan Yellow feather black-bone chicken.The specific primers were designed according to mtDNA D-loop region of Gullus gullus spadiceus (accession No.:NC_007235.1) in GenBank.The sequence was analyzed after PCR amplification and sequencing,and the haplotype number,polymorphism number,haplotype diversity,nucleotide diversity and nucleotide mean difference were counted.The evolution divergence among breeds was calculated by Mega 5.10 software,and the phylogenetic tree was constructed.The results showed that the length of mtDNA D-loop region in four high quality chicken breeds was 591 bp,and 549 bp were used for subsequent analysis.The content of A,T,C and G were 27.2% to 27.3%,30.1% to 30.4%,29.5% to 29.8% and 12.8% to 12.9%,respectively,and the average content of G+C was 42.5%.There were 92 polymorphic sites which contained 14 singleton variable sites and 78 parsimony informative sites,and the percentage of transitions and transversions were 89.13% (82/92) and 10.87% (10/92),respectively.The haplotype diversity ranged from 0.682 to 0.835,and the nucleotide diversity ranged from 0.00849 to 0.01167.There were 32 haplotypes in all sequences,which could be divided into clades A,B,C and E,however,most of the individuals belonged to clades B (51.2%) and E (37.6%).The phylogenetic tree results showed that four high quality chicken breeds could be classified as 4 branches which were consistent with the haplotypes classification results.The results indicated that the four high quality chicken populations from Qingyuan had relatively high haplotype and nucleotide diversity and likely shared two or more common maternal lineages.

To explore the effect of myostatin (MSTN) on the proliferation and myogenic differentiation of bovine skeletal muscle satellite cells,the myogenic differentiation model induced by bovine skeletal muscle satellite cells in vitro was used in this study,three interfering RNAs (si-MSTN-1,si-MSTN-2 and si-MSTN-3) were designed and synthesized in the previous stage,si-MSTN-2(si-MSTN) with extremely significant interference effect was transfected into bovine skeletal muscle satellite cells to interfere with MSTN expression.The effect of interfering with MSTN on the proliferation of bovine skeletal muscle satellite cells was detected by EdU staining assay.In vitro myogenic differentiation of bovine skeletal muscle satellite cells transfected with si-MSTN was conducted,the effect of interfering with MSTN on myogenic differentiation of bovine skeletal muscle satellite cells was analyzed by myotube formation and the expression of differentiation markers.The myotube formation during the differentiation of bovine skeletal muscle satellite cells was observed by microscopy.The expression of skeletal muscle satellite cells differentiation markers MyoG and MyHC were detected using Real-time PCR and Western blotting at mRNA and protein levels.The results showed that EdU positive cell ratio in bovine skeletal muscle satellite cells extremely significantly increased after interfering with MSTN (P < 0.01).The number and diameter of myotubes formed in bovine skeletal muscle satellite cells after MSTN interference were extremely significantly higher than that of control group.At mRNA and protein levels,the expression of MyHC,a marker of myogenic differentiation,was extremely significantly increased after MSTN interference (P < 0.01).The results indicated that MSTN interference could significantly promote the proliferation and the process of myogenic differentiation of bovine skeletal muscle satellite cells promote.The results laid a foundation for further studies on the mechanism of MSTN on the myogenic differentiation of bovine skeletal muscle satellite cells.

The aim of this study was to elucidate the association between tissue expression level and polymorphism of ATF3 gene and meat quality traits in Yanbian Yellow cattle.Using 70 healthy 30-month-old Yanbian Yellow cattle as the research object,Real-time PCR was used to compare the expression of ATF3 gene in 10 tissues including heart,liver,spleen,lung,kidney,hind leg muscle,eye muscle,sirloin,upper brain and subcutaneous fat tissue.The exon 4 of ATF3 gene was amplified,SNP was screened and verified by Sanger direct sequencing and PCR-RFLP methods,and the association with meat quality traits was analyzed.The results showed that the expression of ATF3 gene was detected in 10 tissues of Yanbian Yellow cattle,and the expression of ATF3 gene in subcutaneous adipose was extremely significantly higher than other tissues (P < 0.01).The relative expression of ATF3 gene in sirloin was extremely significantly positively correlated with IMF content (P < 0.01),and the coefficient of correlation was 0.996;There was a significant positive correlation between ATF3 gene expression and IMF content in eye muscle (P < 0.05),and the coefficient of correlation was 0.999.There were no significant correlation between the expression of ATF3 gene and IMF content in the upper brain and hind leg muscle (P > 0.05),and the correlation coefficients were 0.757 and 0.658,respectively.There was a G→C mutation at 1 428 bp of ATF3 gene,contained three genotypes:GG,GC and CC.The fat content and back fat thickness of GC and CC genotypes were significantly higher than GG genotype (P < 0.05),the marbling grade of GC and CC genotypes were extremely significantly better than GG genotype (P<0.01).The results demonstrated that the mutation site could be used as a potential molecular marker for meat quality traits,and ATF3 gene might be a candidate gene for fat deposition in Yanbian Yellow cattle.

The aim of this study was to explore the effects of WNT4 and HOXC13 genes polymorphisms on the fiber diameter trait of Tibetan cashmere goats,in order to find out molecular markers related to fiber diameter trait.380 one year old Tibetan cashmere goats were selected as experimental animals.The single nucleotide polymorphism (SNP) of WNT4 and HOXC13 genes were detected by pooled DNA sequencing method,and genotyped by Sequenom MassARRAY genotype technology.The SNPs loci associations with cashmere mean fiber diameter (MFD),fiber diameter standard deviation (FDSD),coefficient of variation of fiber diameter (CVFD) traits were analyzed using the least squares method in the GLM procedure of SAS 9.1 software.The results showed that two SNPs (SNP1 and SNP2) were identified in exon 3 of WNT4 gene,and two SNPs (SNP3 and SNP4) were identified in exon 2 of HOXC13 gene,respectively,the genetic variation was moderate polymorphism (0.25 < PIC < 0.50).The χ² test indicated that the genotypic distributions of SNP1 and SNP2 in WNT4 gene were deviated from Hardy-Weinberg equilibrium (P < 0.05).Association analysis results showed that 4 SNPs was extremely significantly associated with MFD (P < 0.01),SNP2 and SNP3 were extremely significantly associated with FDSD (P < 0.01),SNP2 was extremely significantly associated with CVFD (P < 0.01).In conclusion, WNT4 and HOXC13 genes had significant effects on the fiber diameter in Tibet cashmere goats,so it could be tried to use the SNPs as one of the molecular markers that affected the fiber diameter of Tibet cashmere goats,providing theoretical basis for the breeding of super fine Tibetan cashmere goats.

The objective of this study was to study the tissue expression distribution of miR-206 and its expression pattern in skeletal muscle growth,and predict its possible mechanism of action.Eight tissues of heart,liver,spleen,lung,kidney,breast muscle,leg muscle and abdominal fat were collected from 4-week-old Jinmao Black chickens.Breast muscle and leg muscle were collected from 2,6,10,14 and 16-week-old Jinmao Black chickens.The expression of miR-206 in eight tissues and skeletal muscle at different growth stages using Real-time PCR.The target genes of miR-206 were predicted and conducted to function annotation by bioinformatics methods.The results showed that the expression level of miR-206 in breast muscle and leg muscle was extremely significantly higher than that in other tissues (P < 0.01).miR-206 was lowly expressed in abdominal fat,heart,liver,spleen,lung and kidney,and specifically expressed in skeletal muscle.The expression pattern showed that the expression of miR-206 was the highest in leg muscle and breast muscle at 2-weeks-old,and showed a downward trend in general.Target gene prediction and functional analysis revealed that miR-206 had 356 target genes,21 of which were involved in myogenesis.Multiple target genes were known to be involved in the regulation of myogenesis,including paired box 7 (Pax7),insulin-like growth factor 1 (IGF1),brain-derived neurotrophic factor 1 (BDNF1),retinoic acid receptor beta (RARB) and frizzled class receptor 7 (FZD7) genes.Pathway analysis showed that four pathways involved in myogenesis were significantly enriched,including MAPK signaling pathway,Wnt signaling pathway,regualtion of actin cytoskeleton and focal adhesion pathway.Combined with the expression results,we speculated that miR-206 might participate in the regulation of chicken muscle myogenesis by targeting Pax7、IGF1、BDNF1、RARB, FZD7 genes and MAPK,Wnt,regulation of actin cytoskeleton and focal adhesion pathways.This study revealed the tissue distribution and expression pattern of miR-206 in the growth of chicken and predicted its possible function and mechanism in the regulation of skeletal muscle myogenesis,which was the important foundation and basis for further study of the mechanism of action and expression regulation of miR-206.

This study was aimed to investigate the association between ovocalyxin-32 (OCX-32) and ovalbumin (OVA) genes polymorphism and egg quality traits,and find the molecular genetic markers associated with egg quality traits in laying hens.PCR-SSCP method was used to detect the polymorphism of OCX-32 and OVA genes in the parents’generation of Hyline Brown laying hens.The correlation between different genotypes and the egg quality traits was analyzed by SPSS 22.0 software,and further verified in the offspring to find the dominant genotype.The results showed that three genotypes (AA,BB and CC),two mutation sites located at 7 018 and 7 116 bp were detected in exon 6 of OCX-32 gene,and three genotypes (AA,BC and BB),two mutation sites located at 4 296 and 4 323 bp were detected in exon 5 of OVA gene.The results of association analysis showed that the egg yolk weight of OCX-32 gene AA genotype of parents was extremely significantly higher than BB and CC genotypes (P < 0.01).The Haugh unit and protein height of OVA gene AA genotype of parents were extremely significantly higher than BC genotype (P < 0.01),which was significantly higher than BB genotype (P < 0.05),the albumen weight of AA genotype was significantly higher than BC genotype (P < 0.05).Verification in the offspring confirmed that AA genotype of OCX-32 gene was the dominant genotype for regulating egg yolk weight,and AA genotype of OVA gene was the dominant genotype for regulating Haugh unit,protein height and protein weight.In summary,OCX-32 and OVA genes had certain effects on egg quality traits,which could be used as molecular breeding markers for laying hens.

The aim of this research was to investigate the effect of cytochalasin B (CB) on the developmental ability of parthenogenetic embryos and cloned embryos in pigs.The optimal concentration and incubation time of CB on early porcine embryo development were screened out by testing different concentrations and treatment time of CB in porcine in-vitroembryo culture medium.At the same time,Hoechst33342 staining was used to observe the difference in cell number of porcine blastocysts during incubation,to study the effect of CB on the development of parthenogenetic embryos and cloned embryos.The results showed that the cleavage rates of parthenogenetic embryos and cloned embryos incubated with 7.5 μg/mL CB in standard culture conditions were 85.00% and 90.23%,respectively,and the blastocyst rates were 35.68% and 42.58%,which were significantly higher than other groups (P < 0.05);While the cleavage and blastocyst rates of the parthenogenetic embryos for 4 h group were 83.80% and 35.39%,and the cloned embryos form cleavage and blastocyst for 6 h group were 83.98% and 55.62%,there were significant differences by corresponding to other groups (P < 0.05),respectively.In addition,embryos stained with Hoechst33342 showed that the average number of parthenogenetic embryos inner cells without CB were 28,and inner cells average numbers in the parthenogenetic and cloned embryos with CB were 36 and 52.There was a significant difference in the number of inner cells between the treated and untreated groups (P < 0.05).The results showed that the parthenogenetic embryos in vitro were treated for 4 h with 7.5 μg/mL CB,which resulted in higher cleavage and blastocyst rate;and the cloned embryos in vitro were treated for 6 h with 7.5 μg/mL obtained the highest of the cleavage and blastocyst rate.The results showed that CB treatment was beneficial embryo early development in vitro and improved the pregnancy rate of cloned embryos.

This study was aimed to investigate the effects of different data structures and animal models on the estimation of genetic parameters of economic traits of Alpine Merino sheep(14 months),and select the best animal model.The best model was used to estimate the genetic parameters of weight,greasy fleece weight,clean fleece yield,clean fleece weight,average fiber diameter,coefficient of variation of fiber diameter and staple length,which could provide the theoretical basis for the breeding of Alpine Merino sheep.The data of 20 720 sheep were divided into data set 1 and data set 2 according to the genealogical integrity and data amount using the correlation function of data sorting in R language.The significance of four non-genetic factors year of identification,birth type (single or twin),group and sex in two data sets were tested by ANOVA with R language.Extremely significant effect (P < 0.01) was put into the animal model as fixed effect.Four models were obtained by combining two data sets and two single-trait animal models.Model 1 and model 2 used data set 1 and data set 2,respectively.The random effects were individual additive genetic effect and residual effect.Model 3 and model 4 used data set 1 and data set 2,respectively,and the random effects were individual additive genetic effect,individual permanent environmental effect and residual effect.Variance component estimation was implemented by ASReml4 software.The Akzo information criterion (AIC) and Bayesian information criterion (BIC) were used to evaluate each model,and Likelihood ratio test (LRT) was used to compare each model.Finally,the optimal model was selected to estimate the heritability.The results showed that,①Fixed effect significance test showed that all traits in data set 1 and data set 2 of identification year and gender were extremely significant (P < 0.01),birth type was extremely significant for weight and greasy fleece weight in data set 1 (P < 0.01),and gender was only extremely significant for weight (P < 0.01).②The direct heritabilities were 0.1614-0.2392,0.1958-0.3254,0.4395-0.5539,0.2003-0.2393,0.4024-0.5897,0.3174-0.6077,0.2960-0.3669 for weight(WT),greasy fleece weight(GFW),clean fleece yield(CFY),clean fleece weight(CFW),average fiber diameter(FD),coefficient of variation of fiber diameter(CVAFD) and staple length(SL),respectively.③Likelihood ratio test showed that there was no significant difference between model 1 and model 3 for all traits (P > 0.05).Model 1 and model 4 showed significant differences in body weight and staple length (P < 0.01).Model 2 and model 3 showed significant difference in clean fleece weight (P < 0.01),but no significant difference in other traits (P > 0.05).There was no significant difference between model 2 and model 4 for all traits (P > 0.05).In conclusion,the optimal model of clean fleece weight was model 1,and the optimal model of WT,GFW,CFY,CFW,FD,CVAFD and SL was model 2.All traits were not significantly affected by individual permanent environment (P > 0.05).Based on the optimal model,WT,GFW,CFY,CFW,FD,CVAFD and SL heritability of alpine merino sheep were estimated to be 0.2392,0.3254,0.4394,0.2893,0.4222,0.3175 and 0.3670,respectively.

It is great reference value to analyze traceability the genetic components of different brands of pork for the protection of commercial brands,traceability and the improvement and development of local pig breeds in China.Four kinds of pig muscle tissues from different sources were selected from the market and the whole genome was resequenced..Combined with the published data of 59 pigs,the ancestral components were analyzed to speculate the possible sources of their ancestors and the contents of their ancestral components.In addition,mitochondrial genomic data were used to determine the maternal origin of four pigs and reveal the possible maternal origin.The 59 reference individuals included 12 breeds,Duroc,Landrace,Largewhite,Pietrain,Erhualian,Jinhua,Jiangquhai,Meishan,Bamaxiang,Luchaun,Wuzhishan and Xiang pigs respectively.The 12 breeds were divided into commercial pig,Eastern pig and Southern pig.In the study,four individuals were evaluated for their ancestral components using the software of ADMIXTURE and RFMix.The results showed that one of the four tested individuals contained 54.8% of commercial pig ingredients and 45.2% of Southern China pig ingredients,the other contained 81.3% of commercial pig ingredients,while the remaining two contained more than 97.0% of commercial pig ingredients.When the ancestral group was set as 12 groups,it was found that one of them contained 64.8% Duroc and 35.2% Luchuan pig;the other contained more complex ancestral components,including 22.5% Landrace,27.6% Large White,8.6% Wuzhishan,5.5% Erhualian,Meishan,Jiangquhai and Bama Xiang;the remaining were about 1.2%.The ancestors of the remaining two pigs were mainly Duroc,Large White and Pietran.In addition,the progeny judgment of mitochondria suggested that the maternal origin of one pig might be from Luchuan or similar local breeds.Through the commonly used method of ancestral analysis,the ancestral components of four pigs were estimated,and find that the genetic components of four pigs were different,and the ancestral components and proportion of different individuals were found.The results showed that the genetic component sources of each test individual could be estimated by the method of ancestral analysis.Using ancestral components as genetic characteristics could provided important genetic reference for brand pork identification and brand protection,as well as guidance and reference for the protection and development of local pig breeds in China.

Intramuscular fat is one of the key factors affecting meat quality,such as tenderness,water holding capacity,flavor and juiciness.The factors that influence intramuscular fat deposition in pigs are complicated,which including breed,age,gender,nutrition and gene.In the present article,relationships between the source of intramuscular fat,the fat content in pig muscle and the three meat characteristics of pork tenderness,pork hydraulics and pork flavor are introduced.It is confirmed that there is a significant correlation between intramuscular fat and meat quality.In Addition,factors affecting intramuscular fat deposition such as variety,weight,gender,dietary energy level,protein level,vitamin A and other related factors are also sketched.Several key genes,such as nutrition level,fatty acid synthase,heart fatty acid binding protein,adiponectin are analyzed.In view of these factors,the preliminary explanation of the effects on intramuscular fat deposition,the mechanism of fat metabolism are reported in the present study.The relationships between the influence factors and intramuscular fat are summarized.Based on the analysis results,it was found that breeding,candidate genes and nutritional regulation are the most effective ways to improve intramuscular fat content.The meat quality of the native pigs and foreign pigs hybridization or genetic modification of native pigs may be more promising research direction in the future.

This study was aimed to understand main viral pathogens infection situation and popular characteristics of piglet diarrhea,and provide scientific basis for effective prevention and control of piglet diarrhea in Guangxi.In this experiment,RT-PCR and PCR methods were used to detect the pathogens of PEDV,PoRV,TGEV,PDCoV,CSFV,PRRSV,PCV2 and PRV of 914 diarrhea samples of piglets from 366 large-scale pig farms in 14 cities in Guangxi from 2016 to 2019,and analyzed these viruses pathogens mixed infection situation and the differences of the positive rates in different years,seasons and regions.According to the survey results,PEDV,PDCoV,PRRSV,PoRV,PCV2,CSFV and PRV all had different degrees of infection,and the average positive rate of the samples were 59.74%,8.32%,7.77%,4.92%,3.72%,3.28% and 2.08%,respectively,and no TGEV was detected.The PEDV had no significant seasonal variations,it was high all the year round.The PEDV still existed in infected piglet diarrhea,even in hot summer.However,the PDCoV had significant seasonal variations,it occurred frequently in both spring and winter.The positive rate of PEDV was high in Guangxi,the single infection rate was up to 50.55%.There were also many cases of diarrhea mixed infection in piglets,among which double infection was more common,and there were also four kinds of infection.The results showed that there were 7 kinds of viral pathogens infecting piglets in different degrees in Guangxi,among which the viral diarrhea caused by PEDV was particularly serious,and the positive rate of new PDCoV was second only to that of PEDV,so it was necessary to pay attention to and strengthen the prevention and control of new PDCoV.

To investigate the cause of death of diseased chickens in large-scale chicken farm in Fujian province,the diseased chickens with a large number of nodular scab in the crown and head skin were identified by PCR.After being identified as FWPV strain,the virus was isolated by inoculating chicken chorioallantoic membrane(CAM).The virus was subcultured with primary chicken embryo fibroblasts (CEF) and passage cells DF-1 cells,and the similarities and differences of the culture characteristics of the isolated strain on the two kinds of cells were observed.The distribution of the virus and the morphology of the virus particles were observed by ultra-thin sections of the CEF infected with the isolated strain.The homologies of TK and FPV175 genes were analyzed.The results showed that the CAM inoculated with antibiotics showed a large area of single white protuberant acne spots,and the CEF and DF-1 cells were inoculated at the same time.The two kinds of cells could produce stable and sustainable passage cytopathic effect,but the time and degree of the lesion were different.Under the transmission electron microscope,the typical fowlpox virus particles were densely distributed in the cytoplasm of the infected CEF,and the sunken cores wrapped by the oval outer membrane were clearly seen.According to the statistics of 23 virion particles,the size of virion particles was (258 to 344) nm×(153 to 238) nm.PCR detection of FWPV TK gene and FPV175 gene showed that those nucleotide sequences of FWPV (accession No.:NC_002188.1) collected by GenBank were 100% and 99.8% homology,respectively.The above results indicated that the isolated strain was FWPV and was named as FWPV-FJ01,which provided a reference for the prevention and control of domestic FWPV.

In order to study the helminth infection intensity of large-scale pig farms in Jiangsu province,937 fecal samples were collected randomly from 12 large-scale pig farms in Xuzhou,Taizhou,Suqian,Yancheng,Yangzhou and Nantong.The samples were examined by centrifugalization,saturated sodium chloride floatation method and saturated magnesium sulfate floatation method.The results showed that parasite infection was found in all 12 farms.The detection rate of worm eggs in fecal samples was 7.6%.Two farms in Xuzhou had the highest infection rate (20.7%),while two farms in Taizhou had the lowest infection rate (1.7%).The infection rates of pig farms in Suqian,Yancheng,Yangzhou and Nantong were 6.5%,8.2%,5.4% and 5.4%,respectively.The infection species of helminth which were identificated successfully included Ascaris suum,Trichuris trichinella,Metastrongylus apri,Oesophagostomum and Strongyloides stercoralis.The infection rates of Ascaris lumbricoides and Oesophagostomum were 5.2% and 3.6%,respectively.At the same time,the mixed infection of Ascaris lumbricoides and Trichuris trichinella,Ascaris lumbricoides and Oesophagostomum,Ascaris lumbricoides and Metastrongylus apri.were found in pig farms of Xuzhou,Suqian,Yancheng,Yangzhou and Nantong.The infection rates of intestinal parasites in pregnant sows and lactating sows were significantly higher than the rates of other production stages.The infection rate of piglets was the lowest.The eggs of Trematoda,Cestoidea and Archiacanthocephala were not detected.The results showed that nematode infections were common in large-scale pig farms in Jiangsu province.Ascaris lumbricoides,Trichuris trichinella and Oesophagostomum were the most highly infected species.Regular insecticidal treatment was helpful for the prevention and control of nematode diseases in large-scale pig farms.This study provided a scientific basis for the prevention and control of parasitic diseases in large-scale pig farms at present.

In order to develop a safer and more effective tuberculosis vaccine for the prevention and elimination of tuberculosis,this study constructed four recombinant BCG vaccines expressing Ag85A,Ag85B,Ag85C and Ag85D genes,respectively.According to the pMV261 vector sequence and the H37Rv sequence registered on Tuberculist,primers were designed,and the recombinant pMV261 plasmid containing Ag85 gene fragment was constructed by PCR amplification and infusion cloning.The recombinant plasmid was identified by PCR,double enzyme digestion and transformed into BCG receptive cells by electroporation.Kan resistance was used to screen the recombinant BCG strain.The expression of Ag85A,Ag85B,Ag85C and Ag85D genes in BCG vaccine were identified by PCR and Western blotting,and then the successfully constructed recombinant BCG vaccines were cultured for follow-up experiment.The results of PCR amplification showed that the target gene fragment of recombinant BCG vaccines rBCG/Ag85A,rBCG/Ag85B,rBCG/Ag85C and rBCG/Ag85D were 1 447,1 402,1 453 and 1 330 bp,indicating that the target gene had been successfully integrated into BCG vaccine.Western blotting results showed that Ag85A,Ag85B,Ag85C and Ag85D genes could be successfully expressed in BCG cells.In this experiment,four recombinant BCG vaccines expressing Ag85A,Ag85B,Ag85C and Ag85D genes were successfully obtained by genetic engineering and named rBCG/Ag85A,rBCG/Ag85B,rBCG/Ag85C and rBCG/Ag85D,respectively.It could provide materials for further inserting other antigen genes into BCG cells to construct recombinant vaccine and vaccine research.

To explore the protective effect of Fagopyrum dibotrys on lipopolysaccharide (LPS)-induced inflammation model of the small intestine in mice,ICR mice were selected in the experiment.Different concentration of Fagopyrum dibotrys and the positive control- Rutin were added in drinking water for 14 days treatment,then intraperitoneal injection of LPS (10 mg/kg) was used to construct the model of acute enteritis.Finally,the results were evaluated by behavior changes,routine blood tests,histological sections and gene expressions of inflammation-related genes.The results showed that the intraperitoneal injection of 10 mg/kg LPS could successfully induced acute enteritis in mice.The addition of Fagopyrum dibotrys could reduce the weight loss of LPS-induced acute enteritis in a dose-dependent manner.Blood routine test showed that intraperitoneal injection of LPS caused a significant decrease in white blood count (WBC),red blood count (RBC),hemoglobin (HGB) and platelet count (PLT) in mice,and the addition of Fagopyrum dibotrys or Rutin could alleviate the blood routine abnormality caused by LPS injection to a certain extent.The hematoxylin-eosin (HE) staining showed that after intraperitoneal injection of LPS,the focal inflammatory cells were found infiltrated into the lamina propria with a large number,as well as the glandular epithelium with a small number.Also,the disorganized intestinal cells,nuclear division and nuclear fragmentation were observed.Furthermore,severely mucosal hyperemia and hemorrhage were observed.To a certain extent,the infiltration of inflammatory cells and mucosa bleeding could be suppressed by adding Fagopyrum dibotrys or Rutin in mice.RT-PCR results showed that intraperitoneal injection of LPS could significantly promote the mRNA expression level of inflammatory factors (i.e.IL-6,TNF-α and IL-1β),and the expression of TNF-α and IL-1β decreased by adding Fagopyrum dibotrys in a dose-dependent manner.The above results showed that Fagopyrum dibotrys or Rutin could effectively alleviate the inflammation of mice small intestine induced by LPS,the 12% adding of Fagopyrum dibotrys could achieve the effect of 100 mg/kg Rutin in mice somehow.

To investigate the effect of n-butanol part of flavonoids from Polygonum hydropiper L.(FNB) against porcine reproductive and respiratory syndrome virus (PRRSV) in vitro,in this study,Marc-145 cells and PRRSV (TJM-F92) were used as the objectives,the toxicity of FNB on cells was detected by CCK-8 method.In addition,the inhibition rate of the FNB on PRRSV after the cells were treated by three methods (drug administration before PRRSV inoculation,drug administration post PRRSV inoculation,drug administration and PRRSV inoculation simultaneously).The results showed that the maximum safe concentration of FNB was 500 μg/mL.Therefore,FNB in the concentration range of 25-500 μg/mL was selected for subsequent experiments.Various concentrations of FNB could inhibit the proliferation of PRRSV on Marc-145 cells to varying degrees and exhibited a dose-dependent relation,which meant the higher the concentration of the drug,the better the antiviral effect.Among them,the last two treatment methods had significant anti-PRRSV effects,the cell viability was 21.55%-65.23% and 24.85%-73.60% respectively in the concentration range of 25-500 μg/mL.However,the infection of PRRSV after first administration couldn’t effectively reduce the infectivity of the PRRSV and the cell survival rate was only 7.00% at the highest dose of 500 μg/mL,so the antiviral effect was not obvious.Although the pretreatment of FNB on Marc-145 cells did not reduce the ability of PRRSV infection,the preventive effect of FNB on PRRSV cells was not satisfactory.However,the effect of FNB on the clinical treatment of PRRSV was considerable.FNB could inhibit the synthesis and release of PRRSV and directly kill of PRRSV,and then effectively inhibit the expression of PRRSV in Marc-145 cells.The results of this study not only provide a reference for the clinical treatment of PRRSV,but also offer theoretical basis for the further development and utilization of Polygonum hydropiper L..

The experiment was aimed to study the abundance and diversity of the intestinal microflora of broiler chickens by feeding water extract from walnut green husks,and to explore the effect of water extract from walnut green husks on the intestinal microflora community structure of broiler.A total of 240 local chickens of 1 day old were randomly divided into 2 groups,the control group and test group,with 4 replicates in each group and 30 chickens in one replicate.The experimental group began to feed the water extract of walnut green husks at 28 days of age.After feeding for 6 weeks,10 fecal samples were collected from each replicate,and the changes in number and structure of intestinal flora were compared by 16S rDNA V3-V4 region high-throughput sequencing.The high-throughput sequencing results showed that all samples contained a total of 17 phyla and 48 genera;The results of diversity index analysis showed that the intestinal flora richness on broiler chicken fed water extracts were increased.From the level of the phylum,the abundance of Firmcutes bacteria increased by 2.208% (P > 0.05),and the abundance of Proteobacteria decreased by 2.218% (P > 0.05).On the genus level,the abundance of Escherichia coli was reduced by 5.35% (P < 0.05).The abundance of the Enterococcus was increased by 9.63% (P < 0.05).The results showed that the abundance and diversity of intestinal flora of broilers increased significantly after feeding the water extract of walnut green husks (P < 0.05).In conclusion,the water extract of walnut green husks could effectively regulate the intestinal flora of broilers and had beneficial effect on intestinal health.

In order to identify the pathogens of clinically suspected duck Pasteurella multocida infection,in this experiment,bacteria isolated and cultured,morphological observation,biochemical identification,16S rRNA gene sequencing analysis,bacterial species-specific identification,capsule type identification and animal regression test were carried out,drug resistance was analyzed by drug sensitivity test and drug resistance gene detection.The results showed that the bacteria isolated from the liver tissue of the diseased duck showed smooth bulging and gray-white colonies on the blood agar medium,which was Gram-negative bacillus,and the Wright's staining showed two-level thick staining;Biochemical identification showed that the isolated strain could ferment glucose,sucrose and mannitol,and the test results of hydrogen sulfide,oxidase and hydrazine were positive.The phylogenetic tree analysis of the 16S rRNA gene sequence showed that the isolated strain was clustered with the genus Pasteurella,homology > 99%.The results of bacterial species specific identification were consistent with Pasteurella multocida.The results of capsule typing only amplified the target gene fragment of about 1 050 bp,which was consistent with capsule serotype A.Animal regression test showed that the isolated strain had strong pathogenicity.The drug sensitivity test showed that the isolated strain was resistant to 12 drugs such as carbenicillin,ampicillin,cotrimoxazole and tetracycline.PCR detection of drug resistance gene showed that the isolate carried Sul1,Sul3,tet(X) and Intl1 four drug resistance genes,consistent with the drug sensitivity phenotype.In this experiment,a duck capsular serotype A Pasteurella multocida was successfully isolated,which provided a reference for the prevention and treatment of duck Pasteurella multocida infection.

In order to establish an sterility test method for ceftiofur hydrochloride intramammary infusion,the sampling amount,diluent type,washing method and enzyme dosage were investigated in accordance with the relevant requirements of the method suitability test in appendix 1101 of Chinese Veterinary Pharmacopoeia 2015 edition.Samples were placed in a sterile funnel,isopropyl tetradecanoate was added to the funnel,the 0.1% sterile peptone solution containing 1% polysorbide 80 was added to the funnel,the water layer was shaken and placed quietly,the water was taken out and treated by membrane filtration and the pH 7.0 sterile sodium chloride-peptone buffer solution was used as the washing solution.Each filter membrane was washed four times with a washing capacity of 100 mL.Penicillinase solution of not less than 6 million units was added to each tube of culture medium.The results showed that the six positive bacteria test groups grew as well as the positive bacteria control group,the test group and the negative control group grew aseptically.These indicated that the test quantity of the test samples had eliminated its bacteriostasis under the test conditions.Ceftiofur hydrochloride had strong bacteriostasis activity.In this experiment,a reasonable method of sterility test was established by membrane filtration method,which could effectively remove the bacteriostasis ingredients in ceftiofur hydrochloride intramammary infusion and make the test results more accurate and reliable.It could be used as a routine aseptic test method for the preparation.

In order to determine the cause of death of Taiwan loach (Paramisgurnus dabryanus ssp.Taiwan) after the transfer of fishing,the pathological anatomy and pathogen isolation of Taiwan loach were carried out.Morphological observation,physiological and biochemical characteristics determination,16S rDNA gene sequencing and identification were carried out for the isolated bacteria,as well as artificial infection test and drug sensitivity test.The results showed that the dominant strains were isolated from the liver,spleen,kidney and intestine of loach and named as XJC after purification and preservation.The strain XJC was positive in glucose fermentation,D-glucose,ornithine,phenylalanine and urea utilization,and the other biochemical indexes were negative,which was consistent with Morganella morganii.The similarity of 16S rDNA gene sequence with Morganella morganii was 99.6%,which was identified as Morganella morganii.The results of infection test showed that XJC had pathogenicity to the loach,and the infected loach had typical pathological changes of intestinal congestion.The strain XJC was identified as the pathogen of loach.The results of drug sensitivity test showed that XJC was sensitive to aminoglycosides,quinolones and the third generation cephalosporins,and equally resistant to tetracycline,penicillin,erythromycin,polypeptide,furazolidone,lincomycin and the first and the second generation cephalosporins.Aminoglycosides and quinolones could be selected for the prevention and treatment of Morgan's disease.The results of this experiment could provide a reference for the diagnosis and control of Morganella morgensis in Taiwan loach.