The study was aimed to clone,bioinformatics of diacylglycerol acyltransferase 2 (DGAT2) gene of Guangling donkey,detected its expression in different tissues,and provided theoretical reference for exploring the mechanism of DGAT2 gene in fat deposition and improving milk fat rate of Guangling donkey.Homologous primers was designed by Primer Premier 3.0 according to the mRNA sequences of DGAT2 gene of Equus caballus (accession No.:XM_023645689.1),Camelus bactrianus (accession No.:XM_010973154.1), Ovis aries(accession No.:XM_027979550.1),and other species published in GenBank.DGAT2 gene sequence of Guangling donkey was amplified with RT-PCR.The encoding sequence of DGAT2 gene was analyzed by bioinformatics methods,and the expression of DGAT2 gene in heart,liver,spleen,lung,kidney,longissimus dorsi muscle,intermuscular fat and subcutaneous fat of Guangling donkey was detected by Real-time quantitative PCR.The results showed that the CDS of Guangling donkey DGAT2 gene consisted of nucleotides of 1 086 bp encoding 361 amino acids.It was submitted to NCBI and the login number was MT993643.The coding sequence of DGAT2 gene showed 99.0%,92.0%,93.5%,92.0%,92.7%,85.3% and 84.1% identity with that of Equus caballus,Bos Taurus,Camelus bactrianus,Sus scrofa,Ovis aries,Homo sapiens and Mus musculus.Phylogenetic tree analysis revealed that Guangling donkey was most closely related to Equus caballus and farthest related to Mus musculus.The DGAT2 protein,with molecular weight of 40.96 ku,fat coefficient of 92.85 and isoelectric point of 9.16,was a stable basic hydrophobic protein with transmembrane region.There were 28 phosphorylation modification sites and 2 glycosylation modification sites in DGAT2 protein,with no signal peptide,it was mainly located in the endoplasmic reticulum.The secondary structure of DGAT2 protein was mainly of 39.89% alpha helix and 36.01% random coil.The DGAT2 gene was expressed in 8 tissues,among which the expression level in subcutaneous fat was significantly higher than that in other tissues (P<0.05),followed by heart,liver and kidney,and the lowest level was in the longissimus dorsi muscle.The experiment results provided a solid theoretical basis for further exploring the role of DGAT2 gene in fat deposition and improving milk quality traits in Guangling donkey.

In order to obtain genome-wide epigenetic information,such as DNA methylation level and patterns of gonad axis tissues in Langshan chicken,the whole genome bisulfite sequencing (WGBS) was used to detect the DNA methylation status across the genome of hypothalamus and ovary tissues.The DNA methylation level and specific methylation pattern of each tissue were also analyzed.The results showed that the global methylation level in the genomes of hypothalamus and ovary were 4.35% and 3.48%,respectively,which were significantly different (P<0.05).6 150 000 and 10 320 000 methylated cytosine (mC) sites were detected in the hypothalamus and ovary,with mCG sites accounting for nearly 69.99% and 87.88%,respectively.And the proportion of non-mCG sites in the hypothalamus was about 2.5 times that in the ovary.Different from each chromosome,the mCHH sites accounted for the highest proportion in the mitochondrial genomes of the two tissues,followed by the mCHG sites.DNA methylation level in the promoter region of the ovarian genome was significantly lower than that in the intron and exon (P<0.01),and significantly higher than that in the intergenic region (P<0.01).The methylation level of promoter region in the genome of hypothalamus was not significantly different from those of intron and exon regions (P>0.05),but significantly higher than that in the intergenic region (P<0.05).Methylation levels of all functional elements in the genome of hypothalamus were significantly or extremely significantly higher than those of the ovary genome (P<0.05;P<0.01).Overall,different DNA methylation patterns and characteristics existed in the hypothalamus and ovary tissues of Langshan chicken.A higher proportion of non-mCG sites in hypothalamus might play an important role in the development of central nervous system.The results provided reference for further research on the regulatory mechanism of DNA methylation in chicken ovary and hypothalamus on their reproductive performance.

The aim of this study was to obtain Chinese Merino sheep full-length sequence of the coding region (CDS) of fibroblast growth factor 10 (FGF10) gene,to analyze its sequence characteristics by bioinformatics method,and to study the expression characteristics of FGF10 gene.It could lay a theoretical foundation for further study of the expression and regulation mechanism of FGF10 mRNA in Chinese Merino hair follicle.The full CDS of FGF10 gene was obtained by PCR amplification and cloned into zero PCR@^TM-Blunt for sequencing verification.Real-time quantitative PCR (qRT-PCR) was used to detect the expression pattern of FGF10 gene.The results showed that the sheep FGF10 CDS was 696 bp in length (Upload the sequence to GenBank and get the accession No.:MT872422),which coded 231 amino acids,the homology of amino acid sequence with cattle and goat was 100%,there was a signal peptide and a transmembrane domain,which was the signal protein of secretory pathway.qRT-PCR analysis showed that FGF10 gene was expressed during the development of Chinese Merino follicles,and the highest expression was found on the 85th day of hair follicle development,which was significantly higher than those of other hair follicle development stages.The complete CDS and expression characteristics of FGF10 gene were obtained.Bioinformatics analysis showed that the CDS of FGF10 gene was conserved among species,and the gene was expressed in the skin tissue of different development periods of hair follicle,which indicated that FGF10 gene might play an important role in the growth and development of sheep hair follicles physical function.

This study was aimed to produce soluble protein of Mink enteritis parvovirus (MEV) capsid protein VP2 and to obtain virus-like particles (VLPs) of MEV.The MEV VP2 gene was optimized,synthesized and cloned into prokaryotic expression vector pET-30a(+).The recombinant vector pET-30a-VP2 was expressed by co-transformation with pTf16 into ER2566,and was induced at different temperatures,L-arabinose concentrations and IPTG concentrations,and analyzed by SDS-PAGE and Western blotting.VP2 was purified by ammonium sulfate precipitation combined with sucrose density gradient centrifugation,then identified by SDS-PAGE.Under the different assembled conditions,the purified protein was self-assemble into VLPs with the removal of sucrose.VLPs were analyzed by dynamic light scattering (DLS) and transmission electron microscopy (TEM).Minks were immunized with homemade MEV VLPs vaccine to identify their immunogenicity.The results showed that recombinant VP2 protein was highly expressed in the supernatant of E.coli after induction with 2 g/L L-arabinose and 0.2 mmol/L IPTG at 25 ℃ for 16 h.SDS-PAGE showed that the fusion protein was about 65 ku.Western blotting confirmed that VP2 had good antigen specificity.VP2 was purified by ammonium sulfate precipitation combined with sucrose density gradient centrifugation and degree of purity could reach more than 90%.DLS and TEM results indicated that VLPs had similar size,shape and hemagglutination (1:2¹⁴) with the authentic virus capsid at 150 mmol/L NaCl,pH 8.0.Immunization with VLPs vaccine could induce high-titer hemagglutination inhibition antibody (the highest was 1:2¹¹) in minks after 21 days,indicating that the VLP vaccine had good immunogenicity and could effectively prevent MEV.VLPs with high immunogenicity could be obtained by expression of VP2 with pTf16 in prokaryotic expression system,which laid a foundation for further research on MEV VLPs vaccine.

The aim of this study was to understand the bioinformatics information of GalR protein,a transcription regulator of Streptococcus suis serotype 4 (SS4),and to express it in prokaryotic cells.The sequence homology and functional domain of GalR protein of SS4 SH1510 strain were searched by bioinformatics analysis website,and the signal peptide,transmembrane domain and isoelectric point were also predicted.At the same time,the GalR gene of SS4 SH1510 strain was amplified,prokaryotic expressed and purified.SDS-PAGE was used to analyze the solubility of the recombinant protein,Western blotting was used to identify the reactivity of the recombinant protein.The amino acid sequence alignment showed that it had 57%,56%,55%,55%,52%,52% and 49% homology with the GalR family proteins of Streptococcus mutans,Streptococcus agalactiae,Pseudomonas aeruginosa,Streptococcus equi subsp.zooepidemicus,Streptococcus pneumoniae, Streptococcus oralis and Listeria,respectively.Domain search showed that the GalR protein had a small DNA binding domain with a helix-turn-helix (HTH) motif at the N-terminus,and a ligand-regulated binding of type Ⅰ periplasmic binding protein folding at the C-terminus.In the middle of these two functional domains,there was a linker of about 18 amino acids.GalR protein had no signal peptide and transmembrane region,and its isoelectric point was 5.10.SDS-PAGE analysis showed that GalR protein was mostly expressed in the supernatant and the molecular weight was 56 ku.Western blotting identification showed that His monoclonal antibody and crude SS4 polyclonal rabbit serum could specifically recognize soluble GalR protein.A comprehensive bioinformatics analysis of the GalR gene was performed and the GalR protein was successfully expressed and purified in this study,which laid a foundation for further study of the role of GalR protein in SS4.

This study was aimed to construct a stable FXR gene knockout cell line and its effect on HeLa cell proliferation by using CRISPR/Cas9 gene editing technique.According to the design rules of CRISPR/Cas9 target,three pairs of upstream and downstream small guide RNA (sgRNA) specifically identifying the first exon related sequence of FXR gene were designed,and the recombinant eukaryotic expression plasmid was constructed using PX459 plasmid as the vector.After enzymatic digestion and sequencing identification,the recombinant plasmid was transfected into HeLa cells,puromycin was used for positive cell screening,then the expression level of FXR in the stable FXR knockout strain of HeLa cells was detected by Real-time PCR,and the FXR knockout effect of cells was identified by Western blotting.Finally,the effect of FXR gene knockout on HeLa cell proliferation was detected by crystal violet staining.The results showed that after sequencing,three pairs of sgRNA were inserted in the correct position and direction PX459 plasmid vector,and successfully constructed the recombinant expression vector PX459-sgRNA.The results of Real-time PCR and Western blotting showed that FXR protein was not expressed in the selected cells after transfection,indicating that a stable FXR knockout cell line was constructed.Crystal violet results found FXR knockout HeLa dyeing depth was significantly lower than the normal HeLa.The D_{595 nm} value of FXR knockout HeLa cell hole was extremely significantly lower than that of wild-type HeLa cell hole (P<0.01),indicating that the proliferation capacity of FXR knockout HeLa cell strain was extremely significantly lower than that of normal HeLa cells.Endogenous FXR gene knockout cell lines were successfully obtained by CRISPR/Cas9 technology,and FXR gene had inhibitory effect on the proliferation of cancer cells.This experiment provided an effective tool for studying the function and mechanism of FXR and laid a foundation for studying the occurrence and development of FXR in related cancers.

The aim of this study was to analyze the sequence characteristics of P48 gene of Mycoplasma bovis isolated strain in Xinjiang,and the structure and function of its encoded protein.Specific primers were designed based on the sequence of P48 gene of PG45 strain (GenBank accession No.:CP002188.1),the sequence of P48 gene was amplified by Overlap-PCR.P48 gene was inserted into pMD19-T vector for cloning and sequencing.Bioinformatics methods were used to predicted and analyzed its nucleotide sequences and amino acid sequences,including the basic physicochemical properties,signal peptide,transmembrane,phosphorylation site,glycosylation site,secondary structure,tertiary structure.The results showed that the sequence length of Mycoplasma bovis isolated strain P48 gene was 1 407 bp,and its homology with Mb PG45 international standard strain was 100%,and it was clustered in one strain.The P48 protein encoded a protein composed of 467 amino acids,no transmembrane structure,which was a stable soluble hydrophilic protein.The P48 protein had a signal peptide,44 potential phosphorylation sites and 3 glycosylation sites.The secondary structure was mixed,and the alpha helix was the most,accounting for 41.76%,followed by the random coil,accounting for 37.69%.Functional prediction showed that the P48 protein had high probability in signal transduction,stress response and receptor.In this study,the isolated strain P48 gene was cloned successfully,and the protein that it encoded was a stable soluble hydrophilic protein.The results provided a theoretical basis for further exploration of its function of P48 gene of Mycoplasma bovis isolated strain.

Molecular detection technology is an important way of pathogen detection,identification and disease diagnosis.Recombinase polymerase amplification (RPA) is a new type of nucleic acid amplification technology.It can amplify DNA or RNA at a constant temperature with a recombinase that is capable of pairing oligonucleotide primers, single-stranded DNA binding (SSB) protein and strand-displacing polymerase.It has advantages of convenient operation,strong specificity,high sensitivity and rapid reaction,which can realize the field diagnosis of diseases.In this paper,the composition of reaction system,reaction principle,the design of primers and probes,and the detection methods of amplified products of RPA technology were introduced.The application of RPA technology in the detection of important viral,bacterial and parasitic pathogens in animals was introduced,the limitations of RPA technology were analyzed,and its development prospect was prospected.

The effects of Dark Plum powder extraction on the contraction of small intestine in vitro were investigated and the possible mechanism was explored.The water extraction was prepared by decocting the following Chinese herbs including 30 g of Dark Plum,50 g of Calyx Kaki,12 g of Medicine Terminalia Fruit,12 g of Coptidis Rhizoma,and 12 g of Radix Curcumae.The technique of isothermal perfusion of small intestine in vitro was used.RM6240 multi-channel biological signal acquisition and processing system was used for information collection and processing.The effects of Dark Plum powder extraction on the spontaneous contraction of isolated gastrointestinal smooth muscles were observed.The effects of 2.18 g/L of Dark Plum powder extraction on the contraction induced by acetylcholine (Ach) were measured.The effects of 2.18 g/L of Dark Plum powder extraction on the Ach-induced both intracellular and extracellular calcium-depended contractions were studied.The amplitude and frequency of spontaneous contraction of isolated gastrointestinal smooth muscles were significantly reduced by Dark Plum powder extraction at the concentration of more than 1.09 g/L (P<0.05).Ach observably induced the amplitude of smooth muscle contractions (P<0.01).Dark Plum powder extraction at the concentration of 2.18 g/L markedly inhibited the amplitude and frequency of spasmodic contractions (P<0.01).In the Ca²⁺ free tyrodes solution,Dark Plum powder extraction evidently inhibited the amplitude and frequency of the Ach-induced both intracellular and extracellular calcium-depended contractions (P<0.01).Dark Plum powder extraction inhibited the spontaneous contraction and spasmodic contraction caused by Ach in a dose-dependent manner,isolated gastric smooth muscle.The mechanism was speculated to be related to inhibiting the extracellular Ca²⁺ inflowing via cell membrane and intracellular Ca²⁺ releasing.

In order to investigate the impact of transportation stress on blood cells and blood biochemical indicators of Ili horses,6 Ili mares were selected for transportation with a mileage of 400 km.The total transportation time were 8 h,the horse serum acute phase proteins (APPs),cortisol (Cor),heat shock proteins (HSPs),interferon-γ (IFN-γ),tumor necrosis factor-α (TNF-α) and blood routine were detected at 0,4,8 h of transport and 12 h after landing.The results showed that when compared with 0 h of transport,horses serum haptoglobin (HP) content increased significantly at 8 h of transport (P<0.05),horses serum Cor content increased significantly at 4 and 8 h of transport and 12 h after transport (P<0.01).Horses serum HSP90 content decreased significantly at the 12 h after landing (P<0.01),but transportation had no significant effect on serum IFN-γ and TNF-α content (P>0.05).The standard deviation in red cell distribution width (RDW-SD),platelet distribution width (PDW),platelet large cell ratio (P-LCR),granulocyte (GRAN) and granulocyte ratio (GRAN%) were all significantly increased at 4 and 8 h of transport and 12 h after transport (P<0.05),and intermediate cells ratio (MID%),intermediate cells (MID),and hemoglobin concentration (MCHC) were all significantly decreased at 4 and 8 h of transport and 12 h after transport (P<0.05).In conclusion,transportation stress had significant effects on horses serum HP,Cor,HSP90 concentration,blood RDW-SD,PDW,P-LCR,GRAN%,GRAN,MID and MCHC,these indicators could be used to evaluate horses sensitivity to transportation stress.

Oxidative stress refers to a kind of stress response in which the oxidation/antioxidation dynamic balance is destroyed due to the increase of active oxygen (ROS) in the body from the stimulation of the external environment.Under normal conditions,the system involved in the production and removal of ROS in the animal body is in a state of dynamic equilibrium.Oxidative stress occurs when ROS production increases or its scavenging capacity decreases due to external environmental stimulation or changes in the body itself.Long-term or excessively strong oxidative stress will result in slowing growth,reducing immune function,occurrence of diseases of aquatic organisms and other hazards,as well as the decline of aquatic product quality and nutritional value.Then,it brings serious economic loss to the aquaculture industy.The Nrf2-Keap1/Are signaling pathway plays a crucial role in resisting exogenous or endogenous oxidative stress.This signaling pathway has been extensively studied in mammals,but hasn't yet seen a systematic review in aquatic organisms.Based on a large number of literatures,from aspects of the mechanism of oxidative stress,the molecular basis of Nrf2 and Keap1,the Nrf2-Keap1/Are signaling pathway,the related research progress of this signaling pathway in aquatic organisms was introduced in this paper,such as fish,shrimp and crab,etc.This paper will provide theoretical parameters and relevant data for the mechanism research of an-oxidative stress in aquatic organisms.

The objective of this study was to investigate the effect of different sources of fiber diets on apparent digestibility,duodenal tissue morphology and cecal fiber digestive enzyme activities of Fujian Yellow rabbits.200 weaning Fujian Yellow rabbits with healthy physique and similar weight were selected and randomly divided into 4 groups,5 replicates per group,10 replicates each.The rabbits in 4 groups were fed with diets composed of 25% alfalfa meal,beet slag,oat hay and barley meal as roughage raw materials,respectively.The feeding experiment lasted for 60 days.On the 54th day,the digestive test was performed using the whole feces collection method.After slaughter,the villous height,crypt dept and their ratio,and cecal cellulase,hemicellulase and pectinase activities were determined.The results showed that:①The digestibility of neutral detergent fiber (NDF),acid detergent fiber(ADF),insoluble fiber(IDF),and soluble fiber(SDF) in alfalfa hay meal and beet slag groups were extremely significantly higher than oat hay and barley meal groups (P<0.01).Among them,the digestibility of IDF and SDF in beet slag group was the highest,which was significantly different from that in alfalfa meal group (P<0.05).②The villus height in alfalfa hay meal and beet slag groups were extremely significantly higher than that in barley meal group (P<0.01),but significantly lower than oat hay group (P<0.05).The crypt depth of alfalfa hay meal and beet slag groups was significantly lower than that of oat hay and barley meal groups (P<0.05),and it was the lowest in beet slag group.The V/C value in beet slag group was highest,which was extremely significantly higher than that of the other groups (P<0.01).③The cecal fiber digestive enzyme activities of alfalfa hay meal and beet slag groups were extremely significantly higher than oat hay and barley meal groups (P<0.01),and the three enzyme activities of beet slag group was extremely significantly higher than that of alfalfa meal group (P<0.01).In conclusion,comparing the four fiber diets,adding 25% beet slag to the diet could improve the apparent digestibility and cecal fiber digestive enzyme activity of meat rabbits,and improve the duodenal tissue morphology of the rabbits.

Intestinal tract is an important digestive and immune organ of the body.Maintaining intestinal health is important for pig growth and disease prevention.The development of high-throughput sequencing technology has greatly promoted people's understanding of intestinal microbial function,and the research of pig intestinal microbiome is gradually becoming a hot spot.At present,although the intestinal microbiome of pigs at a certain growth stage has been well understood,there is still a lack of comprehensive longitudinal studies on the dynamic changes of intestinal microbiome in the whole life cycle of commercial pigs.However,from birth to marketing,the intestinal microbiome of pigs in the whole growth cycle is not constant,but a dynamic development process.In this paper,the longitudinal changes of intestinal microflora and its main influencing factors in different stages from birth to fattening of pigs during lactation,weaning,fattening and pregnancy were reviewed.It was found that the significant changes of intestinal microbial community structure mainly occurred in the weaning period.At the same time,although the composition of intestinal microorganisms was constantly changing over time,there were still some dominant bacteria.This part of microor-ganisms is called the core flora,and the bacteria that only appear in a specific period are only "passers-by" in the gastrointestinal tract,and they are also the most vulnerable to external factors.Intestinal microbiome is related to many factors,such as age,diet,environment,antibiotic use,etc.,among which diet plays an important role in shaping intestinal microflora.This paper provided a theoretical reference for understanding the dynamic changes of intestinal microflora in pigs at different growth and development stages,and using the technology on microorganisms to improve pig growth performance and health level.

The purpose of this study was to isolate the excellent lactic acid bacteria which could be used for both probiotics and silage starter.Silage was collected in large-scale cattle farm.The gradient diluent was coated on MRS plate for anaerobic culture.The dominant strains were screened and their colony morphology was observed.Biochemical test and specific PCR identification were carried out.The growth curve,acid production curve,the influence of culture temperature and initial pH of the medium on the growth of the self-screened lactic acid bacteria,and its bacteriostatic characteristics,antibiotic sensitivity and safety were studied.The results showed that a bacterial colony with round shape,neat edges,smooth surface,milky white,Gram-positive and non-bacillus bacillus was screened.The biochemical results showed that the strain could grow in the anaerobic environment.The results of exercise test,catalase test,nitrate reduction test,gelatin liquefaction test,indole test were negative.The results of 15 kinds of sugar and alcohol fermentation in keeping with the biochemical characteristics of Lactobacillus casei.The strain was identified by PCR method with specific primers of Lactobacillus casei,agarose gel electrophoresis imaging showed a specific amplified band at 727 bp,which was consistent with the biochemical identification results,and the strain was named as RS1.The pH of the fermentation broth of RS1 did not change significantly at 0 to 4 h,and was in the growth delay period.The pH of the fermentation broth decreased rapidly at 4 to 16 h and entered the logarithmic growth phase.The fermentation broth was in the logarithmic growth phase at 16 to 36 h.The pH dropped slowly and entered a stable period.RS1 could grow in the culture temperature range of 20 to 50 ℃,and the suitable growth temperature was 25 to 40 ℃.It could grow under the initial pH of the culture medium 1.0 to 9.0,and the optimum pH was 6.0 to 8.0.The diameter of the inhibition zone of Staphylococcus aureus was 23 mm,and that of Escherichia coli was 17 mm.It was resistant to enrofloxacin and ceftazidime.After 14 days of administering the mice,the growth status of mice was good.These results indicated that Lactobacillus casei RS1 was safe and non pathogenic,and had potential development value in the field of probiotics and biological feed additives.

Wheat bran is rich in a variety of nutrients,meanwhile,its application in feed is limited by the presence of crude fiber,phytic acid,etc.Microbial fermentation of bran has been widely used to reduce the content of anti-nutritional factors and release a variety of active substances,thus greatly improving its application value in feed.This paper reviewed the nutritional components,microbial strains,fermentation process optimization,quality improvement and functional characteristics of the fermentation products of bran.Bran contains crude protein,crude fat,phenolic acid,dietary fiber,and so on.These nutrients exist in the form of complexes,thereby limiting their utilization in the feed.For efficient fermentation of bran,Bacillus,Lactobacillus,yeast and mold are mainly used,which secrete various carbohydrate hydrolyzing enzyme systems and play different roles in the degradation of bran.It's a common practice to apply optimization strategies,especially response surface methodology,to improve the contents of target fermentation products,such as phenolic acids,dietary fiber,soluble sugar and protein.These fermentation products make great contributions to the antioxidant level and the immunity,and improve the intestinal environment and the production performance of the animal.

In order to explore the molecular mechanism of early sexual maturity,low litter size and small body size of Xiang pig at the genomic level,we downloaded the re-sequencing data of Meishan pig and Duroc pig,which had obviously differences in reproduction and body-size as references.SNPs detection and allele frequency difference analysis were carried out on the re-sequencing data of 26 Xiang pigs,24 Meishan pigs and 24 Duroc pigs.The SNPs highly differentiated from Meishan pig and Duroc pig were detected by AS-PCR and Sanger sequencing.Finally,GO enrichment and KEGG pathway analysis were performed for genes with highly differentiated SNPs.The results showed that there were 236 710 SNPs in exons of Xiang pig genome,including 193 of stoplosses,1 238 of stopgains,90 945 of nonsynonymous SNPs and 144 334 synonymous SNPs.A total of 1 046 highly differentiated SNPs were obtained,including 429 of nonsynonymous SNPs,2 of stoplosses,6 of stopgains and 609 of synonymous SNPs,which affected 524 protein coding genes,and a hotspot rich in highly differentiated SNPs in 44-58 Mb of X chromosome was found.The results of allele frequency verification in populations were consistent with the re-sequencing results.8 loci were identified as highly differentiated SNPs in Xiang pig from Meishan pig and Duroc pig.Analysis for the 524 affected genes through GO and KEGG classification,it enriched in the oocyte meiosis,estrogen signaling pathway,estrogen signaling pathway together with other reproduction-related pathways.GO terms were annotated to intracellular estrogen receptor signaling,cilia movement and other reproduction-related items,skeletal system morphogenesis,bone development,osteoblast differentiation and other body-related items.The 18 reproduction-related genes PTGFR,TRO,DNAH17,PKDREJ,KAT8,DNAI2,VDR,TEX14,QSOX1,AR,WNK3,ADCY3,SPEF1,MIGA2,SLC2A8,PSMF1,TBC1D20,IFT172 and 7 size-related genes IBSP,NR6A1,HSPG2,TARS,PDZD2,FGF4,AMER1 with highly differentiated SNPs of Xiang pigs were identified,which might be the key genes to regulate the early sexual maturity,small litter size and small body size of Xiang pigs.

This experiment was aimed to explore the influence of four fixed effects of birth year,season,parity,and lactation stage on milk production traits of Xinong Saanen dairy goat.A total of 645 lactating does from 2006 to 2018 in the Xinong Saanen dairy goat breeding farm of Northwest A&F University were used as the research objects.Milk samples were collected once a month and collected once every morning and evening on the sampling day.Two milk samples from the same individual were mixed in equal proportions,which were determined through milk composition analyzer to obtain the concentrations of eight indexes including milk fat percentage,milk protein percentage,lactose percentage,total solids,non-lipid solid,density,freezing point and acidity.Combined with the milk production records of the farm,the fixed-effect model was used to perform descriptive statistical analysis of the phenotype through SAS 9.4,and then the GLM model was applied to analyze the impact of fixed-effects on milk production traits.The results showed that the average milk yield of Xinong Saanen dairy goats in 300 days was 507.67 kg,the milk fat percentage was 3.58%,the milk protein percentage was 3.20%,the lactose percentage was 4.19%,the total solids content was 12.21%,and the non-lipid solid content was 8.46%.The milk yield was extremely significantly affected by birth year and parity (P<0.01),while the birth season had no significant effect on milk yield (P>0.05).The lactation stage and birth year had extremely significant effects on all eight milk component traits including milk fat percentage (P<0.01).The parity had a extremely significant effect on six milk component traits except milk protein percentage and total solids(P<0.01).The results revealed that the parity and lactation stage were the two main non-genetic factors influencing the milk production traits of Xinong Saanen dairy goats.The third and forth parity had the best milk production performance.The milk quality in the early lactation stage was much better.Our findings revealed the potential importance of the selective breeding of excellent dairy goat population and feeding and management,which would further provide a theoretical basis for the genetic evaluation of economic traits and population genetic improvement of Xinong Saanen dairy goats.

To investigate the regulation of bonemorphogenetic protein and activin membrane-bound inhibitor (BAMBI) gene in transforming growth factor-β (TGF-β) signaling pathway on porcine follicular granulosa cells,in this study,BAMBI interference vector and overexpression vector were designed and constructed.Porcine ovarian granulosa cells were ransfected with the constructed interference (pSIREN-BAMBI-sh1,pSIREN-BAMBI-sh2,pSIREN-BAMBI-sh3) and overexpression (pcDNA3.1-BAMBI) recombinant plasmids,and Real-time quantitative PCR technology was used to screen effective fragments and detect mRNA expression levels of TGF-β signaling pathway downstream genes (TGF-βRⅠ,TGF-βRⅡ,SMAD1,SMAD2,SMAD3,SMAD4,SMAD5) and apoptosis genes (Bax,Bcl-2).Finally,the effects of BAMBI interference and overexpression on the proliferation and apoptosis of ovarian granulosa cells were detected by MTT method and flow cytometry.The results showed that pSIREN-BAMBI-sh2 had the best effect of inhibiting BAMBI expression and the highest interference efficiency.When interfering with BAMBI,the expression of TGF-βRⅡ increased significantly (P<0.05),so that the expression of SMAD2 and SMAD3 increased significantly (P<0.05).When overexpressing of BAMBI,TGF-βRⅡ decreased significantly (P<0.05),so that the expression of SMAD2 and SMAD3 decreased significantly (P<0.05).Up-regulation of BAMBI inhibited the proliferation of granulosa cells and significantly promoted apoptosis (P<0.01).The results suggested that the BAMBI gene significantly affected the phenotype and function of ovarian granulosa cells,and might indirectly affect the reproductive performance of pigs by regulating the TGF-β signaling pathway.

The aim of this study was to optimize the ovum pick-up (OPU) and in vitro fertilization system,and further improve the in vitro embryo production efficiency.The optimal in vitro culture medium was screened out by using fresh ovaries collected from slaughterhouses for in vitro fertilization.The effects of different bull semen and donor cows on the outcome of in vitro fertilization of oocytes collected by OPU were analyzed.The results showed that compared with CR1aa medium,mCR1aa medium had no significant effect on cleavage rate,but the blastocyst formation rate was significantly increased (28.1% vs 20.6%,P<0.05).Three Holstein bulls semen were randomly selected for in vitro fertilization,the proportion of cleaved embryos was not affected,but the blastocyst development rate of in vitro-produced embryos originating from No.1 bull semen (38.7%) was significantly higher than that of No.2 (23.8%) and No.3 bull semen (22.9%) (P<0.05).There was no significant difference in the average number of recovered oocytes obtained from three randomly selected donor cows (H1,H2 and H3),but the developmental ability of embryos after in vitro fertilization was significantly different.The cleavage rates blastocyst rates of H1 and H2 donors were significantly higher than that of H3 donors (P<0.05),and the blastocyst rate of H1 donor was significantly higher than that of H2 donor (P<0.05).In summary,mCR1aa medium significantly increased the blastocyst development rate,and could be used for in vitro embryo production,the individual variability in bull semen and donor cows could directly affect the production efficiency of in vitro embryo production.The results provided a reference for the optimization of OPU-in vitro fertilization technology.

The mammary gland is a highly active organ whose epithelium changes dramatically during puberty and reproductive cycles.These changes are driven by specialized stem and progenitor cells.The research of mammary stem cells is of great significance to animal mammary gland development,lactation and breast cancer.Today,significant advances have been made in the biology of mammary stem cells.Mammary stem cells are histologically undifferentiated epithelial cells that can divide symmetrically to produce two identical stem cells,or asymmetrically to produce one kind of stem cell and one kind of luminal epithelial progenitor cell or basal/myoepithelial progenitor cell.A variety of different types of mammary stem/progenitor cells can be identified by labeling retained cells,dye exclusion,stem cell antigen-1 labeling,cell surface marker labeling,and lineage tracing.The isolation and identification of mammary stem cells by different methods is helpful to understand the heterogeneity.Mammary research is divided into six stages:Embryo,adolescence,sexual maturity,pregnancy,lactation and involuted phases.The types and characteristics of mammary gland stem/progenitor cells are different in different stages of mammary gland development.Studies have shown that the progenitor cells with basal and luminal lineages are produced in embryo phase.Quiescent mammary stem cells may also come from the embryo stage.After birth,quiescent mammary stem cells can be stimulated by ovarian hormones to re-enter the cell cycle,producing a mammary cell lineage and self-renewal.This paper introduced the existence,morphological characteristics and identification methods of mammary stem cells and progenitor cells,briefly described the characteristics of mammary stem cells in embryonic,adolescent and pregnancy,analyzed the current shortcomings and prospects for future application,in order to further study animal breast stem cells.

The purpose of this study was to investigate the changes of reproductive hormones,salivary crystals and follicular development in buffalo saliva.The reproductive hormones in salivary and serum of water buffalo were measured by ELISA kit,salivary crystals were prepared,and the relationship between reproductive hormones and salivary crystals and follicular changes was analyzed.Four methods,including salivary crystallization,rectal examination,salivary crystallization+ B-ultrasound and rectal examination+B-ultrasound were used for verification in production.The results showed that the level of salivary estrogen (E₂) reached a peak of 233.51 pg/mL on the day of estrus,and then began to decrease slowly.The progesterone (P₄) level in buffalo saliva reached 16.17 ng/mL before 2 d of estrus,and decreased to 8.91 ng/mL on the estrus day.The level of follicle-stimulating hormone (FSH) gradually decreased from 37.91 ng/mL to 34.64 ng/mL before 2 d of estrus.However,it gradually increased to 61.20 ng/mL after 2 d of estrus.Luteinizing hormone (LH) gradually decreased from 5.15 ng/mL at 1 d before estrus to 4.12 ng/mL on the estrus day,but increased to 5.77 ng/mL at 1 d after estrus.B-ultrasonography results showed that ovarian follicles grew rapidly from 2 d before estrus to 20 mm on the day of estrus,and the size of follicles was insignificant until ovulation burst.After ovulation,the corpus luteum was formed and maintained until about 4 d before the next estrus,during which the corpus luteum was at its maximum at 14-17 d after estrus,which was consistent with the peak of LH secretion in buffalo saliva.The estrus rate determined by salivary crystallization+B-ultrasound was the highest (86.67%),which was significantly different from other methods (P<0.05).The results of pregnancy diagnosis showed that the pregnancy rate (61.53%) determined by salivary crystallization+B-ultrasound was significantly higher than that by rectal examination,salivary crystallography and rectal examination+B-ultrasound (P<0.05).To sum up,the development of follicles on ovaries was significantly related to the concentration of reproductive hormones in saliva.Saliva crystals on the day of estrus showed typical dendritic shape,and the accuracy of saliva crystals+B ultrasound method for estrus identification was the highest.

The correlation among the daily feed intake,daily weight gain and feed to gain ratio (F/G) of Duroc boars in different body weight ranges were analyzied to evaluate the effectiveness of using higher initial weights to determine the growth and F/G of Duroc boars,to analysis the correlation between F/G in different body weight stages and whole process F/G of Duroc boars,to establish the regression equation between F/G in different body weight stages and whole process F/G,and to increase the annual measurement quantity of the measuring station and improve its utilization rate.The data of daily gain,daily feed intake and F/G of 119 Duroc boars during 30-110 kg were measured by using 8 Osborn feed intake recording equipments.The correlation between F/G at different weight stages and F/G at whole stages was analyzed,and the regression equation was established with the F/G at the whole stages.The results showed that the daily feed intake,daily gain and F/G of Duroc boars were all increased during the period of 30-110 kg,and there was an inflection point at 90 kg during the growing period,and they were all decreased after that.There was a certain correlation between the F/G at different weight stages and the F/G at the whole stages,in which the determination coefficient of the regression equation Y=0.316+0.873X₁ (X₁ was F/G of 40-90 kg) was 0.76.The correction accuracy of regression equations was high,and the F/G of 30-110 kg could be predicted by measuring the F/G of 40-90 kg.Using this coefficient,the time of F/G measurement could be shorten,and it also provide reference for more accurate calculation of F/G,and at the same time effectively improved the utilization rate of the pig breeding station.

The aim of this study was to explore the protein components of midgut contents of adult female haemaphysalis flava collected from hedgehog body surface at four different stages and reveal the types and content changes of protein involved in blood meal digestion.The midgut contents from Haemaphysalis flava at four different stages were detected by LC-MS/MS,and the peptides and proteins obtained were identified and analyzed by using the computer software Mascot 2.2 based on previous established salivary and midgut transcriptome translation library and UniProt database.The results of this study showed that a total of 3 046 unique peptides and 303 proteins were detected and identified.Total of 271 proteins with high credible among 303 proteins identified.Among 271 high credible proteins,23 proteins had abundant contents at first stage,125 types of proteins had zero contents at later stages (from second stage to fourth stage),123 types proteins' contents changed in different degrees.Among 123 proteins with contents changed,there were 24 proteins with contents obvious change,which include 12 proteins that their contents rose obviously and 12 proteins that their contents descended obviously at four stages.It was determined that 148 high credible proteins came from hedgehog serum,and in which 24 high credible proteins may be involved in ticks digestion blood meal.

The purpose of this study was to isolate and culture the pathogen of a clinical diarrheal disease and to explore the biological characteristics of the main antigen proteins encoded by the pathogen.The nucleic acid of Porcine adenovirus type 3 (PADV3) was detected by nested PCR and the virus was isolated and cultured,and was identified by immunoperoxidase monolayer staining (IPMA).The ORF region of fiber gene was amplified by PCR and cloned into pET-28a(+) vector and transformed into E.coli BL21 (DE3) competent cells.Recombinant fiber protein (recfiber) was induced by IPTG and was identified by SDS-PAGE and Western blotting.The purified recombinant protein was immunized to rabbits to prepare polyclonal antibody and determine its immune activity.The results showed that the pathogen was positive for PADV3 nucleic acid,and infected ST cells produced typical "grapevine" cytopathic effect (CPE).The nucleic acid of PADV3 was stable in the passages from P5 to P11,and the P5 of the isolated strain was serologically positive for PADV3.The open reading frame (ORF) of fiber gene was 1 296 bp (GenBank accession number:MT774498),which had the highest nucleotide similarity (86.1%) with PADV3 strain and less than 40% similarity with other strains.The molecular weight of the recombinant fiber protein expressed in prokaryotic cells was about 45.2 ku.The polyclonal antibody of fiber protein showed specific nuclear staining with PADV3 infected cells.A PADV3 strain named PADV3-HY1812 was successfully isolated in this experiment and the polyclonal antibody against fiber protein had good immune activity.

The purpose of this study was to obtain recombinant PRV gE protein and polyclonal antibody against PRV gE protein.The PRV gE gene was amplified from PRV-infected pig tissues (lung,brain and tonsil) by PCR,and then ligated to the cloning vector pMD18-T (pMD-gE) for sequencing and phylogenetic tree analysis.Using pMD-gE as a template,part of the gene encoding gE protein extramembrane domain (gE-outside) was amplified by PCR,then connected to the prokaryotic expression vector pET-30a(+),transformed into E.coli DH5α competent cell to construct recombinant plasmid pET-gE-outside.The recombinant plasmid pET-gE-outside was transformed into the E.coli Rosetta^TM(DE3) pLysS competent cell.After induction by IPTG,the expressed product was analyzed and identified by SDS-PAGE and Western blotting,respectively.The recombinant PRV gE protein was purified by affinity chromatography and used to immunize mice.The titers measure and identification of the mouse anti-PRV gE polyclonal antibody in the serum from the immunized mice after the third immunization for 2 weeks were detected by indirect ELISA and Western blotting,respectively.The PCR and sequencing results showed that the PRV gE gene was successfully cloned in this study,which belonged to the same branch as the domestic virus strain after 2011.The SDS-PAGE and Western blotting results confirmed that the PRV gE-outside protein was correctly expressed by the prokaryotic expression system,with a molecular weight of about 55 ku,and could react with porcine anti-PRV polyclonal antibody.The concentration of gE-outside protein purified by affinity chromatography was 1.23 mg/mL.3 weeks after immunizing mice with gE-outside protein,the mouse anti-PRV gE-outside polyclonal antibody was prepared with a titer of 1:204 800,and had an immune reaction with gE-outside protein.In this study,recombinant PRV gE protein and mouse anti-PRV gE polyclonal antibody were prepared,which could provide technical guidance and material for the study on the mechanism of PRV infection and the establishment of rapid and efficient immunological detection technology.

Bacterial intestinal infections are common diseases during breeding process in livestock and poultry industry,which can easily cause problems such as decline in livestock feeding and production performance,and even death.Antibiotics are usually used to breeding process.However,due to the long-term existence of antibiotic abuse and unreasonable use,drug residues and bacterial resistance problems have become increasingly prominent.Therefore,the development of safe and reliable antibiotic alternatives has become a research hotspot in the livestock industry.Egg yolk immunoglobulin (IgY) is immunoglobulin expressed in egg yolk after vaccination of laying hens,it can be used to the prevention and treatment of corresponding diseases.IgY has many advantages such as large output,stable properties,safety and efficiency.Currently,IgY is widely used in livestock and poultry breeding,and has achieved significant advantages.The article reviewed the molecular structure,physicochemical properties and mechanism of IgY.It summarized and compared the fat removal rate,antibody titer,total protein content and other indicators of five kinds of IgY extraction methods.Finally,the research status and application prospects of IgY in the prevention and treatment of common bacterial infections in livestock and poultry were introduced in detail and it was expected to provide research basis and technical support for the efficient production and disease prevention of IgY.

To screen traditional Chinese medicines to prevent and treat viral disease H9N2 Avian influenza virus (AIV),Qingen extract (QG) was used to evaluate its anti-H9N2 efficiency through the neuraminidase (NA) inhibition and CCK-8 method from the aspects of virus adsorption,proliferation and inactivation,with oseltamivir (Tamiflu,DF) as positive drug control,based on AIV clinical symptoms and basic principles of traditional Chinese medicine.The results showed that the inhibitory effect of QG on NA increased in concentration dependent manner,and IC₅₀ was about 1 130 μg/mL.The maximum safety concentrations of QG and DF were 2 500 μg/mL and 500 μg/mL respectively,and the TCID₅₀ of H9N2 AIV was 10^-4.36/100 μL.Among the concentration range of 156.25-2 500 μg/mL,QG weaken the virulence of H9N2 AIV to a certain extent,and showed anti-attachment and anti-viral replication effects on MDCK cells.In conclusion,QG could inhibit the NA activity of H9N2 AIV,inhibit H9N2 AIV's attachment and replication to target cells.

In order to study the effect of inhibin α (INHα) on reproductive hormone content of Kazakh sheep by active and passive immunity,the INHα protein was expressed and purified from INHα recombinant plasmid strain and then injected into Xinjiang camel to prepare camel anti-INHα polyclonal antibody.The antibody titer was detected and the specificity of the antibody was verified.45 adult Kazakh sheep (3 to 5 years old),with similar estrus and interestrus,were randomly divided into 3 groups,which were respectively used as the INHα polyclonal antibody immunization group (group A),the INHα recombinant protein immunization group (group B) and the control group (group C).These sheep were immuned three times every ten days.The ELISA method was used to detect five reproductive hormones that had important functions in sheep reproduction:follicle stimulating hormone (FSH),luteinizing hormone (LH),pregnancy ketone (P₄),estrogen (E₂),INH (inhibin).Blood biochemical indicators were detected.The results showed that the optimal induction concentration of IPTG was 0.6 mmol/L,and a higher yield of INHα inclusion body protein was induced at 4 h.The INHα protein was highly purified and had good immunogenicity.The prepared INHα antibody had a titer of 1:512 000,and the antibody could specifically bind to INHα protein.It showed that an immunogenic INHα protein was successfully prepared and after further verification.The camel anti-INHα polyclonal antibody with high antibody titer was successfully prepared.After immunization,the LH,P₄ content of group A and FSH,LH,P₄,E₂,INH content of group B compared with group C were not significantly different (P>0.05),while the FSH and E₂ contents of group A were significantly higher than those of group C (P<0.05),the INH content was significantly lower than that of group C (P<0.05).Through the detection of blood biochemical indicators,it was found that the experimental animals had no adverse symptoms after the immunization of the two immune preparations of INHα protein and INHα antiserum.The results showed that both INHα antigens could have a good effect on the secretion of blood reproductive hormones in Kazakh sheep,and the INHα antiserum had a better immune effect.

To identify the accurate species of Paramphistomidae in buffalos of Nanning,Guangxi,trematode identification was performed after collected from livers.In this study,the liver of buffaloes in a slaughterhouse in Nanning was dissected and the pathological lesion was recorded.Afterwards,adult worms in the livers were collected and incubated to lay eggs,then morphology of the adult worms and eggs were observed.The adult worms were also stained with hematoxylin-eosin (HE) and hematoxylin to facilitate its internal structure observation.Finaly,adult worms were identified by molecular biological method.The results showed that there were bleeding spots on the infected liver surface.Besides,congestion,purulent focus,liver enlargement and softer texture was also detectable.The eggs were oval,light gray,covered with round embryo cells,and the yolk cells did not fill the whole egg.The adult was composed of body wall,internal reproductive and digestive system.The body wall was composed of cortex and muscular layer.The reproductive system,digestive tract and some muscle bundles and fibrous tissues also could be seen.The reproductive system included the reproductive organs such as ovary,uterus and testis,while the digestive tract was distributed throughout the body cavity.The amplified product of ITS-2 gene was 441 bp,and comparison with Explanatum explanatum (AB743577.1) in GenBank showed that the similarity was 99.3%.Phylogenetic analysis showed its close relationship with Explanatum explanatum,and they were also located in the same branch.Considering the pathological lesion,egg characteristics,adult morphological and molecular biology characteristics,flukes here in our study was determined as Explanatum explanatum. The results of this study provided a theoretical basis for the classification and identification of trematodes,and further enrich the species of this family.In addition,we have a preliminary understanding of the prevalence of Paramphistomidae in buffaloes in Nanning,Guangxi,and provided a theoretical basis for the accurate control of trematode infection.

The purpose of this study was to obtain Brucella BspD protein,analyze its potential biological functions,and prepare its polyclonal antibody.After the synthesis of BspD gene sequences and the bioinformatics analysis of the amino acid sequence of Brucella BspD,according to the BspD gene sequence of Brucella abortus 2308 (GenBank accession No:NC_007618.1),the target gene was inserted into pMD19-T vector,and the target gene was digested by restriction endonuclease and recombined into pET-28a(+) vector to construct the pET28a-BspD recombinant vector.After double enzyme digestion and sequencing verification,IPTG was used to induce expression strain,SDS-PAGE was used to identify the expression of BspD,His label protein purification column was used to purify BspD protein,BCA kit was used to detecte the protein concentration,purified BspD protein was used to immunize New Zealand White rabbits,Western blotting was used to identify the specificity of polyclonal antibodies.Indirect ELISA was used to detect the titer and reactivity of antibody.The 37 ku BspD protein had been expressed successfully,and the purified BspD concentration was 2 000 μg/mL by BCA method.The Western blotting results showed that the prepared antibody had good specificity,and the titer of BspD polyclonal antibody detected by indirect ELISA was 1:12 800.The polyclonal antibody against BspD had been successfully prepared,but its reactivity was low.According to the analysis of biological information,BspD protein was hydrophilic,existed transmembrane region,had no signal peptide,possessed 17 phosphorylation sites and 11 antigenic epitopes.The secondary structure of BspD protein was mainly α-helix,reaching 86.80%,and there were a few extended chains,random coil,β-folding and other structures.In addition,the tertiary structure of BspD was constructed by Phyre 2,an online software,also confirmed that it was α-helix.The results provided references for further study on the function and molecular mechanism of BspD secreted by Brucella.

Antibody is a class of immunoglobulin protein that specifically recognize and bind antigens.It is also a major component of humoral immunity in the body.The applications of antibody is rapidly expanding as the development of science and technology and the in-depth research of antibody.Nowadays,antibody can be used as affinity ligand or probes for the research on protein-protein interaction,protein-nucleic acid interaction,the orientation and distribution of biological macromolecules,used as immunosuppressant or drugs for the treatment of autoimmune disease,tumor or infectious disease,also can be applied in disease diagnosis,food safety testing and environmental monitoring by combining with a variety of labeling techniques.However,the disadvantages of traditional antibodies,such as high production cost,poor tissue penetration and immune rejection,limiting their wide applications in certain fields.In recent years,scientists have committed to develop search for minimolecule antibodies,and have developed chimeric antibodies,small-molecule antibodies,bi-specific antibodies and other new antibodies.Nanobody (Nb),also known as variable heavy chain domain (VHH) antibody,is a signal domain antibody consisting of only one variable heavy chain domain.Nanobody retains the full antigen-binding ability of the heavy chain antibody.It has the advantages of small molecular weight,strong tissue penetration,high antigen affinity,ability to recognize hidden epitopes,low immunogenicity,stable structure,good water solubility,low production cost and easy industrialization,showing great application prospect in the fields of disease diagnosis,treatment of drugs and infectious disease,detection of small molecule drugs and toxin residues.In this paper,the characteristics of nanobody were introduced firstly,followed by the briefly description of the nanobody preparation process.The applications of nanobody in the fields of disease diagnosis,disease treatment,food safety and environmental monitoring were then reviewed emphatically,and the applications of nanobody in veterinary clinic were finally prospected.

The aim of this study was to study the injury effect of different doses of cyclophosphamide on liver and kidney of mice.40 healthy female Kunming mice aged 5 weeks were randomly divided into 5 groups,injected intraperitoneally with 40,80,120 and 160 mg/(kg·BW) cyclophosphamide for 3 consecutive days,respectively,except the control group.Liver,kidney and serum were collected on 7th day after injection.The paraffin tissue sections and transmission electron microscope sections were prepared,and the histological changes of liver and kidney were observed.The content or activity of CG,ChE,PA,TBA,ALT,AST,BUN,SCr,UA in serum were detected.The results showed that cyclophosphamide could cause pathological injury to liver and kidney of mice in a dose-dependent manner,and the liver showed injury earlier than the kidney.The observed results of paraffin sections and transmission electron microscopy sections showed that the toxic effects of cyclophosphamide on the liver were mainly manifested in hepatic cord disorders,hepatic sinusoidal dilatation,microbiliary cholestasis,inflammatory cell infiltration and fibrous proliferation in the portal area and around the central vein,hemorrhage between tissues,fatty degeneration and balloon-like degeneration of liver cells,apoptosis and necrotic cells increased.Kidney tissue damages were mainly manifested in hemorrhage and fibrosis,renal tubular epithelial cell cytoplasmic vacuolation,mitochondrial damage,nuclear pyknosis,and thickening of the basement membrane of epithelial cells.Cyclophosphamide did not cause serious damage to the bladder.Compared with the control group,serum ChE activites and PA contents in 40-160 mg/(kg·BW) groups,AST activities in 120-160 mg/(kg·BW) groups,BUN contents in 80-160 mg/(kg·BW) groups,and UA level in 80 mg/(kg·BW) group were increased significantly (P<0.05).The other biochemical indicators were not significantly different from the control group (P>0.05).The results showed that cyclophosphamide injection at a dose of 80-120 mg/(kg·BW) could cause obvious pathological damage to liver and kidney tissues and cells in Kunming mice.The results of the study could provide an experimental basis for the selection of appropriate cyclophosphamide in establishment of animal liver immunosuppression model.

The purpose of this study was to facilitate the improvement of Chinese veterinary drug residue standards and the establishment of relevant regulatory system,and provide data reference for China's animal product trade,ensure people's food safety,and promote the better development of China's animal product economy.Firstly,the types of veterinary drug residues in pig tissues and the standards of veterinary drug maximum residue limits (MRLs) specified in the new national standard GB 31650-2019 "National Food Safety Standards Maximum Residue Limits of Veterinary Drugs in Food" was analyzed.Then compared the veterinary drug residue types and veterinary drug MRLs standards in pig tissues specified by the United States,the European Union,Japan and Codex Alimentarius Commission (CAC) with the new national standards.The total number of veterinary drugs,the types of veterinary drugs that were not specified in China but were specified in other standards,and the types of veterinary drugs with other standards that were stricter than Chinese standards were compared to find out difference.The results showed that the new national standards had made more detailed supplements and amendments.On the whole,the MRLs of veterinary drugs in pig tissues stipulated by China were becoming more and more complete,and most of the standards were in line with those specified by the European Union,the United States,Japan and CAC.The gap was getting smaller and smaller.However,there was still a certain gap with these countries or organization in terms of the MRLs for certain types of veterinary drugs.China needed to strengthen exchanges and cooperation with various developed countries and organization,to promote the development of China's veterinary drug residue standards,and accelerate the update of animal product quality and safety standards.

In order to determine the cause of death of piglets in a large-scale pig farm in Guangdong province,the disease materials such as liver,spleen,heart,brain,joint fluid and lymph nodes were collected for bacterial isolation,and the isolated strains were purified and cultured.And colony morphology observation,Gram staining,morphology observation,biochemical test,PCR amplification with serotype specific primers,virulence gene test,drug sensitivity test,growth curve test and animal test were carried out.The results showed that there were round,gray white,smooth surface and neat edge colonies on the agar plate of adult bovine serum.Microscopic examination showed Gram-positive cocci which were arranged in chains.The biochemical test results of this strain were in line with the characteristics of Streptococcus suis,and the PCR amplification results showed that both the conserved gene fragment (gdh and gapdh) of Streptococcus suis and the specific cps2J gene fragment of Streptococcus suis serotype 2 were positive,indicating that the isolated strain was Streptococcus suis serotype 2.The results of virulence gene detection showed that the virulence factor genotype of the strain was gdh⁺/gapdh⁺/epf⁺/mrp⁺/sly⁺/orf2⁺/fbps⁺.The isolated strain reached the logarithmic growth stage after cultured in nutrient broth for 6 h,and the D_{600 nm} value of the bacterial solution cultured to 10 h was 0.310.The results of drug sensitivity test showed that the strain was sensitive to penicillin,amoxicillin,amoxicillin-clavulanate potassium,but resistant to doxycycline.The animal test results showed that BALB/c mice infected with the isolated strain did not die and had no obvious clinical symptoms,and its virulence was low.This study provided important reference information for the prevention and control of Streptococcus in this pig farm.

The purpose of this study was to evaluate the antibacterial activity of porcine beta defensin 2 (PBD-2) against porcine extraintestinal pathogenic Escherichia coli (ExPEC) in vivo and in vitro,and provide more insights for evaluating the value of PBD-2 as an antibiotic substitutes.Firstly,the bactericidal activity of different concentrations of PBD-2 against the porcine ExPEC PCN033 was tested in vitro.Subsequently,female Kunming mice aged 5 weeks and weighing between 18-22 g infected with different doses of ExPEC PCN033 were treated with PBD-2 or PBS (n ≥ 5).The survival rate,the bacteria loads of brain,spleen,lung tissue and blood,the levels of inflammatory cytokines IL-6,IL-12,IL-1β and TNF-α and the pathological changes of brain,spleen and lung tissues were observed.The results showed that 25 μg/mL of PBD-2 could extremely significantly inhibit the growth of ExPEC PCN033 in a concentration-dependent manner (P<0.01).The in vivo study revealed that PBD-2 treatment could effectively reduce the mortality rate of mice infected with ExPEC PCN033.The effects of PBD-2 were enhanced with increase of treatment dose of PBD-2.The effects of PBD-2 via intraperitoneal injection and intramuscular injection route were better than that of the oral route.PBD-2 treatment was less effective than chloramphenicol treatment in reducing the mortality of mice.At the same time,PBD-2 treatment extremely significantly reduced the bacteria loads in the brain,spleen,lung tissue and blood of mice at 21 h post infection with ExPEC PCN033 (P<0.01).At the same time,PBD-2 treatment extremely significantly reduced the levels of IL-6,IL-12 and IL-1β of mice at 21 h post infection with PCN033 (P<0.01).PBD-2 treatment also significantly reduced the degree of pathological damages of the brain,lung and spleen tissues of mice at 21 h post infection with ExPEC PCN033.These results indicated that PBD-2 had good activity against porcine ExPEC in vitro,and had therapeutic effect on swine derived ExPEC infection in mice,indicating that PBD-2 had the potential to develop into therapeutic drugs or antibiotic substitutes.

In December 2019,a series of sudden deaths of protected pigs occurred in a swine farm in Yulin,Guangxi.In order to confirm the cause of death and investigate the pathogenicity and drug resistance of isolated strains,samples and bacterial isolation and culture were conducted in the sick swine farm.Morphological observation,biochemical identification,PCR amplification of gdh gene,identification of serum type and some virulence genes,detection of drug resistance and identification of drug resistance genes were performed on the isolates.One bacterial strain was isolated from two dead pigs separately.On TSB solid medium,the colonies were gray white,smooth,and uniform in size.They were preliminarily identified as Gram-positive Streptococcus and named GXYL-F1 and GXYL-N2,respectively.Both isolates were identified as Streptococcus suis type 9 by specific gene gdh and serum primer.Biochemical identification results showed that the two isolated strains could ferment salicylline,maltose,M.R.and aescinate.The virulence gene of the strain detected by PCR showed that genes gdh,fbps,sao,sbp2' and orf2 were positive,while mrp,epf and sly genes were negative.Drug sensitivity test results showed that GXYL-F1 and GXYL-N2 were all resistant to neomycin,streptomycin,timicoxacin,tylosin,erythromycin,tetracycline,levofloxacin,penicillin and sulfadiazine.In addition,the isolated strain GXYl-F1 was also resistant to enrofloxacin.The drug-resistant gene aadA1,qnrB and qnrS were amplified in both isolates.The study showed that the disease was caused by Streptococcus suis type 9.The above research results provided a reference for this swine farm to develop the control measures of Streptococcus suis type 9 and a new data for the serotype diversity and prevention of technology reaserch.

The purpose of this trial was to explore the effect of thread-embedding therapy on exercise-induced fatigue.The exercise-induced fatigue rats were taken as the research object.36 SD rats were randomly divided into blank group,model group and treatment group,with 12 rats in each group.Thread-embedding therapy was performed on the treatment group before training,to study thread-embedding therapy for regulating energy metabolism in exercise-induced fatigue.After a 7-week training,except for the blank group,the exercise groups performed high-speed exhaustion exercises.The exercise time,energy metabolism-related indicators and fatigue-related biomarkers were measured and skeletal muscle sections were made.The results showed that compared with control group,the blood glucose and glycogen contents of the exercise groups were significantly reduced (P<0.05),and the contents of lactic acid,urea nitrogen and malondialdehyde were significantly increased (P<0.05).Pathological damage was observed under light microscopy of skeletal muscle sections.The ATP content and ATPase activity of the treatment group were significantly higher than those of the model group (P<0.05),and the increase in creatine kinase content was lower than that of the model group.In summary,these results indicated that thread-embedding therapy made the body have more energy substances and fewer metabolites,and might regulate the body's energy metabolism level by improving oxidative stress and increasing energy supply,and delay the generation of exercise-induced fatigue.

The purpose of this study was to explore the changes of the active components of tea tree essential oil after ¹²C⁶⁺ ion injection and evaluate the killing effect of Foot-and-mouth disease virus (FMDV).Tea tree essential oil was irradiated by ¹²C⁶⁺ ion beam of 50,100,150 and 200 Gy,and the changes of main components and mass deposition effects of tea tree essential oil before and after irradiation were analyzed by gas chromatography-mass spectrometer (GC-MS) and infrared chromatography.The contents of terpinene-4-ol and 1,8-eucalyptin in tea tree essential oil were detected.The safety of tea tree essential oil before and after irradiation and the essential oil diluted by 1:2 and 1:4 irradiation were injected into suchling mice to detect the clinical use of tea tree essential oil. The irradiated tea tree essential oil diluted into 1:2, 1:4, 1:6 and 1:8 was thoroughly mixed with the same amount of O-FMDV at 1:500, respectively. The mice aged 4-5 d were subcutaneously injected and observed continuously for 7 d to observe whether the mice were infected with O-FMDV.The results showed that after irradiated tea tree essential oil with 100 Gy ¹²C⁶⁺ ion beam,the content of pinole-4 alcohol increased from 37.90% to 39.03%,the content of 1,8-eucalyptus had no significant change,and the content of hydroxyl compounds significantly increased after irradiated tea tree essential oil.The optimal irradiation dose was 100 Gy,and the tea tree essential oil with radiation dose of 100 Gy (the maximum dilution was 1:4) had the best anti-FMDV effect in suckling mouse.Irradiation of tea tree essential oil with a dose of 100 Gy could improve the killing effect of FMDV.

The aim of this study was to compare the effects of different pre-anesthesia administration regimens combined with emulsified sevoflurane on non-invasive blood pressure and renin angiotensin aldosterone system (RAAS).Ten semi-wild ponies were randomly divided into two groups,KFXES and KFDBES.The ponies were anesthetized (ketamine,Jingsongling and fentanyl were used in KFXES group,while ketamine,butorphanol,dexmedetomidine and fentanyl were used in KFBES group) by intramuscular injection.The ponies maintained spontaneous respiration and maintained anesthesia for 2 h by intravenous infusion of 6% emulsified sevoflurane at constant speed.Before administration,the basic physical signs of ponies were recorded under normal condition,and the vital signs were continuously recorded for more than 2 hours after anesthesia.Noninvasive blood pressure was monitored at 0,30,60,90 and 120 min,and blood samples were taken simultaneously to detect the concentrations of renin,angiotensin,aldosterone and other factors in plasma.The results showed that,in the course of the experiment,the blood pressure of the ponies in KFXES group was significantly different from that before the test (P<0.05).At 0-60 min,the inhibition to RAAS between two groups had no significant difference (P>0.05).When the anesthesia time was more than 90 min,the inhibition of KFXES group on RAAS system was significantly weaker than that of KFDBES group (P<0.05).Therefore,ketamine,fentanyl,butorphanol and dexmedetomidine combined emulsified sevoflurane had better blood pressure stability than ketamine,Jingsongling and fentanyl combined emulsified sevoflurane,and had a greater impact on RAAS after 90 min.The results of this study could provide data support and direction guidance for the optimization of anesthesia method or dose of pony.

By detecting the amounts and kinds of fungi in surface and content of the table eggs at different storage temperatures and time,the dynamic changes of fungal contamination were understood,which provided reference for improving the storage conditions and prolonging the preservation period of eggs.180 fresh eggs from several chicken farms in Southern Hebei were stored respectively at 25 and 4 ℃ for 42 days.The amount and kind of fungi in eggs were detected every 7 days.The national standard GB 4789.15-2016 was consulted for the fungal count detection.The fungi species were identified by morphological observation combined with rDNA-ITS sequence analysis.The results showed that the detection rate and quantity of fungi in eggs increased significantly with time.The detection time of fungi in eggshell was earlier and the detection rate was higher than that in egg liquid,and the detection rate of fungi in egg liquid increased rapidly from 21 to 28 days.At the same time point,the amount of fungi (colony number) of 25 ℃ condition was significantly higher than that of 4 ℃ (P<0.05).168 fungal strains were isolated and identified,belonging to 18 species (genera),of which the dominant genus was Penicillium sp.,and the dominant species was Penicillium oxalicum.There was significant correlation between eggshell and egg liquid in the total number of colonies (P<0.05) and the number of isolated strains (P<0.01).In conclusion,fungal contamination of eggs was frequent in the environment of the farms sampled,the fungal detection rate and total number of colonies in samples increased significantly along with the storage time,refrigeration (4 ℃) was an effective method to prevent spread of the fungi in eggs,the fungal contamination of egg liquid might mainly come from that of the eggshell surface,the diversity of fungi in egg samples was high,and most of the dominant species (genera) were common in the environment,but some pathogenic or toxigenic kinds were also found in the contaminating fungi.