The aim of this study was to clarify CDS sequences and expression characteristics of Bcl-2 and Bax genes in the reproductive axis of female yak,in order to provide theoretical basis for investigating the potential role of Bcl-2 and Bax in regulation of yak reproduction.The samples of hypothalamus,pituitary,ovary,oviduct and uterus of female yak and cattle were collected.The yak Bcl-2 and Bax genes were amplified,cloned and analyzed by RT-PCR and bioinformatics methods.Their mRNA expression levels in different tissues were detected by Real-time PCR.The results showed that the Bcl-2 gene coding region of yak was 690 bp,encoding 229 amino acid.The homology of Bcl-2 gene in yak with Bos taurus was the highest (99.86%),and followed by Capra hircus (98.41%) and Ovis aries (97.97%).The phylogenetic tree showed that the closest genetic relationship was found between yak and Bos taurus.The Bax gene coding region of yak was 579 bp,encoding 192 amino acids.The homology of Bax gene in yak with Bos taurus,Tibetan goat and Jintang Black goat were higher,which was 99.83%,99.48% and 99.48%,respectively,and followed by Ovis aries (99.14%),Equus caballus (95.34%) and Homo sapiens (94.30%).The phylogenetic tree showed that the closest genetic relationship was found between yak and Bos taurus.There were no signal peptides in Bcl-2 and Bax proteins,all of which were acidic unstable hydrophobins.Real-time PCR results showed that Bcl-2 and Bax genes were expressed in hypothalamus,pituitary,ovary,oviduct and uterus tissues of yak and cattle.The expression of Bcl-2 gene was significantly higher in yak ovary (P<0.05) and uterus (P<0.01) than that in cattle.The expression of Bax in yak uterus and ovidut was significantly higher than that in cattle (P<0.05).The Bcl-2/Bax ratio in the yak ovary was extremely significantly higher than that in cattle (P<0.01),and that in uterus and pituitary were significantly higher than cattle (P<0.05).These results indicated that Bcl-2 and Bax were relatively conservative in animal evolution.Bcl-2 and Bax played an important role in reproductive activities.The high expression level of them in yak ovary,uterus,oviduct and pituitary might be related to the anti-apoptotic effect of yak in extreme environment.

The aim of this study was to clone and analyze llsB gene of Listeria monocytogenes LM90SB2 isolated from encephalitis sick sheep in Xinjiang,and further improve the functional study of listeriolysion S.Specific primers were designed according to the full-length sequence of Listeria monocytogenes F2365 gene in GenBank (accession No.:AE017262),llsB gene of Xinjiang isolate LM90SB2 was amplified by PCR,and recycled the target gene and connected with pMD19-T vector.Positive bacteria was identified and screened for sequencing by PCR and double enzyme digestion methods.The nucleotide sequence with the correct sequencing result was analyzed,and the homology and genetic variation analysis were carried out.The results showed that llsB gene of Xinjiang isolate LM90SB2 was 876 bp in length and encoded a total of 291 amino acids.The nucleotide homology of llsB gene were 100% between LM90SB2 isolate and 10-0809,81-0592,81-0558,02-1792,NTSN and 02-1289 isolate strains,respectively,the homology with CⅡMS-PH-1 and NRRLB-57603 were both 99.9%,and the homology with J1816 and R2-502 were 44.7% to 45.0%.The molecular phylogenetic tree showed that llsB gene of LM90SB2 strain was closely related to the strain with serotype 4b,and the clustering was the same branch.Protein secondary structure prediction results showed that LM90SB2 llsB protein was a hydrophilic protein,without signal peptide and transmembrane structure.This experiment successfully cloned LM90SB2 llsB gene,and provided a comprehensive theoretical basis for the function research of LM90SB2 llsB gene.

This study was aimed to detect the expression of suppressor of cytokine signaling 7 (SOCS7) and glial fibrillary acidic protein (GFAP) genes in different tissues of Weining sheep.This experiment took male and female Weining sheep aged 6 months,1 year and 2 years as research objects,the relative expression of SOCS7 and GFAP genes in six tissues of heart,liver,spleen,lung,kidney and brain at different sex and growth stages were determined by Real-time quantitative PCR,and the mRNA level expression pattern of SOCS7 and GFAP genes were explored.The results showed that SOCS7 gene was expressed in different tissues of Weining sheep,the expression of spleen was the highest,followed by lung,kidney,heart,liver and brain,and the relative expression of SOCS7 gene in Weining sheep slightly increased with age.The relative expression of GFAP gene was the highest in brain of Weining sheep,while the rest of the tissues showed a low expression level,which showed a downward trend with age.According to the genders difference,the relative expression of SOCS7 gene in female was generally higher than male,while that of GFAP gene in male was generally higher than female,it suggested that SOCS7 gene might negatively regulate the expression of GFAP gene.

The small intestine tissue and its contents of diarrhea piglets from porcine deltacoronavirus (PDCoV) suspected case were collected for virus isolation in the study,the isolated virus were identified by typical cytopathic effect (CPE),RT-PCR,IFA and genomic sequence analysis.The results showed that a PDCoV strain named CH/GX/1468B/2017 (PDCoV 1468B) was isolated from piglets.The strain could grow and proliferate steadily and effectively in LLC-PK cells with typical cytopathic changes.The strain has been passaged for 15 generations in LLC-PK cells.The infectious titers of the viruses gradually increasing and stabilized above 10^8.10 TCID₅₀/mL.The complete genome sequence of PDCoV 1468B was 25 399 nt in length,excluding the poly(A) tail.Genomic analysis showed that PDCoV 1468B shared 97.2% to 99.4% nucleotide identity with other 23 reference PDCoV strains (GenBank),and the highest sequence identity (99.4%) was found with Vietnam/Binh21/2015 (Vietnamese strain).Genome-wide phylogenetic analysis showed that the PDCoV 1468B strain belonged to group Ⅱ and was closely related to the PDCoV strain in Southeast Asian countries which belonged to the same evolutionary branch.In summary,PDCoV 1468B strain was successfully isolated and sequenced,which would lay a foundation for further research on biological characteristics of PDCoV,such as pathogenicity and vaccine development,and provided data support for genetic evolution of PDCoV.

To obtain the modules used to assembly the cellulosomes,the total microbial genomic DNA of Tan sheep were used to amplify the scaffoldin coding gene ScaC using a pair of primers designed according to the published sequences of Ruminococcus flavefaciens ScaC gene (GenBank accession No.:JN109634.1).The scaffoldin coding gene was amplified,cloned and sequenced,and the nucleotide and amino acid sequences were analyzed.Meanwhile,the expression of the target genes in E.coli was analyzed.The results showed that two scaffolding protein coding genes were successfully cloned using a pair of primers.One of them was 867 bp in length,encoding 288 amino acids,named as ScaC2,and the other was 870 bp in length,encoding 289 amino acids,named as ScaC7.Nucleotide sequence analysis results showed that the similarity of ScaC2 and ScaC7 genes was 74.1%.The similarity of ScaC2 and ScaC7 genes with R.flavefaciens isolate AGY80P318 and DA640P037 ScaC genes were 99% respectively.Conservative amino acid sequence analysis showed that both ScaC2 and ScaC7 amino acid sequences had typical scaffold protein domain,including a type Ⅰ adhesion domain and a type Ⅰ anchorage domain.Both genes were successfully expressed in Escherichia coli BL21 (DE3) and the expression products showed a molecular weight of 33 ku by SDS-PAGE.The two genes could be used as basic materials for the reconstruction of mini-cellulosomes in the future.

The aim of this study was to investigate the prokaryotic expression and bioinformatics character of Pasteurella multocida OmpA gene.Using the genome of Pasteurella multocida HN-01 strain as a template,specific primers were designed to amplify OmpA gene.The recombinant plasmid pET-28a(+)-OmpA was constructed and transferred into E.coli BL21(DE3) competent cells,and the correct recombinant plasmid was identified and expressed by IPTG.The expressed proteins were analyzed by SDS-PAGE and Western blotting,and the OmpA gene sequence was analyzed by bioinformatics softwares.The results showed that the OmpA fragment of Pasteurella multocida was about 1 044 bp,and its coding region sequence was 89.72% homologous to HN-06 strain.After induction,the optimal induction condition of pET-28a(+)-OmpA recombinant strain was 1 mmol/L IPTG at 37℃ for 6 h,and the expressed recombinant protein was about 40 ku in the form of inclusion bodies.Western blotting results showed that about 40 ku of recombinant protein carried His-tag.Bioinformatics analysis found that the OmpA had the formula C₁₆₈₄H₂₆₁₉N₄₅₇O₅₀₅S₃ and was a basic hydrophobin.The amino acids 1 to 21 of the polypeptide chain was signal peptide regions and had various structures.In summary,OmpA might had a special structure,consistent with the structural characteristics of many outer membrane proteins.In this study,the prokaryotic expression system of Pasteurella multocida OmpA gene was constructed,and the OmpA recombinant protein was stably obtained after optimized induction conditions,which provided a theoretical basis for further exploring the pathogenic mechanism of Pasteurella.

To understand the slaughter performance and meat quality of Yuxi Black pigs,and compare these indexes with Henan indigenous Queshan Black pigs,eight Yuxi Black pigs (120.28 kg±6.96 kg) and eight Queshan Black pigs (117.80 kg±8.76 kg) under the same conditions were randomly selected to investigate the body size indexes,slaughter performance,meat quality and blood indicators according to related rules.The body size indexes (body height,body length,chest circumference and cannon circumference,etc.),slaughter performance (carcass weight,carcass length,skin rate,bone rate and slaughter rate,etc.),meat quality (meat color,marbling score,pH,intramuscular fat and crude ash,etc.),blood indicators (white blood cell count (WBC),red blood cell number (RBC),platelet number (PLT) and thrombocytocrit (PCT),etc.) and the amino acids content in muscle between Yuxi Black pigs and Queshan Black pigs were analyzed.The results showed that the body length,carcass length,skin rate and head weight of Yuxi Black pigs were significantly lower than that of Queshan Black pigs (P<0.05),whereas the backfat thickness,lean meat rate,spleen weight,heart weight and lion eye area of Yuxi Black pigs were extremely significantly or significantly higher than that of Queshan Black pigs (P<0.01;P<0.05).For the meat quality,the marbling score,pH_{45 min},pH_{24 h},and the contents of crude protein and crude ash of Yuxi Black pigs were significantly or extremely significantly lower than that of Queshan Black pigs (P<0.05;P<0.01),while the contents of moisture and phosphorus were significantly or extremely significantly higher than that of Queshan Black pigs (P<0.05;P<0.01).For the blood indicators,the PLT and PCT of Yuxi Black pigs were extremely significantly lower than that of Queshan Black pigs (P<0.01),but there was no significant difference of other blood indicators between Yuxi Black pigs and Queshan Black pigs (P>0.05).The contents of Arg,Val and His in Yuxi Black pigs were significantly or extremely significantly higher than that of Queshan Black pigs (P<0.05;P<0.01),and the content of Gly was extremely significantly lower than that of Queshan Black pigs (P<0.01).In conclusion,there was no significant difference in body size indexes,meat quality and blood indicators between Yuxi Black pigs and Queshan Black pigs,the slaughter performance of Yuxi Black pigs was slightly better than Queshan Black pigs,both of which could provide consumers with quality pork.

DGAT gene includes diacylglycerol acyltransferase 1 (DGAT1) and diacylglycerol acyltransferase 2 (DGAT2) genes,among which the former belongs to ACAT gene family and the latter belongs to MGAT gene family,respectively encoding DGAT1 and DGAT2,which are microsomal enzymes that control the synthesis of triglycerides and transmembrane proteins localized in the endoplasmic reticulum.The membrane topological structure has the ability to interact with other proteins and organelles,affects fat metabolism and lipid deposition in tissues,participates in the regulation of energy synthesis and catabolism in animal body,and affects the metabolism of triglycerides in heart and liver.Meanwhile,the polymorphism of DGAT gene affects the content of fat and lactation.Therefore,it is of great significance to understand the structure and biological function of DGAT gene for the healthy growth,development and production of livestock and poultry.In this paper,the basic structure,biological function and related mechanism of DGAT gene were briefly introduced,and some basic applications of DGAT gene in animal husbandry,such as the regulation of production in mammals,fat deposition and milk fat content,were analyzed.

The study was conducted to examine the effects of replacement of soybean meal with fermented cottonseed meal (FCM) at different levels on growth performance,slaughter performance,and serum biochemical indexes of Cobb broilers.240 one-day-old male Cobb broilers were randomly divided into four groups with six replicates per group and 10 chicks per replicate.Chicks in control group were fed basic diet without FCM,while that in experimental groups were fed experimental diets with 3%,6% and 9% FCM replacing soybean meal,respectively.The experiment was carried out in two stages:The early growth stage (1-21 d) and the late growth stage (21-42 d).At the beginning and end of each stage,the broilers were weighed and blood samples were collected,and the growth performance,slaughter performance and serum biochemical indexes of broilers at each stage were measured.The results showed that:①Compared with control group,the F/G of 1 to 21 d in experimental group significantly decreased (P<0.05),and the ADFI of 21 to 42 d in 9% FCM group was significantly decreased (P<0.05).②Compared with control group,the percentages of half-eviscerated yield,breast muscle and leg muscle at two stages were increased and subcutaneous fat thickness were significantly decreased (P<0.05).The chest and leg muscle percentages of 6% FCM group were significantly higher than those of the control group and 9% FCM group on 42 d (P<0.05).The dressing percentage of experimental group increased on 42 d and the differences between the 3% and 6% FCM groups and the control group were significant (P<0.05).③ At 21 days of age,the concentration of serum TG and GLU in experimental groups were decreased comparing with that in control group,while TP content had the converse trend,and the 6% group had the best effect.At 42 days of age,the concentration of serum Ca in experimental groups were increased.In conclusion,adding appropriate amount of FCM in diet could improve the growth performance and slaughter performance of Cobb broilers,reduce the contents of serum TG and GLU,and increase the contents of serum TP and Ca to some extent.

In this experiment,6 healthy Chuanzhong Black goats ((25±2.5) kg) were selected and rumen fistula was installed for rumen fluid collection.The ANKOM RFS in vitro gas production system was used to select the diet formula based on compound alkali storage of spent mushroom substrate.The test formula was prepared according to the nutrient requirement of NRC goats.The addition ratio of the compound alkali storage of spent mushroom substrate was 0,20%,30%,40% (CK,SMS2,SMS3,SMS4),and each level was repeated 3 times.The gas production (GP) at 0,6,12,18,24,30,36,42 and 48 h was measured,and the volatile fatty acids (VFA),microbial protein (MCP),ammonia nitrogen (NH₃-N),pH and in vitro true digestibility (IVTD) in fermentation broth at 48 h were determined.The results showed that the GP_{48 h},IVTD,MCP and TVFA of the experimental groups SMS2,SMS3 and SMS4 compared with control group,were increased by 6.87%(P<0.01),9.40%(P<0.01),4.22%(P<0.05);10.98%(P<0.01),16.11%(P<0.01),18.17%(P<0.01);4.01%(P<0.05),8.59%(P<0.05),10.08%(P<0.05) and 12.96%(P<0.05),24.93%(P<0.01),31.09%(P<0.01),respectively.The pH of four groups ranged from 6.66 to 6.70,NH₃-N ranged from 16.96 to 18.45 mg/100 mL,and A/P ranged from 3.68 to 4.33,they were all within the appropriate range.In conclusion,compound alkali storage of spent mushroom substrate could replace part of the roughage,and the recommended dosage was 30% for the fattening goat diet.

The study was designed to investigate the effects of Clostridium butyrate and Lactobacillus on immune performance,serum antioxidant indexes and intestinal function in young pigeons.384 female young pigeons about 80 days old were randomly divided into 4 groups with 6 replicates per group and 16 young pigeons per replicate.Among them,group A was fed with basal diet,and groups B,C and D were added 1×10⁸ CFU/kg Clostridium butyrate,5×10⁹ CFU/kg Lactobacillus,5×10⁹ CFU/kg Lactobacillus+1×10⁸ CFU/kg Clostridium butyrate in the basal diet,respectively.The preliminary trial period was 7 d and the trial period was 28 d.The results showed that:①Compared with group A,the spleen index and bursa of Fabricius index in groups B,C,D were increased but the difference was not significant (P>0.05).②Compared with group A,the serum CAT,SOD,T-AOC and GSH-Px of young pigeons in groups B,C,D were significantly increased (P<0.05),the content of MDA were significantly decreased (P<0.05);Compared with groups B,C,the serum CAT,T-AOC and GSH-Px of group D were significantly increased (P<0.05).③Compared with group A,the activity of trypsin in duodenum of groups B and D were significantly increased (P<0.05),and the activity of lipase of group D was significantly increased (P<0.05).④Compared with group A,the duodenal crypt depth in group D was significantly decreased (P<0.05),The ratio of villus height to crypt depth of duodenum in groups B and D,and that of ileum in group D were significantly increased (P<0.05).In conclusion,adding Clostridium butyrate and Lactobacillus to diets could improve the intestinal morphology,increase the activity of intestinal digestive enzymes and enhance the serum antioxidation ability of young pigeons,thereby enhance the immunity of young pigeons.

To better understand the genetic information and provide theoretical basis for the breeding and conservation of Zaosheng cattle,13 pairs of microsatellite primers were used to analyze the genetic diversity by PCR amplification and capillary electrophoresis in 48 Zaosheng cattle.The correlation between different genotypes at 13 microsatellite loci and seven traits (birth weight,6-month-old weight,adult weight,body height,body length,chest circumference and cannon circumference) of Zaosheng cattle were analyzed.The results showed that the average number of alleles at 13 microsatellite polymorphic loci was 9.54,the average effective allele was 4.792,the average observed heterozygosity was 0.498,the average expected heterozygosity was 0.783,and the average polymorphic information content was 0.712.The correlation analysis results showed that D12S4 and D15S10 were correlated with birth weight;D18S5 and D12S4 were correlated with 6-month-old weight;D14S31 and D11S15 were correlated with adult weight;D18S5,D15S10 and D19S2 were correlated with body height;D15S10,D14S31 and D19S2 were correlated with body length;D18S5,D12S4 and D19S2 were correlated with chest circumference;D18S5 and D19S2 were correlated with cannon circumference.This results found that 6 loci (D18S5,D12S4,D15S10,D14S31,D19S2 and D11S15) were associated with the traits of Zaosheng cattle among the selected 13 microsatellite loci.The microsatellite loci selected in this experiment had rich polymorphisms in Zaosheng cattle population,and were associated with economic traits,which could be used for genetic resources evaluation and early breeding improvement of Zaosheng cattle.

The objective of this study was to study the spatio-temporal expression of a long non-coding RNA (lncRNA) and its predicted target gene,which are highly expressed in chicken testis,and its regulation in asthenospermia Beijing-You chickens.A high-expression lncRNA (MSTRG.15568.9) was screened by the transcriptome of normal and the asthenospermia Beijing-You chickens.Its potential target gene (sperm-associated antigen 4,SPAG4) was revealed by cis prediction,and RT-qPCR was used to further expression analysis.3 normal cocks with similar body weight were randomly selected at 0,5,20,30,45,and 60 weeks of age to detect the difference in expression between MSTRG.15568.9 and SPAG4 at different ages;At 30 weeks of age,3 normal cocks were selected,and 8 tissues including testis,liver and spleen etc. collected to detect the expression of MSTRG.15568.9 and SPAG4.At 45 weeks of age,3 normal and 3 asthenospermic cocks were selected,and the difference in expression levels between MSTRG.15568.9 and SPAG4 were compared.The results showed that MSTRG.15568.9 and SPAG4 had significant spatio-temporal expression differences,and the expression trends of them were basically the same.The expression trends of MSTRG.15568.9 and SPAG4 were similar in testis tissues of different ages.The expression level of MSTRG.15568.9 at 20 weeks was significantly higher than that at 0,5,30,45 and 60 weeks (P<0.05).The expression levels at 0 and 5 weeks of age were significantly lower than those at 20,30,45 and 60 weeks (P<0.05);SPAG4 was highest at 45 weeks of age,followed by 20 weeks (P<0.05).The expression levels of MSTRG.15568.9 and SPAG4 in testis and liver were significantly higher than that in spleen and kidney (P<0.05).The expression levels of MSTRG.15568.9 and SPAG4 in testis of cocks with normal sperm motility were significantly higher than those in asthenospermia cocks (P<0.05).In conclusion,MSTRG.15568.9 and SPAG4 had obvious tissue expression specificity,and the expression of SPAG4 might be related to MSTRG.15568.9,and both could be involved in spermatogenesis and sperm motility regulation.The specific mechanisms need to be further explored.This study could provide references for the identification of functional genes related to the regulation mechanism of chicken asthenospermia.

The purpose of this study was to investigate the effects of glycine (Gly) supplement on the survival rate,nuclear development,mitochondrial distribution and cortical granules distribution in mink oocytes during vitrification and in vitro maturation at germinal vesicle (GV) stage.The experiment was divided into three groups:Control group without vitrification,vitrification group and Gly supplement (1 mmol/L) vitrification group.Mink GV oocytes were cultured for 3 h after vitrification and in vitro maturation,respectively.Immunofluorescence labeling was used to detect the differences of mitochondrial distribution in GV oocytes and the changes of cortical granules distribution in MⅡ oocytes.The results showed that there was no significant difference in the survival rate of oocyte in Gly supplement vitrification group at 3 h after vitrification compared with vitrification group (P>0.05),but it was significantly lower than control group (P<0.05).The meiotic recovery rate of oocytes in Gly supplement vitrification group was significantly higher than vitrification group (P<0.05),but there was no significant difference with control group(P>0.05).The results of immunofluorescence showed that the normal distribution rate of mitochondria in GV oocytes of Gly supplement vitrification group was significantly higher than vitrification group (P<0.05),but the normal mitochondria distribution rate in GV oocytes of Gly supplement vitrification and vitrification groups were significantly lower than control group (P<0.05).The results of cortical granules distribution showed that when mink GV oocytes matured in vitro after vitrification to MⅡ stage,the proportion of cortical granules in Gly supplement vitrification group was significantly higher than vitrification group (P<0.05),but the proportion of cortical granules in Gly supplement vitrification and vitrification grous were significantly lower than control group (P<0.05).The results showed that the Gly supplement in mink oocytes could increase the meiotic recovery rate,and reduce the loss of mitochondria and cortical granules after vitrification.

This study was aimed to establish the parentage testing system of Dezhou donkey.13 microsatellite loci were selected as markers.A total of 53 blood samples of Dezhou donkey were collected,including 16 offsping,13 candidate fathers and 24 candidate mothers samples.The genomic DNA was obtained with phenol and chloroform method.The microsatellite loci were amplified by PCR and conducted gene scanning,the genotyping result was read using Peak Scanner Software v1.0.The genetic diversity of microsatellite loci were analyzed,the parentage relationship between individuals were identified using the likelihood-based (Cervus 3.0 software)and exclusion-based methods.The results showed that the average of alleles number,observed heterozygosity (Ho),expected heterozygosity (He) and polymorphism information content (PIC) were 6.846,0.689,0.671 and 0.625.The difference between expected heterozygosity and observed heterozygosity varied from 0.002 to 0.088.Their combined exclusion probability reached 0.990.The microsatellite loci showed high polymorphic and high exclusion probabilities,which made them suitable for genetic analysis and parentage testing.Most similar parents of 16 offspring donkeys were identified using Cervus 3.0 software.The genotypes of the 16 donkeys and their most likely parents were compared to exclude unrelated individuals.The parents of 11 donkeys were identified.In this experiment,13 microsatellite loci were used as the core markers,and the likelihood-based and exclusion-based methods were used as the main analytical method for the parentage testing system of Dezhou donkey,which provided the useful information for breeding work.

This study was aimed to investigate the difference of spermatogenic epithelial cycle and the morphological changes during testicular development before and after puberty.The testicular correlation indexes at 15,30,60 and 90 d were measured,and the morphology of testicular tissue was observed to judge the puberty,and divided the spermatogenic epithelial cycle in Congjiang Xiang pigs.The results showed that the testicular index at 30 d was extremely significantly higher than that of 15 d (P<0.01).The growth rates of testicular weight,long axis and short axis were 298.05%,66.42% and 65.45%,respectively,the testicular weight growth rate was relatively stable at the two stages of 60 and 90 d.Morphological observation showed that the dissociative sperm appeared in the seminiferous tubules at 30 d of Congjiang Xiang pigs,completing the first spermatogenesis and entering the puberty phase.The seminiferous tubules area and seminiferous epithelium thickness at 30 d were extremely significantly increased relative to 15 d (P<0.01),the growth rate were 136.12% and 40.19%,respectively,it was in a stable growth state at 60 and 90 d.Statistical analysis result of the testicular cells showed that the number of setoli cells (SC) was not affected by the increase of age (P>0.05),but the number of germ cells (GC) at 30 d extremely significantly increased compared with that of 15 d (P<0.01).Correlation analysis result showed that there was a significant positive correlation between the increase of germ cell number and the increase of the seminiferous tubule area and spermatogenic epithelium thickness (r=0.994;0.96).The spermatogenic epithelium was divided into 3 and 8 stages before and after the puberty stage by the difference in the combination of germ cells.Germ cells in the first meiotic period were mainly at the first stage of meiosis,there were A and B types spermatogonia,SC,primary spermatocyte (Ps),preleptotene (PI) and leptotene (L) in the spermatogenic epithelium before and after puberty,but the round spermatids (R),elongating spermatid (E) and spermatozoa (S) were only found in the spermatogenic epithelium after puberty.This study found that Congjiang Xiang pigs reached the puberty when it was 30 days old,the testicular development was dominated by the rapid increase in the area of germ cells and seminiferous tubules.This results had important guiding significance for excavating early-maturing traits,breeding pigs,development and utilization in Congjiang Xiang pigs.

Fat is an important energy storage material in animals,and is closely related to important economic traits such as lean percent.The development of fat is a complex and accurate process which is regulated by a variety of lipogenesis-related genes,transcriptional regulators and epigenetic factors.Long non-coding RNAs (lncRNAs) is a type of non-coding RNAs with a transcription length greater than 200 nt,which can regulate the expression of target genes through many pathways such as in transcription level,post-transcriptional level and epigenetic modification level,and then regulate life activities of animals.In recent years,the research on lncRNAs has gradually increased,its functional scope covers almost all aspects of life activities,and some lncRNAs have been shown to play important regulatory roles in the process of fat development.For example,brown fat lncRNA 1 (Blnc1) was shown to promote brown and beige adipocyte differentiation through a ribonucleoprotein complex that acts through early B-cell factor 2 (Ebf2) to enhance expression of thermogenic genes such as uncoupling protein 1 (Ucp1),and lnc-BATE1 is required for BAT adipogenesis and maintenance through binding with the heterogeneous nuclear ribonucleoprotein U (hnRNPU).In addition,lncRNA SRA can promote preadipocytes to adipocytes differentiation and further regulate the function of fat through binding to peroxisome proliferator-activated receptor gamma (PPARγ) and enhance PPARγ activity and many other pathways.The authors reviews the basic characteristics,action mechanism and research methods of lncRNAs,as well as the research results of lncRNAs related to fat development at home and abroad,in order to provide a reference for further exploring the regulation mechanism of lncRNAs on fat development.

In order to explore the changes of related genes during adipocyte differentiation of Small-tail Han sheep,white inguinal adipose tissuees of two-month-old Small-tail Han sheep were collected and preadipocytes were isolated by enzymatic digestion in vitro.After the cultured preadipocytes were covered with cell plates,the cells were induced to differentiate into mature adipocytes by induction solution Ⅰ and induction solution Ⅱ,respectively.Oil red O staining was used to verify adipocytes and detect lipid droplets.Total RNA was extracted from 70% and 90% of the proliferative cells,48 h of induction Ⅰ,48 h of induction Ⅱ and 48 h of complete culture medium (2,4,6,8,10 d,respectively),and was retranscribed into DNA.Real-time quantitative PCR (RT-qPCR) was used to detect genes PPARγ,C/EBPα,LPL,SREBP1,KLF5,KLF6,FABP4,STAT5,ACSS2,IGF1,ADD1,FOXO1,ACACA,DGAT1 and CPT1A that had been shown to be involved in the differentiation of human and mouse preadipocytes to explore their expression in the differentiation of Small-tail Han sheep.The results showed that the preadipocytes were successfully separated and induced to become mature adipocytes with obvious lipid droplets inside the cells;RT-qPCR assay showed that the expressions of above genes had significant fluctuations in the cell differentiation stage,and the peak time was different;The peak expression of C/EBPα and FOXO1 appeared on the 6th day,which may play an important role in the early stage of cell differentiation.PPARγ,LPL,SREBP1,KLF5,KLF6,FABP4,STAT5,ADD1 and ACSS2 gene peaks appeared on the 8th day,but the expression multiples and trends were different;The expression of ACACA gene fluctuated up and down.The peaks of IGF1 and DGAT1 appeared on the 10th day.The expression of CPT1A was declining;The expression multiple of FABP4 gene was significantly higher than that of other genes.This study completely detected the expression regulation of key genes in the differentiation process of Small-tail Han sheep preadipocytes.The results could provide theoretical reference for exploring the molecular mechanism of Small-tail Han sheep adipose differentiation,discovering new key genes involved in adipose differentiation and improving the intermuscular fat content of Small-tail Han sheep.

This study was aimed to investigate the expression of SH2B1 gene in the different tissues and backfat at various growth stages of pigs,and predict the effect of miR-276-3p which regulated SH2B1 gene on the expression of backfat in pigs.Real-time quantitative PCR was used to detect the relative expression of SH2B1 gene of six tissues in pigs,and in backfat of pigs at 30,60,90,120 and 180 d.The target regulation miRNA of SH2B1 gene was predicted and the regulation of SH2B1 gene by miR-276-3p was detected by Real-time quantitative PCR.The results showed that SH2B1 gene was expressed in all six tissues,with the highest expression in fat and the lowest expression in muscle.The expression of SH2B1 gene was expressed at various stages of growth and development in pigs.The expression level was low at the early stage (30 and 60 d) and high at the middle and late stages (90,120 and 180 d).The expression of SH2B1 gene was significantly higher at the middle and late stages than that at the early stage (P<0.05).The relative expression of miRNA-276-3p and SH2B1 gene in backfat tissue of pigs that in high or low backfat thickness group (HBFT or LBFT) was detected by Real-time quantitative PCR.The results showed that miRNA-276-3p and SH2B1 gene were differentially expressed in backfat tissue of pigs.The expression of miRNA-276-3p in high backfat thickness group was significantly lower than that in low backfat thickness group (P<0.05),while the expression of SH2B1 gene in high backfat thickness group was significantly higher than that in low backfat thickness group (P<0.05).miRNA-276-3p negatively regulated SH2B1 gene and affected the fat deposition on the back of pigs.This results provided a reference for further studying the molecular mechanism of fat deposition and backfat thickness difference in pigs.

The aim of this study was to initially explore the proliferation of Japanese encephalitis virus (JEV) infected with PK15 cells,screen the lncRNAs associated with viral infections,and predict its subcellular localization and target genes.The expression of viral structural protein E was detected by immunofluorescence,the proliferation of virus in PK15 cells was detected by TCID₅₀ method,and Real-time PCR was used to detect the expression level of lncRNA after viral infection.Subcellular localization of lncRNA was carried out in the NONCODE database,target gene prediction and signal pathway analysis were performed by starBase,NONCODE,KEGG and other databases.The results showed that after infection of PK15 cells by JEV,the viral titer increased exponentially from 24 to 36 h,and the virus titer reached 10^-5.75 TCID₅₀/mL at 36 h after infection.There was no significant change in the expression of lncRNA A,B and C in PK15 cells after 12 h of infection (P>0.05),and the expression level of lncRNA D was extremely significantly decreased (P<0.01).The expression levels of lncRNA A,B and C increased extremely significantly after 24,36 and 48 h of infection (P<0.01),and the expression level of lncRNA D extremely significantly decreased (P<0.01).lncRNA A was mainly localized in the cytosol,lncRNA B was distributed in the nucleus and cytoplasm,lncRNA C was mainly located in the cytoplasm and might also be distributed in the nucleus,lncRNA D might be widely distributed in the cell.Through target gene prediction and signal pathway analysis,the target genes of lncRNA A,B and C were mainly OAS1,OAS2,OASL and COX1,etc.The target genes of lncRNA D were mainly DST,ND1,ND2,ND4 and so on.Signal pathway analysis revealed that lncRNA might participate in the proliferation process after viral infection through signaling pathways such as TNF,NF-κB and TLR.This study laid a foundation for further exploration of the effect of host cell lncRNA on viral proliferation.

The aim of this study was to reveal the growth and development law of the slow-feathering line of Guizhou Yellow chicken.100 chickens (50 males and 50 females) were selected randomly,and were weighed at the end of 0,2,4,6,8,10,12 and 14 weeks old.Three nonlinear growth curve models of Bertalanfy,Logistic and Gomperta were used in this experiment,and the body weight changes of Yellow chicken slow-feathering line at 0-14 weeks were analyzed by fitting.The results showed that the three models could all well fit the growth curve of Yellow chicken,and the R² were all above 0.99.Logistic model fitted the growth curve of both cock and hen best,and the fitting results were nearest to the measured values.The inflexion age was 10.42 weeks old,the inflexion weight was 1 550.81 g in the male,and the inflexion age was 9.14 weeks old,the inflexion weight was 976.67 g in the female.The results preliminarily revealed the growth and development regularity of slow-feathering line of Guizhou Yellow chickens,which could provide a scientific basis for breeding,production and industrialization development of slow-feathering line of Guizhou Yellow chickens.

In order to understand the complete gene sequence,structural characteristics and genetic variation of fowl adenovirus genotype 4 (FAdV-4) W strain,the liver and kidney of infected chicken were identified by PCR.The isolation of SPF chicken embryo and electron microscope observation showed that the virus was FAdV-4 and TCID₅₀ was 10^-7.2/0.1 mL.The virus was about 70 nm in diameter and without envelope under electron microscope.Chorioallantoic membrane inoculation of 10-day-old SPF chicken embryos could significantly inhibit the development of chicken embryos and produce dwarf embryos,and the death rate was 100% within 10 days after inoculation with chorioallantoic membrane.The complete genome of W strain was amplified and sequenced.The virus was 43 591 bp,which had 60 open reading frames.The nucleotide sequence homology was 98.4% to 100% between W strain and referenced FAdV-4 strains published in GenBank.The homology was 98.4% with standard non-pathogenic virus strain ON1 (accession No.:GU188428.1).The ORF19 (lipase gene) and ORF27 were not found.It was found that W strain was closely related to the domestic isolates by analyzing the Hexon,Fiber-1 and Fiber-2 genes of the main structural proteins in recent years.It had a great difference with the ON1 strain and other foreign strains.The results showed that the strain had high pathogenicity and was different from foreign isolates,which provided the basis data for further analysis of the virulence enhancement mechanism of FAdV-4.

In order to control the upstream and downstream processes of H9N2 subtype avian influenza virus (AIV) vaccine,the methods for purification of H9N2 subtype AIV and quantification of viral HA protein were established by combining sucrose density gradient centrifugation and SDS-PAGE gray analysis.Firstly,the harvested viruses were clarified,concentrated and purified by differential centrifugation,PEG6000 precipitation and sucrose density gradient centrifugation,respectively.Subsequently,the virus centrifugal zone formed after centrifugation was collected by HPLC,and identified by SDS-PAGE and Western blotting.The linearity and repeatability of the collection method were also investigated.On this basis,the clarification process of viruses was optimized to improve the virus recovery.Finally,the degree of co-migration of viral protein bands of purified H9N2 subtype AIV isolated by reduced SDS-PAGE was observed and the optimal condition for PNGase F deglycosylation treatment was decided.The gray of four main viral protein bands (NP,HA1,M1 and HA2) in SDS-PAGE was analyzed by Image J software to determine the content of hemagglutinin.The results showed that the protein concentration of the virus centrifugal zone collected by HPLC had a good linear relationship with the volume of virus supernatant in the range of 8 to 32 mL (R²=0.994).The intra-batch and inter-batch coefficients of variation were 1.29% and 4.11% respectively,which indicated a good repeatability of this collection method.After the optimization of clarification process,the final recovery of virus was 79.55%.When the protein concentration of H9N2 subtype AIV was 1 000 μg/mL,the SDS-PAGE showed clear and well-separated viral bands after deglycosylation treatment with PNGase F.The calculated total HA accounted for 46.18% of total viral proteins by SDS-PAGE gray analysis.This study established methods for purification of H9N2 subtype AIV and quantification of viral HA protein,providing a simple and accurate detection method for the development and production of H9N2 vaccine in the future.

To construct and screen the recombinant modified vaccinia virus Ankara (MVA) strain expressing green fluorescent protein (GFP),primers were designed and synthesized.GFP gene was amplified by PCR and inserted into the ORF086-087 site (70303-70304 bp) of MVA based on the principle of homologous recombination.Fluorescence microscope was used to select single plaque with GFP as the reporter gene,and the recombinant strain was identified by fluorescence technique,PCR and Western blotting.The results showed that a large number of single plaques expressing GFP could be observed under fluorescence microscope,GFP gene successfully intergrated into recombinant strain MVA-GFP genome.Western blotting result showed that GFP was successfully expressed in infected cell.In this study,the recombinant strain MVA-GFP expressing GFP was constructed by genetic engineering technology,which provided materials for further screening unmarked recombinant strain and vaccines by inserting foreign target genes into GFP site.

The aim of the experiment was to construct a recombinant adenovirus expressing Mycoplasma suis ENO gene and analyze its immune effect.Recombinant cloning plasmid pMD-19T-ENO and adenovirus shuttle vector AdV4-GFP were double digested to construct recombinant adenovirus shuttle plasmid AdV4-M/ENO;The recombinant adenovirus shuttle plasmid AdV4-M/ENO,which was linearized by PacⅠ,was transfected into 293 cells to obtain recombinant adenovirus Ad4-M/ENO.Expression of Mycoplasma suis ENO gene in 293 cells was identified by PCR and indirect immunofluorescence assay (IFTA),293 cells were cultured and the titer of recombinant adenovirus was determined.30 BALB/c mice were divided into three groups:Recombinant adenovirus Ad4-M/ENO group,AdV4-GFP empty vector control group and PBS control group,respectively,vaccinated separately.ELISA was used to detect IgG,IgG₁,IgG_2a antibody levels and IFN-γ and IL-4 cytokine levels in serum,and the levels of CD4⁺ and CD8⁺ in the spleen of mice were detected 2 weeks after the third immunization.The results showed that the recombinant adenoviral shuttle plasmid AdV4-M/ENO gene fragment size was 1 182 bp;The recombinant adenovirus Ad4-M/ENO was successfully packaged and expressed in 293 cells with a titer of 1×10⁹ PFU/mL.The serum levels of IgG,IgG₁,IgG_2a,IFN-γ,IL-4 and lymphocyte subsets CD4⁺ and CD8⁺ in BALB/c mice immunized with recombinant adenovirus Ad4-M/ENO were significantly or extremely significantly higher than AdV4-GFP empty vector control group and PBS control group (P<0.05;P<0.01).The results showed that the recombinant adenovirus expressing Mycoplasma suis ENO gene was successfully constructed in this experiment,and the recombinant adenovirus could induce specific humoral and cellular immune responses in mice.

Porcine epidemic diarrhea virus (PEDV) is a globally distributed alpha coronavirus,which can cause epidemic diarrhea in piglets.Porcine epidemic diarrhea outbreak will bring huge economic losses to the breeding industry.Type Ⅰ interferon (IFN-Ⅰ) is a key mediator of innate antiviral responses during viral infection.Most coronaviruses produce strategies to limit IFN response by limiting IFN production and activation of IFN responses.However,the molecular mechanism by which PEDV antagonizes the antiviral effect of IFN is not fully understood.After the pig is infected with PEDV,the body's innate immunity is not effective against PEDV.PEDV escapes host innate immunity by limiting or blocking the function of IFN and hiding its pathogen-associated molecular patterns (PAMP).In this process,structural and non-structural proteins of PEDV and some proteases play a key role.The nucleocapsid (N) protein can inhibit the production of IFN-Ⅰ and help the virus escape the antiviral natural immunity of the body.Papain-like protease blocks the natural immune signaling pathway by deubiquitinating enzyme activity.PEDV also evades host innate immunity by blocking the production of IFN-Ⅰ induced by double-stranded RNA (dsRNA).These studies provide a theoretical basis for understanding the role of IFN in the pathogenesis of PEDV and the mechanism by which PEDV escapes IFN against viruses,and help to understand the relationship between the virus and the host's innate immunity,it also provides a theoretical basis for the prevention and treatment of PEDV and the development of PEDV vaccine.

To know the prevalence of gastrointestinal parasites in Hu sheep from stall-feeding farms,a total of 553 fresh fecal samples from part stall-feeding farms in Henan were collected and examined the presence of eggs or oocysts using centrifugation method,saturated salt solution floatation technique,Sheather's sugar flotation technique,Lugol's iodine-solution staining method and McMaster's method.The results showed that the overall infection rate of gastrointestinal parasites was as high as 97.47%,and 75.23% of the samples were mixed infection,samples mixed infections with up to 5 species of parasites.A total of 7 species of parasites,Coccidia,Cryptosporidium,Giardia,Amoeba,Whipworm,Nematode and Moniezia were found to be 90.42%,0.90%,4.88%,65.64%,12.48%,42.13% and 4.88%,respectively.The highest infection intensity was Coccidia,and its highest number of oocysts per gram(OPG) of feces was 652 000,followed by Nematode,and the highest number of eggs per gram (EPG) of feces was 7 000.There was no significant age and gender difference in infection rate of gastrointestinal parasites in Hu sheep (P>0.05).The seasonal epidemic dynamics showed that the parasitic infection rates in spring,summer and autumn were different from those in winter.In conclusion,parasitic infections in the digestive tract of Hu sheep were more common,and effective comprehensive prevention and control measures should be taken to ensure the healthy development of the flock.

In order to provide a target animal model of exudative dermatitis of piglet for exploring the pathogenesis and therapeutic reagents,in this study,the toxin type of isolated Staphylococcus hyicus 437-2 was identified by PCR and BLAST sequence alignment,its drug resistance was detected by disk diffusion method,and the clinical pathogenicity,minimum pathogenic concentration,pathogenesis and histopathological changes caused by the strain were analyzed by artificially infecting piglets with different concentrations of bacterial liquid.The results showed that the strain carried the ExhD virulence gene,which showed 99% gene sequence homology with the isolated Germany strain (GenBank accession No.:AM94662.1) and Russian isolate (GenBank accession No.:AM950188.1).The drug resistant assay displayed S.hyicus 437-2 was a multi-drug resistant strain,which showed resistance to penicillin,sulfamethoxazole,streptomycin,azithromycin,erythromycin,tetracycline,bacitracin and norfloxacin.And the pathogenicity test of S.hyicus 437-2 on piglets showed that the low dose group displayed dermatitis symptoms only at the injection site throughout the experimental period,while significant exudative dermatitis symptoms appeared in the middle and high dose groups after subcutaneous injection for 2 d,which showed detailedly as oily skin behind the ear,rough fur,a large piece of suede and yellow liquid exuded,and thick dark brown skin.The results of pathological sections showed that organs in the low dose group were not damaged,while the skin tissue displayed excessive keratinization on the ear skin,inflammatory cell infiltration,and no cell separation.Meanwhile,the piglets in the middle and high dose groups all showed a certain degree of damage,the cell epidermal layer of the skin appeared obvious cell separation.This results indicated that this strain had strong pathogenicity and could successfully replicate exudative dermatitis on piglets.In addition,this result further indicated that the minimum bacterium concentration required for the establishment of exudative dermatitis model was 1×10⁹ CFU/mL.

As one of the most common diseases in large scale pig farms,piglet diarrhea has brought serious problems and enormous losses to swine industry worldwide.Enterotoxigenic Escherichia coli (ETEC) is the main pathogen of piglet diarrhea,adhesin and enterotoxin are main virulence factors of ETEC.ETEC colonizes the small intestinal epithelial cells via adhesin,then enterotoxin is secreted and causes a large amount of water and electrolytes filling the enteric cavity,resulting in piglets diarrhea.At present,vaccination is the most effective way of controlling ETEC diarrhea.However,the commercial ETEC vaccines can not provide efficient protection against ETEC infection and have the obvious regional restriction.Therefore,it is of great significance for developing a safe,efficient and broad-spectrum ETEC vaccine.Now,many new experimental ETEC vaccines have been reported which mainly focused on the adhesin and enterotoxin.New type of ETEC vaccines with different advantages and good immune protective effect (such as subunit vaccine,ghost vaccine,plants vaccine and outer membrane vesicles vaccines) have been also developed by domestic and foreign researchers.In this paper,the research progress of domestic and foreign scholars in the field of ETEC vaccine were summarized,and the advantages and disadvantages,experimental and applied studies of various swine ETEC vaccines were summarized to provide references for future studies on ETEC vaccine.

Endometritis is a widespread postpartum reproductive disease in dairy cows,it can lead to prolongation of calving interval,decrease of milk production,increase of elimination rate,abandonment of milk source during treatment,increase of management and treatment cost,and is also one of the important reasons leading to infertility of dairy cows.At present,there are many diagnostic techniques and treatment methods for preventing and treating cow endometritis at home and abroad,including colposcopy,Metricheck device evaluation,uterine cytology examination,antibiotic therapy,traditional Chinese medicine therapy,integrated traditional Chinese and Western medicine therapy,hormone therapy,ozone new therapy and so on.Colposcopy is a visual examination method,which can directly evaluate the vaginal endocrine characteristics of dairy cows to determine the severity of endometritis.The Metricheck device is designed to evaluate the endometritis and reproductive performance of postpartum dairy cows according to the content and characteristics of vaginal endocrine products.In cytological examination of uterus,the ratio of endometrial cells to neutrophils (PMN) was determined by uterine cell brushing,lavage or biopsy,and the optimal threshold of PMN was set for diagnosis of endometritis in dairy cows at different postpartum stages.Antibiotic therapy can treat cow endometritis through intrauterine infusion,subcutaneous injection and intravenous injection of antibiotics,the treatment cost is low and the effect is quick and it is the most commonly used treatment for cow clinical endometritis at present.Chinese medicine therapy screens different components of Chinese medicine compound preparation,through regulating the body immune system of dairy cows,improves blood circulation in uterus,inhibits the reproduction of pathogenic bacteria,and promotes the recovery of normal physiological function of uterus.The combination of traditional Chinese medicine and Western medicine has the advantages of dialectical treatment of traditional Chinese medicine and the characteristics of quick effect of Western medicine,it has wide application value in clinical treatment of dairy cow endometritis.In hormone therapy,prostaglandin can induce luteal lysis,eliminate progesterone production and immunosuppression,stimulate uterine involution,and reduce the risk of long-term uterine infection and inflammation in postpartum dairy cows.Ozone new therapy is to make ozone into foam preparations,oil products and other products through intrauterine perfusion to kill pathogens.These diagnostic and therapeutic techniques have achieved different results in the treatment of endometritis in dairy cows,and have a good guiding role in evaluating reproductive performance and production performance of postpartum dairy cows.This article introduced the research progress of the application of these diagnostic and therapeutic techniques in dairy cow endometritis,so as to provide reference for the prevention and treatment of the disease.

The aim of this experiment was to evaluate the anti-inflammatory effect and immune effect of Spirulina.An inflammation model of mice was constructed by xylene induced mice ear swelling,and the effect of Spirulina on anti-inflammatory was investigated by using dexamethasone in positive control group and mice ear swelling as observation indexes.An immune-suppression model of mice was set up with cyclophosphamide,and the immune effect of Spirulina was investigated by comparing the organ indexes,levels of IL-2,IL-6,IL-1β,TNF-α and IFN-γ,and spleen and thymus histopathology in both normal and immune-suppressed mice treated with different doses of Spirulina.The results showed that:In the anti-inflammatory experiment,the ear swelling inhibition rate of mice treated with 0.3% spirulina was extremely significantly higher than that of dexamethasone group and other Spirulina treatmens (P<0.01),and viscera index of all Spirulina groups had no significant difference (P>0.05);In the immune function experiment,compared with blank control group,the spleen index and thymus index were extremely significantly decreased (P<0.01),and the liver index was extremely significantly increased (P<0.01) in cyclophosphamide positive control group,and the thymus indexes of Spirulina treated groups had no significant difference (P>0.05).Serum IL-2,IL-6,TNF-α and IFN-γ levels had no significant difference among all the groups (P>0.05).At the same time,the decrease of thymus corpuscles and the degeneration and necrosis of lymphocytes and reticular cells were observed in the positive control group by histopathological observation,while the structure of spleen corpuscles and thymus corpuscles in Spirulina treatments were more clear and complete,and the lymphocytes were increased.In conclusion,Spirulina could reduce the immunosuppression of dexamethasone and cyclophosphamide on mice,and repair the damage of spleen and thymus in mice.Spirulina played a certain role in anti-inflammatory and alleviating immunosuppression.

The aim of the experiment was to construct the expression system of canine interferon alpha 6 (CaIFN-α6) in Pichia pastoris,optimize and screen the expression system in order to obtain recombinant canine interferon alpha 6 with high activity.According to the sequence of CaIFN-α6 gene and the codon preference of Pichia pastoris,the whole gene sequence of CaIFN-α6 was optimized and synthesized.The recombinant expression plasmid of pPICZαA-CaIFN-α6 was constructed using Xho Ⅰ and Not Ⅰ and linked to the vector pPICZαA.The recombinant expression plasmid was transformed into E.coli DH5α competent cells.The plasmid pPICZαA-CaIFN-α6 was extracted and linearized.The recombinant strain was prepared by electroporation into yeast competent cell X33.The recombinant CaIFN-α6 was purified by methanol induction,supernatant collection and ultrafiltration concentration.The concentration of purified CaIFN-α6 protein was 1.5 mg/mL by BCA.Western blotting analysis showed that CaIFN-α6 protein had good reactivity.SDS-PAGE showed that the purity of CaIFN-α6 protein was above 95%.The titer of CaIFN-α6 protein was 2.37×10⁷ IU/mL by MDCK/VSV detection and the specific activity was 1.58×10⁷ IU/mg.The results showed that CaIFN-α6 was successfully expressed in Pichia pastoris pPICZαA expression vector system with high biological activity,which provided a good support for the clinical prevention and treatment of canine viral disease in the later stage.

In order to investigate the hidden infection of Haemophilus parasuis (Hps) in slaughtered pigs,this study collected 80 diseased lungs from a large-scale pig slaughterhouse in Shaanxi province.The positive lungs were aseptically sampled,and the suspected Hps was obtained by streaking through the plate.The morphology of the isolates,analysis of the culture characteristics,observation of the satellite phenomenon,biochemical test and sequence analysis of the isolates nucleic acid fragments were carried out,and the Hps isolates were purified.The sensitivity of Hps isolates to commonly used drugs was analyzed by drug sensitive paper method.The results showed that 39 Hps positive samples were detected from 80 lungs,and the positive rate was 48.75% (39/80).Three suspected Hps were isolated from 39 Hps-positive lungs.The isolate was Gram-negative,short rod under the microscope,grew well on TSA plates supplemented with fetal bovine serum and NAD,exhibited a typical "satellite phenomenon" growth around S.aureus,could ferment glucose,sucrose,fructose,galactose,maltose and arabinose without fermenting mannitol.The PCR amplification sequence was more than 98% homologous to Hps.The results of drug susceptibility test showed that the three isolates were highly sensitive to antibacterial drugs such as ciprofloxacin,enrofloxacin and florfenicol,and were not sensitive to antibacterial drugs such as penicillin,lincomycin and trimethoprim.The Hps isolates obtained in this study provided the basic materials for vaccine development,and provided a reference for the selection of sensitive drugs to prevent and control Hps infection in pig farms.

In order to study the effects of ultrafine powder of Radix Isatidis on immune regulation in rats,cyclophosphamide was used to construct the model of immunodeficiency.80 SD rats were randomly divided into 4 groups:Model group,ordinary powder group,ultrafine powder group and blank control group with 20 rats in each group.40 mg/kg cyclophosphamide was injected intraperitoneally for 5 d.From 6th day,The blank control group and model group were given normal saline,and the other groups were given 5 g/kg ordinary powder or ultrafine powder.At 7th and 14th day,the blood samples and liver,spleen and thymus tissues were collected.The white blood cell count (WBC),organ indexes of spleen and thymus,C3b-R,ICR,phagocytosis indexes were detected.Real-time PCR was used to detect the transcription levels of TNF-α and IL-2 mRNA.The expression of TNF-ɑ and IL-2 protein were detected by Western blotting.The results showed that the WBC,body weight,thymus and spleen indexes in model group were significantly lower than those in the blank control group (P<0.05).On the 14th day,the percentage of macrophage phagocytosis of rat in ultrafine powder group reached 54.7%,the index of macrophage phagocytosis was 0.58.The C3b-R of rat at 14th day in ultrafine powder group was 9.44%,significantly higher than that of the ordinary power group (8.97%)(P<0.05).And the ICR of ultrafine powder group was 6.09%,lower than that of the ordinary power group (6.33%)(P>0.05).Compared with the ordinary powder group,the ultrafine powder of Radix Isatidis could increase the expression of TNF-ɑ and IL-2 in liver on the 14th day.In conlusion,ordinary powder and ultrafine powder of Radix Isatidis could both enhance the nonspecific immune function of rats,and ultrafine powder was superior to the ordinary powder.

In order to find out the cause of death of carp in a carp farm in Guizhou,this experiment carried out bacterial isolation and culture,morphological observation of bacteria,physiological and biochemical identification of bacteria,16S rRNA gene sequencing,bacterial drug sensitivity test,drug resistance gene detection,animal infection test,virulence gene detection and so on.The results showed that the bacteria isolated from the diseased tissues of dead carp were round,gray-white colony with smooth surface and were Gram-negative brevibacterium.Biochemical identification showed that the isolated bacteria were motile,could ferment glucose to produce acid and gas,and were positive for arginine dihydrolase,lysine decarboxylase,M-R and V-P tests.The bacterial 16S rRNA gene universal primers were used for PCR amplification,and obtained a 1 421 bp fragment.Sequencing results showed that the isolate had 75.3% to 100.0% homology with 16 strains of Aeromonas hydrophila.Drug susceptibility test results showed that the isolate was highly susceptible to florfenicol,ceftriaxone,tobramycin,ofloxacin and enrofloxacin,and was resistant to compound sulfamethoxazole,sulfamethoxazole and carbenicillin.PCR result showed that the isolate carried Sul1,Sul2 and Intl1 resistance genes,which were consistent with the drug sensitivity phenotype.Artificial infection test showed that the isolate was pathogenic.Virulence gene test results showed that the isolate carried aer,hly and act 3 virulence genes.In conclusion,a sulfonamide-resistant strain of Aeromonas hydrophila from carp was isolated in this study,which provided a scientific basis for disease control and rational drugs use for carp in this farm.

The aim of this experiment was to study the hypoglycemic effect of different proportions of polysaccharide from Soirulina platensis (PSP) and Ginkgo biloba extract (GBE).SPF Kunming male mice were selected for the experiment.Diabetic model was made by intraperitoneal injection of 0.2 mL at a concentration of 200 mg/(kg·d) for 5 consecutive days.The model was successfully constructed when the fasting blood glucose was greater than 11.1 mmol/L by tail-cut blood sampling.Then mice were randomly divided into blank control group,model group,positive control group,group P (PSP),group F (Ginkgo biloba flavone),group L(Ginkgo biloba lactones) and composite treatment groups that were PSP and GBE mixture of 1:1 (CP₁ group),2:1 (CP₂ group) and 1:2 (CP₃ group),10 mice in each group.Each mice in the control and CY groups was given 2.5 mg/(kg·d) glibenclamide,and mice in the other groups were given 200 mg/(kg·d) corresponding drugs once a day for 30 d continuously.The blood glucose value,body weight,spleen index,thymus index,liver glycogen and other indicators were measured.The results showed that compared with the model group,the body weight of mice in the drug groups was extremely significantly increased (P<0.01).The efficacy of compound drug groups was higher than that of single drug groups.Spleen and thymus indexes and liver glycogen content were extremely significantly increased (P<0.01),while the blood glucose level was extremely significantly decreased (P<0.01).Among them,the CP₂ group showed the greatest drop in blood glucose,and the hypoglycemic rate was 52.14% and the increase of hepatic glycogen was 61.88%.In conclusion,polysaccharide from Soirulina platensis and Ginkgo biloba effective components had synergistic effect on hyperglycemic mice induced by alloxan and had obvious hypoglycemic effect.

Antibacterial drugs have played an important role in the prevention and treatment of animal diseases.However,non-standard and over-dose drug use and even abuse have made the antibacterial drug residues in animal foods increasingly prominent,causing widespread concern.The detection of antimicrobial drug residues is an important part for animal foods safety.According to the purpose and needs of the detection,it can be divided into on-site testing and laboratory testing.Colloidal gold immunechromatography is the most widely used on-site detection method.In addition,there are many laboratory detection methods,which can be divided into qualitative detection methods and quantitative detection methods for antibacterial drug residues.The former mainly includes 2,3,5-triphenyltetrazolium chloride method (TTC method), paper method (PD method) and activity detection method,etc.The latter mainly includes high performance liquid chromatography,liquid chromatography-mass spectrometry,ultra-performance liquid chromatography-mass spectrometry,capillary electrophoresis,biosensor and immunoassay,etc.These techbiques can meet the needs of different testing purposes and experimental conditions.In this article,the authors summarized the on-site detection and laboratory detection techniques of antibacterial residues in meat,eggs and milk which could lay a solid foundation to further study the rapid,sensitive,high-throughput and low-cost detection of antimicrobial drug residues in food.