This study was aimed to analyze the expression profiles of long non-coding RNA (lncRNAs) in bovine muscle tissue from different periods and screen out lncRNAs which might involve in muscle development.The bovine muscle tissue RNA samples from 3-month-old embryos,6-month-old embryos and 9-month-old calf were collected for high-throughput sequencing.The identified lncRNAs were analyzed and filtered with bioinformatics methods,Veen analysis was performed on lncRNAs target gene and three periods differential mRNAs,Go and KEGG enrichment analysis were performed on Veen results to select lncRNAs and target mRNAs which related to muscle development.The results showed that 212 851 novel unannotated lncRNAs were identified by high-throughput sequencing,of which 9 913 were differentially expressed in different periods.These lncRNAs distributed on each chromosome and belonged to four types,which included intergenic,sense strand,antisense strand and bidirectional,and the sense strand lncRNAs had the largest number and the widest distribution range on each chromosome.The structural features analysis revealed that these lncRNAs had the characteristics of short open reading frame,low expression level,weak coding ability,wide distribution and the huge numbers.55 muscle myogenesis related differentially expressed lncRNAs among 3-month-old embryos,6-month-old embryos and 9-month-old calf were screened out by GO and KEGG enrichment analysis,and 38 candidate target mRNAs were also filtered.This study presented the expression pattern of lncRNAs in bovine muscle tissue from three periods by predicting the interaction between lncRNAs and mRNA,and greatly reduced the research range of bovine muscle development related lncRNAs.The study also provided a critical target for revealing the function of bovine muscle development.

This study was aimed to investigate the molecular sequences and prokaryotic expression product characteristics of interferon-beta (IFN-β) gene coding region in Congjiang Xiang pig.Congjiang Xiang pig was selected as the research object,the total RNA was extracted from liver tissue of Congjiang Xiang pig,and the mRNA was reversely transcribed into cDNA.The encoding region of IFN-β gene was amplified by specific primers,ligated into pET-28a vector,obtained the recombinant plasmid pET28a-CJpoIFN-β,and the CDS sequence of IFN-β gene was analyzed using bioinformatics softwares.The pET28a-CJpoIFN-β was transformed into Escherichia coli BL21(DE3) competent cells.After induction by IPTG,the product of prokaryotic expression was analyzed by SDS-PAGE and Western blotting.The results showed that the CDS sequence of IFN-β gene in Congjiang Xiang pig (CJpoIFN-β) was 561 bp,which encoded 186 amino acids;CJpoIFN-β protein was secretory protein,and the first 21 amino acid residues was predicted to be the signal peptide.The secondary structure of CJpoIFN-β protein was mainly alpha helix (77.42%) and random coli (17.74%).The nucleotide sequence alignment results showed that the IFN-β gene homology was 99.5% to 100.0% between Congjing Xiang pig and others porcine,the lowest homology was 35.2% between Congjing Xiang pig and poultry.Phylogenetic tree result showed that CJpoIFN-β was closely related to that of Meishan pig and Bama pig,the amino acid homology was 100.0% in Congjiang Xiang pig and Bama pig,Meishan pig;And the homology was 99.5% between Congjiang Xiang pig and Guizhou Baixiang pig,there were three amino acid differences in E43Q,K73R and C161R.Western blotting results showed that His-tag recombinant protein could be recognized by His monoclonal antibody,the band was about 24 ku.The results of this experiment provided a reference for further study on the biological activity of IFN-β gene and speeding up the effective utilization of the resources of Congjiang Xiang pig.

This study had investigated the localization and expression of fibroblast growth factor 21 (FGF21) with its receptors FGFR1 and FGFR2 in the formation phase of embryonic mice hair follicles.E13-E18 embryonic mice back skin were used to determine the mRNA and protein relative expression levels of FGF21,FGFR1 and FGFR2 by immunohistochemistry,Real-time PCR and Western blotting analysis.The results showed that:①FGF21 expressed in epidermis,basal layer and connective tissue at E13;Expressed in epidermis and placode at E14;Slightly expressed in hair bud at E15;Expressed in hair peg at E16 and E17;Mainly expressed in immature hair follicle at E18.FGFR1 and FGFR2 expressed in epidermis,basal layer,placode,hair bud,hair peg,immature hair follicle and connective tissue at E13-E18.②The results of Real-time PCR and Western blotting showed that the relative expression levels of FGF21 mRNA and protein were higher at E14 than that of any other stages;The relative expression levels of FGFR1 mRNA and protein were higher at E16-E17;And the relative expression of FGFR2 mRNA and protein showed ascending trend from E13 to E18 and was highest at E18.In summary,during the embryonic hair follicle formation and development,FGF21 might play a role in inducing hair follicle activation;FGFR1 might promote hair peg formation;FGFR2 might be necessary to induce hair follicle formation and hair follicle maturation.

This study was aimed to clone ompW gene of Pasteurella multocida in goat and carry out bioinformatics analysis.According to the ompW gene sequence of Pasteurella multocida strain HN07 (accession No.:CP007040.1) in GenBank,one pair of primers were designed using DNAMAN 5.0 software,and the high-fidelity enzyme PrimeSTAR^® Max DNA Polymerase was selected for PCR reaction to obtained the target gene fragment,the nucleotide and amino acid sequences of ompW gene were analyzed by bioinformatics softwares.The results showed that the length of PCR product was about 615 bp,which encoded 204 amino acids.The nucleotide homology analysis result showed that ompW gene of Pasteurella multocida in goat was highly homologous with porcine,bovine and avian-derived Pasteurella multocida,but not highly homologous to Pasteurella multocida in rabbit.The phylogenetic tree result showed that the relationship was the closest between ompW gene of Pasteurella multocida in goat and porcine.The bioinformatics analysis results showed that the molecular formula of ompW protein was C₁₀₀₇H₁₅₆₇N₂₅₇O₂₈₃S₃,the molecular weight was 21.90 ku,the theoretical isoelectric point (pI) was 9.16,which was a basic protein with a hydrophobic index of 96.57 and a total average hydrophobicity (GRAVY) of 0.173 (> 0),it belonged to a hydrophobic protein.The first 21 amino acids were signal peptides,the 5th to 27th amino acid regions had a transmembrane region,there were N-glycosylation and phosphorylation sites,no O-glycosylation site,and there were multiple B cell,CTL cell and Th cell epitopes.The alpha helix,extended chain,beta turn and random coil of the secondary structure accounts for 17.65%,35.29%,3.92% and 43.14%,respectively.The tertiary structure was a beta barrel monomer belonging to one of the members of the outer membrane protein family.These results provided a theoretical basis for further elucidating the mechanism of anti-host immune stress and the development of vaccines in the process of infecting host of Pasteurella multocida.

Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of reproductive failure in sows and respiratory disorders in piglets.The spread and outbreak of PRRSV has led to a serious threat to global pig industry.Currently,the pathogenic mechanism and interaction with the hosts of PRRSV remains unclear.With the rapid development of high throughput sequencing,the transcriptome sequencing has been widely applied in the research of PRRSV.It offers a new way for the explanation of the pathogenic mechanism and interaction with the hosts of PRRSV.This article reviews the application of transcriptome sequencing technology in the response,the function of virus proteins,the strains,the vaccine and the resistance breeding of PRRSV research,it summarizes the differentially expressed genes (DEGs) and pathways enriched in the transcriptome sequencing of PRRSV infection,and may provide a reference for PRRSV research in the future.

In order to evaluate the effect of inlA/inlB/inlC genes on biological characteristics of Lm,the inlC gene deletion mutant was constructed by fusion PCR method,and then pKSV7-△inlC shuttle vector was constructed,which was transformed into competent cells Lm681-△inlAB.Homologous recombination was conducted using temperature (42℃)and chloramphenicol (10 μg/mL) resistance to achieve the inlC gene deletion strain,and then the homologous recombination was screened for identification and some biological characteristics were studied.PCR and sequencing results confirmed that Lm681-△inlABC was successfully obtained.The results showed that the growth characteristics of the deletion mutants were the same as the wild-type strain Lm681 and there was no significant difference.The hemolysis activity of the mutant strains were consistent with wild-type strain Lm681.In the mouse infection experiment,the mortality rate of wild-type strain Lm681,Lm681-△inlAB and Lm681-△inlABC were 80%(8/10),60%(6/10) and 40%(4/10),respectively.Results of mice virulence determination showed that the median lethal dose of wild-type strain,Lm681-△inlAB and Lm681-△inlABC were 4.36×10⁴,1.35×10⁶ and 2.95×10⁷ CFU,respectively.Furthermore,the colonization ability of the inlA/inlB/inlC genes deleted strain was extremely significantly lower than wild-type strain in liver,spleen and brain (P < 0.01).Collectively,these results indicated that inlA/inlB/inlC genes might play an important role in pathogenicity of Lm infection and provided scientific basis for the study on the mechanism in host cell invasion by Lm.

The aim of this study was to clone the CDS of casein family genes (CSN1S1,CSN1S2,CSN2 and CSN3),and determine their expression in various tissues.Each of 3 healthy,about 4 years old Leiwuqi yak were selected.After slaughter,the tissues of mammary gland,heart,liver and skeletal muscle were collected for the total RNA extraction.The primers for casein family genes were designed and used for gene cloning,and then the sequences were analyzed using bioinformatics.The mRNA expression level of casein family genes were determined using Real-time PCR.The cDNA length of CSN1S1,CSN1S2,CSN2 and CSN3 genes were 919,832,805 and 715 bp,which containing 645,669,690 and 585 bp CDS and encoding 214,222,259 and 194 amino acids,respectively.The yak casein family was closest associated with that of Bos grunniens,followed by Bubalus bubalis,and distant with Sus scrofa.The tissue expression analysis showed that casein family genes were widely expressed in multiply tissues,which had the highest expression level in mammary gland,and followed in skeletal muscle.In addition,there was no difference among CSN1S1,CSN1S2 and CSN2 genes (P > 0.05),but the expression level of CSN2 gene was significantly higher than that of CSN3 gene in mammary gland (P < 0.05).These data provided basic information for the functional research of casein family genes in the regulation of protein metabolism in yak mammary gland,and laid a foundation for the development and utilization of yak genetic resources.

To select a suitable transfection reagent and condition for primary bovine skeleltal muscle satellite cells,the pcDNA3.1-EGFP and pEGFP-C1 plasmids with green fluorescent protein(EGFP) were used as exogenous gene to transfect primary bovine skeletal muscle satellite cells by Lipofectamine 3000,X-tremeGENE HP and FuGENE HD in this study.Then the transfection efficiency was evaluated by fluorescence microscopy,Real-time quantitative PCR and Hoechst 33342 staining.The results showed that transfection efficiency of pcDNA3.1-EGFP was better than pEGFP-C1 for the same period and transfection reagent.Among different transfection reagents,the effect of transfections in Lipofectamine 3000 and X-tremeGENE HP were superior to FuGENE HD.Myotubes were most obvious with Lipofectamine 3000 transfection after differentiation 72 h,followed by FuGENE HD,X-tremeGENE HP transfection leaded to more cell death.The expression of EGFP was extremely significantly higher than FuGENE HD after transfection with 1.0 μL Lipofectamine 3000, 1.5 μL Lipofectamine 3000 and X-tremeGENE HP (P < 0.01), but there was no significant difference in three groups (P > 0.05). The cell transfection efficiency was basically consistent with the result of EGFP quantification, the transfection efficiency of 1.0 and 1.5 μL Lipofectamine 3000 was extremely significantly higher than FuGENE HD (5.59%) and X-tremeGENE HP (10.87%) (P < 0.01), respectively up to 26.07% and 24.77% with no significant difference (P > 0.05). The transfection efficiency of X-tremeGENE HP was also extremely significantly higher than FuGENE HD (P < 0.01).Comprehensive consideration the amount of transfection reagents,cytotoxicity and economic benefits,this study preliminarily assumed that the transfection of primary bovine skeletal muscle satellite cells should adopt Lipofectamine 3000 transfection reagent,and the transfection efficiency was the highest when the ratio of Lipofectamine 3000 to plasmid DNA was 1:1.This experiment could provide important references for transfection of primary cells myogenic.

Melanocytes are melanin-producing cells that are differentiated from their precursor melanoblasts.Melanoblasts are formed in two migration pathways in embryonic stage.The first is dorsolateral pathway which neural crests take when they are specified as melanoblasts,the second is ventral pathway where Schwann cell precursors (SCPs),a sub-cellular neural stem cell,are formed,and induced to differentiate into melanoblasts,and melanoblasts from SCP are the major source melanocytes in the skin.The formation of melanocytes is closely related to animal hair color and other important economic traits,and the increase or decrease of melanocyte formation,ectopic formation or ectopic invasion may lead to the corresponding disease,but Silky fowl,a vertebrate which has typical ectopic melanocytes is healthy,and is an economic breed for food and medicinal usage.Exploring the formation mechanism of melanocytes and its influencing factors,as well as the ectopic formation of melanocytes not only provides informative theoretical basis for the breeding of animal economic traits but also provides a very important theoretical basis for the treatment of related diseases.This paper would explore the cell origin of melanocyte and the factors affecting the melanocytes formation from different origin,and discuss the formation mechanism of melanocyte-related traits,especially the black characteristics of Silky fowl.

In order to analyze the pathogenic biological characteristics of Streptococcus suis type 4(SS4).10 strains of SS4 from different regions in China were subjected to multilocus sequence typing.Seven conserved housekeeping genes aroA,gki,dpr,mutS,recA,thrA and cpn60 of Streptococcus suis were amplified by PCR.The sequencing results were uploaded to MLST database to find the sequence type,and the clustering analysis map was constructed to clarify the genetic relationship in strains.The seven major virulence genes gdh,mrp,epf,sly,fbps,gapdh and orf2 were identified by PCR,and the distribution of virulence factors of SS4 was analyzed by virulence factor spectrum.BALB/c mice were subjected to animal pathogenicity test with 10 strains of purified bacteria,and the strongest strains were selected according to the lethal number of mice and subjected to New Zealand rabbit pathogenicity test.The results of multilocus sequence typing showed that 6 strains were ST850,3 strains were ST1006,and 1 strain was ST94.According to the location analysis,the strains in Guangdong and Jiangsu had high homology,and the strains in Jiangsu and Shanghai showed differentiation,which showed genetic diversity.gdh,gapdh and orf2 genes were detected in 10 strains, sly gene was detected in 7 strains,and fbps gene were detected in 4 strains.According to the virulence factor spectrum,a total of 3 virulence genotypes were found,6 strains were gdh⁺sly⁺gapdh⁺orf2⁺ type,3 strains were gdh⁺fbps⁺gapdh⁺orf2⁺ type,and 1 strain was gdh⁺sly⁺fbps⁺gapdh⁺orf2⁺ type.Animal pathogenicity test results showed that 10 strains could all cause the death of BALB/c mice.The LD₅₀ of SH1510 was as low as 1×10⁸ CFU,which was the most pathogenic to mice,and SH1510 could cause typical neurological symptoms and death in New Zealand rabbits.The above results provided new data for the genetic evolution and virulence studies of Streptococcus suis,which had enriched the study of Streptococcus suis.

The objective was to assess the effects of Saccharomyces cerevisiae (SC) supplementation on plasma metabolomics of steers fed diets with different concentrate to forage ratios (CTFR).10 Simental×Local breed steers (450 kg ±50 kg) fitted with rumen fistulas were assigned to control and treatment groups.Steers in the two groups were fed the same basal diets but steers in treatment group received supplementation with SC (8×10⁹ CFU/h per day through the ruminal fistula) following a 2-period crossover design.Each period consisted of four phases,each of which lasted for 17 d,16 d for dietary adaptation,and 1 d for rumen sample collection.From the 1st to the 4th phase,steers were fed in a stepwise fashion with increasing CTFRs (30:70,50:50,70:30,90:10).UPLC-QTOF-MS was used to detected plasma metabolomics.PCA,PLS-DA and OPLS-DA were used to filtrate biomarkers.The results showed that biomarkers among different dietary CTFR groups were cytosine,dihydrothymine,palmitic amide,stearamide,oleamide and D-4-phosphopantothenate.When dietary CTFR was 30:70, biomarkers between SC supplementation and control groups were sphinganine,phytosphingosine and PC (18:0);When CTFR was 50:50, biomarkers between the two groups were 13E-docosenamide,1-monoacylglycerol and PC (16:0).When dietary CTFR was 70:30,biomarkers were 1-monoacylglycerol,diacylglycerol, triacylglycerol and PC (18:0,17:0),while the biomarkers were 13E-docosenamide,glycocholic acid and PC (18:2,20:2) when CTFR was 90:10.The results showed that with concentrate level increasing, fat metabolism was improved, but rumen wall was damaged;SC supplementation could improve phospholipid and fat metabolism, which was beneficial to maintain animal health status and improve production performance.

In order to explore the effects of drinking magnetized water (DMW) on the digestion and metabolism of sheep fed with pellet diet,two experiments were designed in this study.In experiment 1:Four 1.5-year-old Small-tail Han ram with (44.7±1.8) kg body weight were selected and assigned to 4 groups by 4×4 Latin square design,in which they were fed with ground diet (treatment 1),ground diet+DWM (treatment 2),pellet diet (treatment 3) and pellet diet+DMW(treatment 4),respectively,to study the effect of DMW on the voluntary intake,digestion and metabolism of sheep fed with the ground and pellet diets.In experiment 2:Six 1.5-year-old non-pregnant Small-tail Han ewe with (46.3±2.1) kg body weight were selected and divided into 3 groups by 3×3 Latin square design,in which all of sheep were fed with pellet diet with voluntary intake (treatment 1),voluntary intake+DMW(treatment 2) and restricted feeding+DMW (treatment 3),respectively,to study on the effects of DMW on the digestion and metabolism of sheep with restricted feeding.The results showed that:①In experiment 1,the dry matter voluntary intake,apparent digestibility of the dry matter,crude protein and energy,and nitrogen retention of ram with DMW were increased by 14.7%(P < 0.05),2.6%(P > 0.05),2.2%(P > 0.05),2.2%(P > 0.05) and 22.7%(P < 0.01),respectively,when fed with ground diet;And that were increased by 14.3%(P < 0.05),4.2%(P < 0.05),4.8%(P < 0.05),6.0%(P < 0.01) and 14.2%(P < 0.05),respectively,when fed with pellet diet.② In experiment 2,the apparent digestibility of the dry matter,organic matter,crude protein,cellulose,hemicellulose,non-enzyme-hydrolyzable cellulose and hemicellulose,energy and nitrogen retention of feeding-restricted ewe with DMW were increased by 19.1%,19.0%,9.7%,18.4%,16.2%,341.5%,226.7%,17.4% and 14.8%(P < 0.01),respectively,compared with the ewe that had voluntary intake.It was concluded that under the circumstance of voluntary intake of sheep increased by feeding pellet diet,it would be increased further by DMW;Under the circumstance of voluntary intake,DMW did not affect the digestibility of sheep fed with ground diet but increased the digestibility of dry matter,crude protein and energy and nitrogen retention of sheep fed with pellet diet;In restricted feeding,the nutrients digestibility of sheep was significantly increased by DMW.

The purpose of this study was to investigate the effects of adding tannins and feeding cellulase on rumen microbes in the growth and fattening period of Hu sheep using high-throughput sequencing technology,and provide useful data for the better utilization of tannins and cellulase in ruminant production.Thirty-six meat Hu sheep with good growth and average body weight (19.85±1.45) kg at 3 months old were randomly divided into control group and 3 treatment groups.The control group was fed with basal diet,and three treatment groups were added 0.1% tannin (tannin group),0.1% feeding cellulase (cellulase group) and 0.1% tannin +0.1% feeding cellulase (mixed group) in the basal diet respectively,3 replicates per group,each repeat 3 sheep.The test period was 70 days,in which the transition period was 7 days,the pre-test period was 7 days,and the positive feeding period was 56 days.At the end of the experiment,the rumen fluid of Hu sheep was collected,and the total DNA of the bacteria was extracted and PCR amplification was conducted.The amplified products were sequenced by Illumina Hi Seq 2500 for high-throughput sequencing.The results showed that:① A total of 957 440 pairs of microbial samples were obtained from 12 rumen samples,with an average of 55 997 clean tags,after filtering chimeras,a total of 502 965 effective tags were generated.AvgLen of rumen microbial samples from each group of sheep were between 419 and 420;②The Ace index and Chao1 index of rumen microorganisms in each group had no significant difference (P > 0.05);Compared with the control group,the Shannon index of rumen microorganisms in tannin,cellulose and mixed groups were significantly increased (P < 0.05).The Simpson index of rumen microorganisms in cellulase group was significantly lower than that in the control group (P < 0.05).③At the level of phylum:The abundance of Bacteroidetes of tannin group was significantly lower than that of the control group (P < 0.05);The abundance of the Firmicutes in tannin,cellulose and mixed group were lower than that of the control group,but the difference was not significant (P > 0.05).④On the genus level,the abundance of Prevotella_1 in tannin group was droped,and there was significant difference compared with the control and cellulase groups (P < 0.05),but there was no significant difference with mixed groups (P > 0.05).The abundance of Rumen-bacterium in tannin and cellulase groups were higher than that of the control and mixed group,and the difference with mixed group was significant (P < 0.05).In conclusion,adding tannin and cellulase at the same time could improve the diversity and abundance of rumen flora,affect the structure of rumen flora,and alleviate the inhibition of tannin on Bacteroidetes and Firmicutes.Mixed addition could alleviate the inhibition of cellulose decomposition by adding tannin alone.The dominant bacteria in the rumen of each group of Hu sheep were Bacteroidetes and Firmicutes.On the genus level,the dominant bacteria in the rumen of each group were studied Rikenellaceae_RC9_gut_group,Bacterium,Prevotella_1 and Rumen_bacterium.

Perilla seed is a traditional Chinese medicine with antipyretic,analgesic and anti-inflammatory effects.It is rich with α-linolenic acid which is involved in maintaining the normal structure and function of immune cells and inhibiting the production of inflammatory factors.Perillaldehyde can inhibit the expression of pro-inflammatory factor genes and enhance immunity capability of animals.Flavonoids,phenolic acids and other active substances can reduce infiltration of inflammatory cell and the formation of edema,induce apoptosis of diseased cells,and also have antioxidant effects.This paper mainly introduces the main anti-inflammatory active ingredients and anti-inflammatory effects of α-linolenic acid,perillaldehyde,iso-perillaldehyde,luteolin and rosmarinic acid in Perilla seed.The research progress of its application in livestock production was reviewed,and the regulation of Perilla seed in animal intestinal flora,production performance,immune function and meat quality was analyzed.In addition,the problems of differences of ingredients contents with different varieties breeds and the strong adverse effects were also discussed,aiming to provide a theoretical reference for the development and utilization of Perilla seed.

This experiment was conducted to study the detoxification effect of amination on aflatoxin B₁ (AFB₁) by different ammonia treatments and its effect on the in vitro fermentation of dairy cows.This experiment was divided into two sections.Trial 1:The corn and rice mixture (1:3) artificially infected with 2 550 μg/kg AFB₁ were ammoniated,and they were randomly divided into control group (group Ⅰ,untreated) and test groups Ⅱ (0.1 Mpa,1 h),Ⅲ (0.1 Mpa,2 h),Ⅳ (0.1 Mpa,3 h),Ⅴ (0.2 Mpa,1 h),Ⅵ (0.2 Mpa,2 h),Ⅶ (0.2 Mpa,3 h),Ⅷ (0.3 Mpa,1 h),Ⅸ (0.3 Mpa,2 h),Ⅹ (0.3 Mpa,3 h) with 3 duplicates per group according to the difference in ammonia pressure and period.The detoxification effect of different ammonia treatments on AFB₁ and nutrient composition of the mixture were determined.Trial 2:The trail was divided into four treatments:Control group (group Ⅰ,untreated) and test groups Ⅱ (0.3 Mpa,1 h),Ⅲ (0.3 Mpa,2 h) and Ⅳ (0.3 Mpa,3 h) with 3 duplicates per group to explore the effect of ammoniated AFB₁ on the in vitro fermentation of dairy cows.The gas production,pH,in vivo dry matter degradation rate (IVDMD),ammonia nitrogen (NH₃-N) and volatile fatty acid (VFA) of the fermentation broth were measured after 2,4,8,12,24 and 48 h fermentation.The results showed that,in trail 1,the CP content of the test groups were significantly higher than that of control group (P < 0.05);The AFB₁ content of the group Ⅹ (0.3 Mpa,3 h) was significantly lower than that in the other groups (except group Ⅶ)(P < 0.05).In trail 2,the pH of fermentation broth of group Ⅳ (0.3 Mpa,3 h) was significantly lower than that of control group (P < 0.05),and the gas production,VFA,NH₃-N content and IVDMD (except 48 h) were significantly higher than that of control group (P < 0.05),while the effects of groups Ⅱ and Ⅲ were not obvious.In summary,ammonia treatment could significantly reduce the content of AFB₁,increase the CP content,and then improve the efficiency of ruminal fermentation,in which the treatment of 0.3 Mpa,3 h had the best effect.

This study was aimed to examine the effects of supplemental feeding compound probiotics on feces fiber content,VFA concentration and plasma cytokines levels of 3-6 months old Yili horse.Ten Yili horses of 3 months old with the average body weight (90.23±11.02)kg were divided into 2 groups (control and trial groups),5 horses per group.The trial group was fed the basal diet supplemented with 0.075 g/d(2×10¹¹ CFU/g) Bacillus subtilis and 0.133 g/d(3×10¹⁰ CFU/g).Lactobacillus plantarum.Feeding trial lasted for 90 d.The results showed that the contents of ADF and strach of feces in trial group were significantly or extremely significantly lower than that of control group(P < 0.05;P < 0.01),which were reduced by 3.50% and 11.32%,respectively.The acetic acid and propionic acid levels of feces in trial group were significantly or extremely significantly higher than that of control group (P < 0.05;P < 0.01),which were increased by 14.65% and 64.29%,respectively.The TNF-α level of plasma in trial group was extremely significantly higher than that of control group (P < 0.01),which was increased by 16.17%.This results indicated that supplementation of compound probiotics could reduce the fiber content of feces,increase the concentration of VFA and the plasma cytokine levels of 3-6 months old Yili horse.

The effects of 4 kinds of traditional Chinese medicine on immune performance and intestinal flora in immunosuppressed mice were studied.70 6-week-old male Kunming mice were chosen and assigned to 7 groups:Blank control group,model group,YPF group,Fructus Mume group,Herba Taraxaci group,Codonopsis Radix group and Pericarpium Granati group.A pathological model of immunosuppression was established by cyclophosphamide (Cy).From the 3rd day of administration,75 mg/(kg·BW) Cy was intraperitoneal injected in groups 2 to 7,once every another day for 4 times.During the injection,the normal administration was continue to perform for 7 d,and 0.9% saline was administered in model group until the second day after the Cy injection was stopped.The effects of 4 traditional Chinese medicines on body weight,spleen index,thymus index,blood indicator,sIgA,IgG and intestinal flora of immunosuppressed mice were examined.The results showed that compared with model group,the body weight of immunosuppressed mice were significantly increased by Fructus Mume,Herba Taraxaci,Codonopsis Radix and Pericarpium Granati (P < 0.05),and the effect of Fructus Mume was the best.The immune organ index of immunosuppressed mice were significantly increased by 4 kinds of Chinese medicines (P < 0.05),but could not reach the normal level,of which the spleen index of Herba Taraxaci group was the highest.The WBC were improved and the RBC were increased significantly (P < 0.05),but could not reach the normal level.The concentration of immunoglobulin in each Chinese medicine group was increased,the sIgA and IgG in the Codonopsis Radix group were significantly higher than that in the blank control group (P < 0.05),buts no significant different with YPF group (P > 0.05).The number of Escherichia coli was significantly decreased (P < 0.05),the number of Bifidobacterium and Lactobacillus was significantly increased (P < 0.05) and the richness and diversity of intestinal flora of immunosuppressed mice were increased,of which the Codonopsis Radix and Herba Taraxaci groups were better.In conclusion,Fructus Mume,Herba Taraxaci,Codonopsis Radix and Pericarpium Granati could antagonize the effects of Cy on body weight,immune function and intestinal flora in mice,and could be used in the development of immune-enhancing drugs.

Seaweed is a general name for algae in the ocean.It is distributed in the shallow sea area below the low tide line and is an occult plant in the plant kingdom.There are more than 30 000 species of algae known worldwide.Algae that have been developed and utilized by humans are mainly divided into four categories:Red algae,brown algae,green algae,and cyanobacteria.With the tight supply of conventional feed ingredients and rising prices,the development of unconventional and integrated functional feed has become a long-term task to solve the effective supply of feed resources in the livestock industry.As a high-quality unconventional feed resource,seaweed has the characteristics of wide distribution,large yield,strong adaptability,etc.,and contains a large number of biologically active substances and nutrients that terrestrial plants lack,and these characteristics determine its enormous value in feed economic development.The authors reviewed the regional distribution,characteristics,classification and functions of various nutrients and their applications in animals of seaweed.Seaweed is rich in nutrients such as proteins,amino acids,minerals,polyunsaturated fatty acids,and functional substances such as polysaccharide polyphenols and terpenoids.The authors also pointed out that seaweed has great development potential in animal husbandry production and application,as well as the problems that should be paid attention to the process of using seaweed as feed ingredients,and explore its application value and potential market in multiple functional fields of animal production.This review was respected to provide a reference for the application of algae resources in animal diets.

This study was conducted to explore the effect of oligo-chitosan supplemented in sow diet on growth,antioxidant capacity and immunologic function of suckling piglets.24 healthy Yorkshire sows with similar parity,body weight and expected date of confinement were assigned into 3 groups with 8 replicates per group and 1 sow per replicate.Sows in the control group were fed the basal diet and the others in the experimental groups Ⅰ and Ⅱ were fed the basal diet supplemented with 50 and 100 g/t oligo-chitosan,respectively.The experiment lasted from the 85th day of pregnancy to the 21st day after delivery.The results showed as follows:①The diarrhea rate of suckling piglets in group Ⅱ was significantly lower than those in group Ⅰ and the control group (P < 0.05),while there was no significant difference between experimental group Ⅰ and the control group (P > 0.05).The body weight at 21 days of age of suckling piglets in groups Ⅰ and Ⅱ were much higher than that in control group (P < 0.05).②Compared with the control group,the activity of LDH and the concentration of TG in group Ⅰ,activities of α-AMY and LDH,and concentration of LDL in group Ⅱ were significantly decreased (P < 0.05).③The activity of SOD,levels of cortisol,IL-10,IL-1β,TNF-α and IFN-γ in group Ⅱ were significantly higher than those in group Ⅰ and control group (P < 0.05),while the concentration of MDA was significantly lower than that in the control group (P < 0.05).④The concentrations of immunoglobulin IgG,IgA and IgM of sow milk in groups Ⅰ and Ⅱ were much higher than those in the control group (P < 0.05).Compared with the control group,the serum IgG and IgA levels of suckling piglets in group Ⅰ and IgA level in group Ⅱ were significantly increased (P < 0.05).In conclusion,the addition of oligo-chitosan at a certain dose in sow diet could improves the antioxidant and immunity capacity,reduce the diarrhea rate,and thus improve the growth performance of suckling piglets,indicating oligo-chitosan was an effective feed additive.

To explore the function of acetaldehyde dehydrogenase 1A1 (ALDH1A1) gene,16-month-old Yanhuang cattle were used as subjects,and total RNA were collected from the heart,liver,lung,kidney,stomach,duodenum,subcutaneous fat and longissimus dorsi after slaughter.According to the ALDH1A1 gene sequence published in GenBank(accession No.:NM_174239.2),primers were designed using Oligo 7.0 software,ALDH1A1 gene was amplified by RT-PCR,and the amplification product was connected into pMD18-T vector for clonging and sequencing.The complete CDS sequence of ALDH1A1 gene in Yanhuang cattle was obtained,the nucleotide sequences and its protein structures were analyzed using bioinformatics softwares.Then using the total RNA from different tissues of Yanhuang cattle as a template,the quantitative expression of ALDH1A1 gene in different tissues of Yanhuang cattle was detected by Real-time quantitative PCR.The results showed that the length of CDS sequence of ALDH1A1 gene was 1 506 bp,which encoded 501 amino acids.The homology of the ALDH1A1 gene sequence with bison and cattle was ≥ 99.7%,and its homology with cats and leopards were 89.4% and 89.6%,respectively,which was in accordance with species evolution rules.The molecular weight of ALDH1A1 was 54.805 ku,the isoelectric point was 7.16;A relatively strong hydrophilicity accounted for 86.4%;acidic and basic amino acids accounted for 11.4% and 12.2%,respectively.ALDH1A1 was soluble proteins,which was not secretory proteins,but there was no typical signal peptide cleavage site.There were 31 amino acid phosphorylation sites (score > 0.5).The secondary structure of ALDH1A1 protein in Yanhuang cattle contained alpha helix,extended chain,beta turn and random coil,which accounted for 42.12%,16.17%,8.18% and 33.53%,respectively,which were identical to the predicted structure of the tertiary structure of ALDH1A1 protein.Real-time quantitative PCR results showed that ALDH1A1 gene was extremely significantly expressed in liver,stomach,subcutaneous fat,duodenum and kidney tissues (P < 0.01),and significantly expressed in longissimus dorsi (P < 0.05) of Yanhuang cattle,respectively.This study results provided a reference for further research on the function of ALDH1A1 gene and the screening of meat gene in Yanhuang cattle.

SMAD3 gene is one of the members of the SMADs gene family,and the SMAD3 protein which encoded by SMAD3 gene,is the special intracellular signal transduction molecule of TGF-β super gene family.Not only SMAD3 gene participates in regulating many aspects of important physiological processes via the superfamily of TGF-β,such as disease,immune regulation,growth and development,wound healing,development and maintenance of cartilages and bones,but also involves in regulating physiological activities in animal,for example,the proliferation,differentiation,maturation,adhesion,atresia,apoptosis of germ cells,and secretion of steroid hormone.Therefore,it's of great significance to understand the structure and function of SMAD3 gene for the researches relating to animal growth,development and reproduction.This review sketched the structure,function,mechanism of SMAD3 gene,and analysized its research progress of the application in livestock production.It showed that a large number of researches about the application of SMAD3 gene in animal production was mainly focus on aspects of growth regulation,development,cell immunity and apoptosis,hormone secretion and reproductive performance of vertebrate (pigs,cattle,sheep,chickens,etc).Regulations of SMAD3 gene in disease,growth and development,fat deposition and reproductive performance were still hot in domestic and abroad.However,the precise molecular regulation mechanism of this gene on disease occurrence,growth and reproductive performance remains to be further researched.

As an efficient assisted reproductive technology,in vitro embryo production is of great significance to the conservation and effective utilization of germplasm resources and genetic improvement.However,compared with embryos produced in vivo,the embryos produced in vitro develop slowly,the cleavage rate is low and the rate of apoptosis increases in the process of culture,which are accompanied by placental hypertrophy and prolonged pregnancy after transplantation.Preterm delivery,low birth weight,and macrosomia syndrome (LOS) occur after birth which are closely related to abnormal gene expression and epigenetic modification.The progress of epigenetic modification in the production of embryos in vivo and in vitro was briefly reviewed in this paper.The differences of epigenetic modification of imprinted genes,DNA methylation and histone acetylation between mammalian embryos in vivo and in vitro were introduced.The application of microarray technique in the production of embryos in vitro and in vivo was briefly described in order to obtain the key imprinted genes that lead to the difference of embryo quality between in vivo and in vitro,and explored the reasons why the quality of embryos produced in vitro was lower than that of in vivo,and then improved the in vitro embryo production system.

To determine whether the miR-23a regulate Smad3 gene,wild-type and mutant-type dual-luciferase reporter vectors containing Smad3-3'-UTR (psiCHECK^TM-2-W-Smad3-3'-UTR and psiCHECK^TM-2-M-Smad3-3'-UTR) were constructed using Not Ⅰ and Xho Ⅰ,miR-23a mimics,miR-23a inhibitor and their negative control were transfected with or without dual luciferase reporter vectors in PK-15 cells,luciferase activity were assayed,the mRNA and protein expression levels of Smad3 gene were determined by Real-time PCR and Western blotting,respectively.The results showed that the luciferase activity in PK-15 cells transfected with wild-type dual-luciferase reporter vectors and miR-23a mimic was significantly lower than negative control (P < 0.05).In PK-15 cells transfected with miR-23a mimic,the expression levels of Smad3 mRNA and Smad3 protein were significantly down-regulated (P < 0.05).While there was no significant difference in the expression level of Smad3 protein between PK-15 cells tansfected with miR-23a inhibitor and its negative control (P > 0.05).The results indicated that miR-23a could regulate directely the expression of its target gene Smad3.

In order to understand the prevalence and antigenic variation of canine parvovirus (CPV) in Kunming,and select and develop vaccines pertinently,six suspected CPV feces were collected from some pet hospitals in Kunming for virus isolation and culture in this study.The obtained suspected virus solution was subjected to PCR,IFA,hemagglutination titer measurement,TCID₅₀ measurement,and VP2 gene sequence analysis.The results showed that the isolated virus could produce obvious cytopathic effect (CPE) in F81 cat kidney cells.The length of PCR product was 164 bp.The F81 cat kidney cells infected by IFA showed green fluorescence and the blood coagulation titer was 1:512.The TCID₅₀ of the virus was 10^-4.375/0.1 mL,and the length of VP2 gene was 1 755 bp.The VP2 structural protein of the isolate was sequenced and analyzed,and the isolate was determined to be CPV-2a subtype,and there were five variant amino acid sites compared with the standard strain,which belonged to the mutant strain.The sequencing results were compared with the commercial vaccine strains and the domestic reference strains.The results showed that the nucleotide homology with the attenuated vaccine Intervet/vaccine/06 and Pfizer/vaccine/06 were both 98.7%;The sequence homology was 98.2% to 99.5%,which had the highest homology with ANTU-1 and WH02/06 strains.It was closely related to ANTU-1 strain,WH02/06 strain and BJ01 strain,and was located in the same cluster of evolutionary tree.This study was of great significance for monitoring the genetic variation trend of CPV and the development of vaccines.

This study was aimed to screen the sub-unit vaccine candidate antigen protein of outer membrane protein (Omp) of Fusobacterium necrophorum (Fn),and lay the foundation for the development sub-unit vaccine of Fn.Fn Omp sequence was predicted by Uniprot,and the expression protein was screened based on the predicted results.Specific primers were designed according to the corresponding gene sequence of Fn Omp in GenBank,the QL03 strain was used as template for PCR amplification,prokaryotic expression,SDS-PAGE and Western blotting analysis.The proteins immunogenicity were analyzed by bactericidal assays and vaccination efficacy assessment in mice to screen the best candidate protein.The results showed that 121 proteins were obtained through the predicting outcomes,named 1-Fn to 121-Fn in turn,and 8-Fn,11-Fn,41-Fn,95-Fn and 102-Fn were screened to be candidate proteins,the fragments of 672,1 164,570,1 059 and 729 bp were amplified by PCR.SDS-PAGE and Western blotting results showed that 8-Fn expressed unsuccessfully,and the sizes of the others expressed proteins were 60,39,59 and 44 ku,which named P11-Fn,P41-Fn,P95-Fn and P102-Fn,and all proteins could react with Fn positive serum.The bactericidal assays results showed the 4 recombinant proteins had certain bactericidal effects,and the anti-P102-Fn serum was the best,the sterilizing rate reached 40.49%.The vaccination efficacy assessment results in mice showed the 4 recombinant proteins could protect mice from the challenge of QL03,and the P102-Fn was the best,the protection rate reached 60%.These results indicated that P11-Fn,P41-Fn,P95-Fn and P102-Fn were all of effective immunogenicity,P102-Fn could induce an immune response and it might be useful as an antigen for vaccine.

In order to understand the antimicrobial sensitibity to sulfonamides and distribution of sulfonamides resistance genes of Pasteurella multocida strains isolated from ducks in Fujian,Guangdong,Jiangxi,etc,89 Pasteurella multocida strains were tested for antimicrobial susceptibility to trimethoprim-sulfamethoxazole by agar dilution method, PCR method and multi-locus sequence typing (MLST) were used for detection of sulfonamides resistance genes and to analyze the genetic relationship among the sulfonamides-resistant strains.The results showed that 33 strains (37.1%) were resistant to trimethoprim-sulfamethoxazole.Among the 33 sulfonamides-resistant strains,the detection rates of sul1,sul2 and sul3 genes were 100%(33/33),97.0%(32/33) and 21.2%(7/33).The simultaneous detection rate of sul1 and sul2 genes were 97.0%(32/33).The simultaneous detection rate of sul1,sul2 and sul3 genes were 21.2%(7/33).All of the 33 sulfonamides-resistant strains belonged to the ST129 sequence type.The results indicated that there was sulfonamides resistance to Pasteurella multocida isolated from ducks,and all the sulfonamides-resistant strains,which belonged to the same sequence type ST129,carried sul1 resistant gene,most of sulfonamides-resistant strains carried sul1 and sul2 resistant genes,suggesting that it should be rationally used sulfonamides to prevent and cure the infection of Pasteurella multocida isolated from ducks in some areas of Fujian,Guangdong and Jiangxi,etc.

To understand the molecular evolution and mutation of SzM gene of Streptococcus equi subsp.zooepidemicus (S.zooepidemicus) from horse in Xinjiang and provide basis for the prevention and treatment of infectious disease,lymph gland samples from sick horse were collected for bacterium culture,biochemical identification,and drug sensitivity test was also tested.The SzM gene was amplified from genomic DNA of S.zooepidemicus isolate by PCR based on the primers designed according to the published sequence in GenBank.Then the amplified SzM gene was cloned,sequenced and analyzed,and phylogenetic tree was constructed.The results showed that the isolate was Gram-positive Streptococcus,which was named as S.zooepidemicus ZMSY15-1.Drug sensitivity test showed that S.zooepidemicus ZMSY15-1 was resistant to penicillin and sulfadiazine sodium salt,and sensitive to other 14 microbials.The amino acid homologies of SzM gene of ZMSY15-1 with different strains at home and abroad were 56.0% to 70.0%.In addition,the phylogenetic analysis indicated that these test strains belonged to 4 different clades,the ZMSY15-1 isolate shared identities of 59.9% with swine isolates and they belonged to two different groups.The ZMSY15-1 isolate was closely related to US horse NH55426.The results could enrich the information data of SzM gene of S.zooepidemicus in domestic horses,and provide experimental data for the pathogenic mechanism and prevention control of S.zooepidemicus.

In order to quickly and accurately detect porcine reproductive and respiratory syndrome (PRRSV),one TaqMan-MGB Real-time RT-PCR assay for detection of PRRSV was established using specific primers and probe based on PRRSV gene.The developed Real-time RT-PCR method could detect PRRSV classic strain,highly pathogenic mutant strain,as well as the new NADC30-like strain at the same time.The specificity,sensitivity and repeatability of the established method were detected.120 clinical samples were detected using the developed Real-time RT-PCR method and the conventional PCR method.The results showed that TaqMan-MGB Real-time RT-PCR method was specific for PRRSV classic strain,highly pathogenic mutant strain,as well as the NADC30-like strain,and the test results were negative with CSFV,PEDV,TGEV,RV,PRV,PPV and PCV2.The method showed a good linear relationship within the template ranges from 10¹ to 10⁸ copies/μL, and its sensitivity was 100 times that of routine PCR assay.The correlation of the standard curve was 0.9999,and the efficiency was 93%.The limit of detection concentration was 10¹ copies/μL.The coefficient of variation in the intra-and inter-assays were 0.17% to 0.90% and 0.65% to 2.34%,respectively.The positive detection rate of PRRSV in clinical samples using the developed Real-time RT-PCR method (59.2%) was higher than that using the conventional PCR method (44.2%).The establishment of Real-time RT-PCR method provided a rapid and accurate detection means for early diagnosis and epidemiological investigation of the disease.

In order to totally understand the prevalence of bovine viral diarrhea (BVD) in a large-scale cattle farm in Jilin province,a total of 157 clinical serum samples,18 stool samples,17 liver and semen tissue samples were collected.Serum antibody detection was performed using BVDV antibody detection kit.BVDV antigen detection was performed on serum and clinical samples by Nano-PCR method and BVDV type 1 specific primers,the antigen positive results were sequenced and analyzed;The antigen positive samples were ground and diluted,filtered and sterilized by 0.22 μm filter membrane,and connected to MDBK cells for virus isolation and culture.The antigen detection was carried out again after three generations;The infection of MDBK cells was detected by immunofluorescence technique;Phylogenetic tree and homology analysis were drawn by Mega software.The results showed that the positive rate of clinical serum BVDV antibody in the cattle farm was 77.1%,the positive rate of serum antigen was 12.1%,and the positive rate of sample antigen in clinical feces was 74.3%.After the diseased material was connected to the cells,the cytopathic effect could not be observed.PCR results of the obtained strains were consistent with the results of sample antigen detection.The immunofluorescence assay results showed that BVDV venom was normally adsorbed in MDBK cells,and there was obvious fluorescence reaction.The main strain of BVD and BVDV JL-1 strain were analyzed by antigen sequencing,the homology was as high as 99.0%,and both were BVDV type 1 strains.This study had conducted a comprehensive survey of the prevalence of BVDV in the cattle farm,laying the foundation for the purification work.

The present study was aimed to express the VP2 protein of Senecavirus A (SVA) and prepare its polyclonal antibodies.The full-length coding sequence of the SVA VP2 gene was amplified by RT-PCR using the SVA RNA of GD01/2017 strain as the template.The amplicon was then cloned into the prokaryotic expression vector pET-30a(+) to construct a recombinant plasmid pET-30a-SVA-VP2.After verification by DNA sequencing,the plasmid was transformed into E.coli Rosetta (DE3) competent cells which were then induced by IPTG.The recombinant SVA VP2 protein was purified from the supernatant of the lysate of pET-30a-SVA-VP2 plasmid-transformed E.coli Rosetta (DE3) cells using the Ni-NTA agarose under native conditions.The purified VP2 protein was used to immunize the New Zealand White rabbits to prepare its polyclonal antibodies,which were then purified from the rabbit sera by affinity chromatography using the Protein A Sepharose CL-4B.The reactivity of the polyclonal antibodies with SVA was analyzed by an indirect immunofluorescence assay.The results showed that the recombinant SVA VP2 protein was expressed in E.coli Rosetta (DE3) cells in the form of both soluble and inclusion bodies with a molecular weight of about 47 ku,and that the titer of the rabbit anti-VP2 polyclonal antibodies was 1:64 000.The prepared polyclonal antibodies merely reacted with SVA and had no cross-reactivity with other common porcine pathogens,such as porcine reproductive respiratory and syndrome virus,porcine circovirus type 2,encephalomyocarditis virus,classical swine fever virus and pseudorabies virus.It indicated that the prepared polyclonal antibodies against SVA VP2 protein had good specificity.The successful preparations of recombinant SVA VP2 protein and its polyclonal antibodies provided valuable biomaterials for the establishment of serological detection method for SVA.

This paper was aimed to study on isolation,identification and drug resistance analysis of a bacterial (NZ-2017),which was isolated from local sick hybrid sturgeons.Morphology,physiological and biochemical characteristics and phylogenetic analysis of 16S rRNA gene were used to identify its species.The filter paper diffusion method test,antimicrobial resistance genes test and animal regression test were carried out to analyze its pathogenicity.The results showed that NZ-2017 was a gram-negative bacillus,which was 99.70% homologous with Plesiomonas shigelloides 16S rRNA gene fragment by sequencing and BLAST analysis.With the results of physiological and biochemical identification,NZ-2017 was confirmed as Plesiomonas shigelloides.The drug sensitivity test results showed that NZ-2017 was sensitive to ofloxacin,norfloxacin,ciprofloxacin,doxycycline,and resistance to kanamycin,amoxicillin,tylosin and sulfamethoxazole.The antimicrobial resistance genes test results showed that there were three antimicrobial resistance genes (Sul1,Sul2 and Intl1),which was compatible with phenotype.The animal regression test result showed that NZ-2017 had pathogenicity to mouse.This study conducted an accurate diagnosis of the cause of the epidemic of sturgeons in Guizhou,and provided a basis for the prevention and controls to this bacterial disease of fish in this region.

In order to investigate the taxonomic status and phylogenetic relationship of Cylicocyclus leptostomum (C.leptostomum),this study used a digital microscope to observe the morphology of C. leptostomum,the ribosomal DNA internal transcribed spacer (rDNA-ITS) sequence was amplified by PCR,and 12 other ITS sequences of Strongylid nematodes were downloaded from GenBank,and the phylogenetic tree was constructed by maximum likelihood (ML) using Strongylus equinus as the outer group.The results showed that the nematode was medium-sized,the cyst was cylindrical,the width was greater than the depth,with small teeth at the bottom;The front end of the pouch wall was thin,and the base of the back end had obvious hoop-shaped thickening;The outer leaf crown was composed of 20 to 24 leaflets,and the inner leaf crown was composed of 50 to 60 leaflets;The esophagus funnel was smaller;Male reproductive cone was longer,conical;The female tail was straight,tail tip was finger-shaped;The total length of the measured ITS sequence was 837 bp,of which ITS1 was 366 bp,5.8S was 153 bp,and ITS2 was 318 bp.The GC content of ITS1 (46.0%) was significantly higher than that of ITS2 (39.8%);By BLAST homology alignment analysis,the ITS sequence of this study was 99.85% homologous with the same nematode sequence registered in GenBank (accession No.:AJ004849 and Y08587),and the homology with C.ashworthi,up to 99.0%,the homology with other nematodes in the genus was only 93.33% to 98.45%.The interspecies differences in ITS sequences were much larger than intraspecies differences.Phylogenetic analysis showed that C.leptostomum was closely related to C.ashworthi,and it was relatively distant from other nematodes in the genus.In summary,the ITS sequence could be used as a molecular genetic marker for identifying parasitic nematodes.It was confirmed that the specimen was a C.leptostomum,and the ITS sequence of C.leptostomum was reported for the first time in China,which would lay a foundation for further studies on this species.

To explore whether gene rearrangement rabies virus (RV) vaccine could stimulate the production of RV antibody IgG in mice,and compare the IgG level and safety performance in different immune modes and different immune doses of attenuated RV vaccine,28 35-day-old Kunming mice with (20±3)g were chosen and assigned to 7 groups:Group 1 (low dose oral group,50 μL compound adjuvant +50 μL RV),group 2 (middle dose oral group,150 μL compound adjuvant +150 μL RV),group 3 (high dose oral group,300 μL compound adjuvant +300 μL RV),group 4 (low dose injection group,50 μL compound adjuvant +50 μL RV),group 5 (middle dose injection group,150 μL compound adjuvant +150 μL RV),group 6 (high dose injection group,300 μL compound adjuvant +300 μL RV) and group 7 (oral control group,300 μL compound adjuvant +300 μL SRV9 strain).Immunisation was performed at 0,7 and 14 d,respectively,and the IgG level of mice in each immune group was detected by ELISA kit,and the liver,kidney,lung,brain tissues of the immune groups were collected to evaluate the safety of the vaccine at the end of immunization test.The results showed that IgG was produced in each immune group.The rate of IgG produced by intramuscular injection was higher than that of oral immunization,but intramuscular vaccination could lead to death of mice.The IgG level produced by 600 μL oral immunization group was significantly higher than 100 and 300 μL oral immune groups and parental strains SRV9 control group (P < 0.05),which indicated that increasing oral immunization dose would increase the level of antibody produced by oral immune and would not cause adverse reactions.Histopathological examination found that the liver,kidney,lung and brain tissues of each oral immune group were normal and did not see pathological changes,indicating that the gene rearrangement RV had less side effects and had better safety.It provided a new direction for the development of a new type of RV attenuated oral vaccine.

The aim of the experiment was to study the bacteriostatic activity and stability of the antimicrobial peptide BSN-37.The minimum inhibitory concentration (MIC) of BSN-37 against several bacteria were detected with serial dilution method.The bactericidal dynamics of BSN-37 against Escherichia coli (E.coli) CVCC1568 were measured by colony counting method.The salt ion stability,acid-alkali stability,serum stability,pancreatic enzyme stability,thermal stability,repeated freeze-thaw stability,maintain stability at room temperature,and efficacy stabilization stability were analyzed through diffusion method.The results showed that the MIC of the antimicrobial peptide BSN-37 against Gram negative bacteria (E.coli and Salmonella) was 1.5625 to 25 μg/mL,and the MIC of BSN-37 against Gram positive bacteria (Bacillus subtilis and Bacillus pumilus) were 1.5625 to 12.5 μg/mL.But BSN-37 had no bacteriostatic activity against fungi (Candida albicans).The antimicrobial peptide BSN-37 could completely kill E.coli CVCC1568 within 90 min.In addition to the Fe²⁺ and pancreatic enzymes could affect the bacteriostatic activity of antimicrobial peptide BSN-37,at other conditions including the high temperature (75 to 100℃),150 to 250 mmol/L high concentration of salt ions (Na⁺,K⁺,Ca²⁺ and Mg²⁺) and 20% to 25% of saturation concentration of Cu²⁺,pH 4 to 10,20% to 25% of serum,10 to 12 times of repeated freezing and thawing,preservation for 10 to 12 days at room temperature,BSN-37 could still maintain good bacteriostatic activity.Efficacy maintain time of BSN-37 was more than 7.5 days.The results provided a theoretical basis for the clinical application of antimicrobial peptide BSN-37 which had a potential to replace antibiotics as new antimicrobial agents.

To explore the therapeutic effects of deer hard antler button (DHAB) water extracts on hyperplasia of mammary glands (HMG) in rats and to explore its mechanism of action and dosage,the model of HMG was established through intramuscular injection of estradiol benzoate (0.5 mg/(kg·d)) and progesterone (5.0 mg/(kg·d)) in 70 SD rats.There were 7 groups with 10 rats in each group,which were divided into blank group,HMG model group,positive drug tamoxifen group (0.036 mg/(kg·d)),4 DHAB groups (high-dose,H,2.625 g/(kg·d);Medium-high-dose,MH,1.575 g/(kg·d);Medium-low-dose,ML,1.05 g/(kg·d);Low-dose,L,0.63 g/(kg·d)).Each group was treated with intragastric administration for 45 d.The diameter and height of nipple and body weight were recorded weekly.Blood samples were taken and thymus,spleen,uterus and ovary weights of the rat breasts were measured at the end of the test.Histopathological sections and HE staining were performed and sex hormone estradiol (E₂) and progesterone (P) were detected by radioimmunoassay.The results indicated that different doses of DHAB water extracts could reduce the diameter and height of nipple,increase thymus and spleen indexes and reduce uterine index,reduce the number of breast lobules,acinar numbers and secretion contents,and decrease E₂ and increase P levels in serum of HMG rats.All these indexes were significantly or extremely significantly different from the model group (P < 0.01;P < 0.05).In conclusion,DHAB water extracts had significant therapeutic effects on HMG in rats,and the medium-high-dose was the best.The action mechanism of DHAB water extracts was enhancing immunity and regulating serum E₂ and P levels.

The aim of the experiment was to study the pharmacological effects of compound Myrobalan oral liquid against Escherichia coli (E.coli) diarrhea in calves.We used neutralize enterotoxin test,anti-exudation test,antipyretic test,analgesic test,intestinal peristalsis test and immunomodulation test to verify the pharmacological effects of compound Myrobalan oral liquid.The results showed that in the neutralize enterotoxin test,there was extremely significant difference between compound Myrobalan oral liquid and negative control groups (P < 0.01).In the anti-exudation test,all drug groups could reduce the exudation of Evans blue to varying degrees,the inhibition rates of the aspirin group,compound Myrobalan oral liquid high,medium and low dose groups were 58.1%,42.8%,31.6% and 14.9%,respectively.Among them,the compound Myrobalan oral liquid low dose group was significantly different from blank group (P < 0.05),while other groups were extremely significantly different from blank group (P < 0.01).The compound Myrobalan oral liquid medium and low dose groups had extremely significant difference compared with aspirin group (P < 0.01),but the compound Myrobalan oral liquid high dose group had no significant difference(P > 0.05).In the antipyretic test,the high-dose group of compound Myrobalan oral liquid quickly inhibited the increase of body temperature 1 h after administration,and the increase of body temperature was inhibited 2 h after the administration in medium dose group;3 h after the administration,the body temperature was significantly reduced in high and medium dose groups compared with model group;The difference between low dose and model groups was not significant.In the anti-analgesic test,there were extremely significant differences in aspirin group,compound Myrobalan oral liquid high and medium dose groups compared with blank group (P < 0.01),the writhing inhibition rates were 64.55%,52.73% and 32.73%,respectively,the pain latency were 9.59,6.33 and 5.74 min;And there was no significant difference in low dose group (P > 0.05).In the intestinal peristalsis test,compared with blank group,compound Myrobalan oral liquid high dose and atropine sulphate groups had obviously inhibitory effect on intestinal peristalsis (P < 0.01),and the promotion rates were 44.18% and 52.55%,respectively;The effect of medium dose group of compound Myrobalan oral liquid was significant different (P < 0.05),low dose group of compound Myrobalan oral liquid was not significant (P > 0.05).In the immunomodulation test,the white blood cell (WBC) count,spleen and thymus organ indexes in model group were significantly lower than those in blank group (P < 0.05),and the WBC count,spleen and thymus organ indexes in compound Myrobalan oral liquid group were significantly higher than those in model group on the 14th day (P < 0.05).In conclusion,compound Myrobalan oral liquid had neutralize enterotoxin,anti-exudation of tissue fluid,antipyretic,analgesia and inhibitory effect on intestinal peristalsis of E.coli diarrhea,and could enhance the immune function and other pharmacological effects.

The effects of Yupingfeng polysaccharides (YPF-P) on the morphology and T cell subsets of Peyer's patches (PPs) were studied to explore its mechanism of immune response to mice intestinal mucosa.Ninety-six SPF mice were randomly divided into 6 groups:Blank control group(0.2 mL saline),YPF-P positive control group (200 mg/kg YPF-P),immunosuppressive group (80 mg/kg cyclophosphamide) and 100 (low),200 (medium) and 400 mg/kg (high) YPF-P groups,feeding for one week.Small intestine PPs were sampled,section and HE stained.The changes of PPs morphological structure were analyzed by image analysis technology.Flow cytometry was used to study the changes in subsets of T lymphocytes isolated from murine PPs.The results showed that YPF-P promoted the growth and development of small intestine PPs in mice.Cyclophosphamide could significantly reduce the area of PPs,the cross-section area of the small intestine and their ratio(P < 0.01).YPF-P could alleviate the damage in the small intestine caused by cyclophosphamide to some extent,increase CD3⁺,CD4⁺ T lymphocytes and CD4⁺/CD8⁺ in PPs lymphocytes (P < 0.05;P < 0.01).The results showed that YPF-P could enhance the intestinal mucosal immune function of cyclophosphamide-induced immunosuppressive mice,and promote the proliferation of related T lymphocytes in PPs,which could enhance the intestinal mucosal function of mice.

Artificial insemination (AI) technology is an advanced assisted reproductive technology,it can improve the efficiency of breeding,but due to the unique reproductive characteristics of cats,the development of cat AI technology is limited.In recent years,many researchers and clinical veterinaries have used mature AI techniques from other species,combined with feline unique characteristics to optimize and innovate cat AI technologies such as semen collection,semen quality assessment,semen preservation,stimulation of female cat ovulation,and artificial insemination,for example:A new and practical urinary catheterization technique for collecting cat semen have been developed;Based on the traditional semen quality assessment,researchers began to use fluorescent staining,flow cytometry,and in vitro fertilization technique to evaluate the quality of sperm;Researchers explored the effects of different extenders formulas and freezing procedures on the preservation of male cats semen and tried to use ultra-rapid cryopreservation to preserve the genetic material of male cats,and also explored the differences in the use of different hormones to induce estrus and ovulation in female cats;As well as the influence of different parts,methods and timing of the insemination on the pregnancy of female cats and so on.Although these new technologies have already demonstrated their potential for practical application in experimental animals,there are still some problems,such as complicated operations,high costs and lower-than-expected results.As a result,the application of AI in cats is limited,and is not as widely used as dogs or other species.In the future, further researches and explorations are needed to meet the requirements of clinical use.This paper reviewed the research progress on various aspects of cat AI technology in order to provide reference for further research and application of this technology.