The objective of this study was to clone the complete CDS region of porcine ankyrin repeat and suppressor of cytokine signalling box containing protein 2 (ASB2) gene,analyze the CDS sequence and the basic characteristics of proteins by bioinformatics methods,and explore its expression profiles in different tissues of one-day-old Jinfen White pig and satellite cell myoblast induction.Based on the predicted nucleotide sequence of pig ASB2 gene in GenBank.Primers were designed to amplify the CDS region of ASB2 gene by RT-PCR using cDNA of longissimus dorsi muscle as template.The amino acid sequence and the structure and function of ASB2 were analyzed using bioinformatics software.The expression profiles in different tissues and during the procession of myogenic differentiation of satellite cells were investigated by Real-time quantitative PCR.The results showed that the complete CDS region of porcine ASB2 gene was 1 824 bp in length,which encoded a total of 607 amino acids.The nucleotide sequence of porcine ASB2 gene had the highest similarity with those of goat and cow.Bioinformatics analysis results showed that ASB2 was a hydrophilic protein,including 11 O-glycosylation sites,1 N-glycosylation site,and no signal peptide.There were 11 ANK motifs and 1 SOCS motif.The secondary structure of the protein was predicted to be 43.99%,40.36%,10.05% and 5.60% with random coil,alpha helix,beta turn and extended strand.Real-time quantitative PCR results showed that the highest expression level was found in porcine psoas muscle,and followed by longissimus dorsal muscle and heart,which were extremely significantly different from those in other tissues (P<0.01).During the procession of myogenic differentiation of satellite cells,the expression of ASB2 gene was increased at the beginning,reached the peak after 2 d induction,then decreased,which suggested that ASB2 gene might be involved in regulating muscle growth.The results of this experiment provided a reference for further exploring the function and mechanism of porcine ASB2 gene.

The purpose of this study was to establish a highly sensitive 3D digital PCR (3D-dPCR) method for the detection of equine herpesvirus 1 (EHV-1),which could accurately and quantitatively detect the samples with low EHV-1 content and realize the early diagnosis and prevention of equine rhinopneumonia.According to the conserved region of EHV-1 glycoprotein B gene,we designed specific primers and probes,optimized the concentration and annealing temperature of primers in the 3D-dPCR reaction system,analyzed the sensitivity,specificity and repeatability of this method,and established the 3D-dPCR method of EHV-1.In this study,the best concentration of primer and probe of 3D-dPCR was 0.4 and 0.4 μmol/L respectively,the best annealing temperature was 60 ℃,R² of the absolute quantitative curve of the method was 0.998,the linear relationship was good,the sensitivity was about 10 times higher than that of Real-time PCR,and the minimum detection limit was 5.83 copies/μL.There was no cross reaction with EHV-4,Theileria equi and the nucleic acid of equine arteritis.The results showed that the positive rate of 3D-dPCR was 66.7%,which was higher than that of Real-time PCR for EHV-1 in OIE (64.2%).The results of 3D-dPCR were consistent with those of Real-time PCR,and the sensitivity of 3D-dPCR to the samples with low virus content was higher,which could effectively detect suspicious samples.The results showed that the established 3D-dPCR method was more sensitive,specific and reproducible for the detection of clinical samples with low copy number,and could be used for the accurate and quantitative detection of EHV-1.

In order to explore the structure,function and expression characteristics of Na⁺/I^- symporter (NIS) protein of mammary gland in mouse,NIS gene was amplified from mammary gland in mouse,after sequencing,NIS protein was analyzed by bioinformatics software in this study.The transcription and expression level of NIS gene were detecteded in different stages of mammary gland development by Western blotting,immunohistochemistry and semi-quantitative PCR.The results showed that the CDS sequences of NIS gene was 1 857 bp,and the homology was 80.40% between Homo sapiens and Mus musculus,the conservation was stronger.There were 618 amino acid residues of NIS protein in mouse,with a molecular weight of 65.7 ku,the molecular formula was C₂₉₉₆H₄₇₀₁N₇₄₉O₈₃₃S₃₂,the theoretical isoelectric point was 7.44,the half-life was 30 h,the extinction coefficient was 102 510,the unstability coefficient was 29.31,and the fat solubility coefficient was 105.91.It was rich in hydrophobic amino acids, and was stable hydrophobin.The secondary structure of NIS protein contained alpha helix (41.42%),extended chain (18.28%),beta turn (4.05%) and random coil (36.25%),which was consist with the tertiary structural results.Western blotting results showed that the specific hybridization bands appeared in all four periods,with a size of 65.7 ku in line with expected results.The immunohistochemistry results showed that the expression of NIS protein increased gradually from puberty and pregnancy to lactation,reached the peak in lactation,there was a significant difference (P<0.05),and returned to a similar puberty state in degenerative period.The level of NIS mRNA was consistent with NIS protein.This study provided a reference for further exploring the role of NIS protein in mammary gland development and lactation regulation.

The purpose of this study was to clone the N gene of canine coronavirus(CCV),express the N protein in vitro,and prepare a polyclonal antibody against this protein for the diagnosis of CCV and its antigen detection.Referring to the N gene sequence of CCV in GenBank (accession No.:KY063618.2) and the N gene of CCV epidemic strain was selected,and the recombinant expression plasmid pET-B2M-N was constructed by codon optimization and gene synthesis.Finally,an effective gene was selected to construct the recombinant plasmid,which transformed into E.coli DH5α constructer.After sequencing,positive clones were transformed into E.coli BL21 (DE3) strain and induced by IPTG.Finally,IPTG concentration of 0.5 mmol/L was used to induce at 30 ℃.The results showed that the recombinant plasmid pET-B2M-N containing canine coronavirus N gene was successfully constructed.After optimizing the induction conditions,the recombinant protein with a size of about 49 ku was successfully expressed.The recombinant protein was mixed with Freund’s adjuvant to immunize Japanese White rabbits G767 and G768 for several times,and the G768 antibody titer was up to 1:512 000 assayed by indirect ELISA.The antibody could be purified to 10 mg/mL.The charactered of polyclonal antibodies detected by ELISA,Western blotting and immunofluorescence,indicating that the expressed recombinant N protein had good immunogenicity and reactogenicity.This study provides a basis for the detection of canine coronavirus antigen and antibodies and the establishment of target CCV diagnostic kit.

Genome-wide association analysis (GWAS) was used to identify candidate genes and molecular markers which genes related to meat color traits in ducks and explored the genetic basis of meat color traits.In this study,three meat color phenotypes (including redness a^*,yellowness b^* and brightness L^*) were collected in 555 ducks from an F2 segregating population generated by Pekin duck×mallard crosses and performed GWAS using whole-genome resequencing data to detect SNPs loci associated with meat color traits.The results showed that the meat color L^*,a^* and b^* displayed low heritability which was 0.23,0.12 and 0.13,respectively,and the coefficient of variation for meat color yellowness (b^*) was 26.18%.Correlation analysis results showed that high positive phenotypic and genetic correlations between muscle yellowness and muscle redness (r=0.52),and low negative correlations between muscle fiber diameter and muscle yellowness (r=-0.16).GWAS was performed using a mixed linear model and potential significant SNP association with muscle yellowness (-log₁₀ P=8.28) was found.Then the loci of interest more closely was examined by calculating pairwise linkage disequilibrium (LD).42 SNPs spanning a region from 0.37 Mb to 0.43 Mb on chromosome 9 where contained 7 annotated genes were highly correlated (pairwise r²>0.4).The mRNA expression of five genes were expressed in breast muscle.By functional annotation of this 5 genes,SELENOT was identified as candidate gene for yellowness b^*,which provided an important reference for the genetic improvement of meat quality in ducks.

To obtain the native conformation and antigenicity of σB and σC proteins of avian reovirus,the σB and σC proteins was expressed by Bac-to-Bac baculovirus expression system,and its reactivity was identified.The σB and σC genes of avian reovirus were amplified by designing two pairs of primers according to the sequence published on NCBI,inserted into donor plasmid pFast-dual to obtain recombinant donor plasmid,then transformed into E.coli DH10Bac,and the recombinant bacmid plasmid was obtained.Recombinant baculovirus was transfected into sf9 insect cells using cellfection reagent to obtain the production of recombinant viral contained the sequence of σB and σC genes.sf9 insect cells were inoculated by recombinant viral to explore the MOI and harvest timing expression condition,and the expression condition was optimized.The results of Western blotting and indirect immunofluorescence assay (IFA) showed that the recombinant σB and σC proteins were expressed successfully in sf9 insect cells and had good biological activity.This study provided a further research direction of ARV vaccine of virus-like particles and ARV subtype antibody ELISA detection kit.

This experiment was aimed to study the experimental model of broiler ascites syndrome (AS) by multi-factor method and its effect on diastolic-systolic factors of vascular endothelium in lung tissue.A total of 158 healthy Ross broilers were randomly divided into normal group,low temperature model group and multi-factor model group.At 25,35 and 45 days of age,the clinical symptoms,post-mortem change and the pathological change of lung tissue were observed,the body weight,the incidence of AS and ascites heart index (AHI) were measured,and the change of NO,ET-1 and NOS were detected,and the expression of ETAR and VEGF genes in lung tissue were detected by Real-time quantitative PCR.The results showed that the AHI of broilers at each day-age in the low temperature and the multi-factor model groups was extremely significantly higher than that in the normal group (P<0.01),the AHI of broilers at each day-age in the multi-factor model group was significantly higher than that in the low temperature model group (P<0.05).The body weight of broilers at 35 and 45 days of age in the low temperature model group and each day-age in the multi-factor model group were significantly or extremely significantly lower than that in the normal group (P<0.05,P<0.01).The incidence of AS at 35 and 45 days of age in the low temperature model group and each day-age in the multi-factor model group were extremely significantly higher than that in the normal group (P<0.01).At 25 days of age,the incidence of AS in the multi-factor model group was extremely significantly higher than that in the low temperature model group (P<0.01).At 35 days of age,lung tissue of broilers with AS structure was damaged,endothelial cells were shed,and smooth muscle arrangement was disturbed.Compared with the normal group,the contents of NO and ET-1,iNOS activity,ETAR and VEGF mRNA expression in lung tissue at 35 and 45 days of age in the low temperature and the multi-factor model groups were significantly or extremely significantly increased (P<0.05,P<0.01),the ratio of NO to ET-1 and the activity of cNOS and TNOS were significantly or extremely significantly reduced (P<0.05,P<0.01).Experimental ascites syndrome model in broiler could be successfully established induced by multi-factor method with low temperature,high sodiu and high energy feed.The multi-factor model method could reflect the diversity of disease factors better than the low temperature model method.The expression disorder of diastolic-systolic factors of vascular endothelium in lung tissue was involved in the occurrence of broiler AS.

The aim of this study was to compare the efficiency of transfected pig kidney epithelial cells (PK15) by three transfected methods,so as to lay the foundation for studying the function of foreign genes with PK15 cells as a model.In this study,PK15 cells as research subject were transfected with liposome,electroporation and lentivirus methods.The transfected efficiency was evaluated based on the expression of green fluorescent protein observed by fluorescence microscope,the expressions of EGFP and NR2F2 genes were detected by Real-time quantitative PCR and the cell survival rate was detected by CCK-8 reagent.The results showed that the transfected efficiency of PK15 cells transfected with electroporation and lentivirus methods was extremely significantly higher than that of liposomes (P<0.01),whereas the difference between electroporation and lentivirus methods was not significant (P>0.05).The Real-time quantitative PCR results showed that the expressions of EGFP and NR2F2 genes in PK15 cell transfected with lentivirus method were the highest and the lowest with liposome method,which was in accordance with the transfected efficiency.CCK-8 results showed that the cell survival rate transfected by electransfection was the lowest,which was extremely significantly lower than that of control group (P<0.01).The cell survival rate transfected with the liposome was significantly lower than that of control group (P<0.05).The difference of cell survival rate between lentivirus and control group was not significant (P>0.05).Based on the transfection efficiency and cell viability of this study,the lentiviral transfection method was the suitable way for transfection of PK15 cells,which provided a perfect method for high-efficiency transfection of PK15 cells in the future.

The aim of this experiment was to study the effects of probiotics,oligomeric chitosan and acidulant use alone or in combination with complex enzymes on growth performance,immune organ index,carcass traits and meat quality of Yellow-feathered chicken aged from 1 to 66 days old.1 440 1 day old rapidly growing Lingnan Yellow-feathered male chicken were randomly assigned into 8 groups according body weight:Control group (antibiotic-free),antibiotic group (225 mg/kg aureomycin+200 mg/kg eramycin),probiotics 1 group (500 mg/kg Bacillus subtilis+200 mg/kg Lactobacillus+1 000 mg/kg active yeast),probiotics 2 group (200 mg/kg mixed bacteria product),oligomeric chitosan group (50 mg/kg oligomeric chitosan),acidulant group (2 000 mg/kg benzoic acid+2 000 mg/kg citric acid),antibiotic-free combination 1 group (50 mg/kg oligomeric chitosan+1 000 mg/kg acidulant+500 mg/kg complex enzyme),antibiotic-free combination 2 group (probiotics 1 group+antibiotic-free combination 1 group),each group consisted of 6 replicates with 30 broilers.The experiment was carried out in three stages,and the experimental period was 66 days.The results showed that:1 to 21 days,compared with control group,the feed/gain ratio (F/G) of oligomeric chitosan and antibiotic-free combination 1 groups was significantly reduced (P<0.05).Compared with antibiotic group,the body weight (BW) and average daily gain (ADG) of probiotics 1 and 2 groups were significantly decreased,ADG of antibiotic-free combination 2 group was significantly decreased,and F/G of probiotics 1 and antibiotic-free combination 2 groups was significantly increased (P<0.05).The thymus index of oligomeric chitosan and antibiotic-free combination 1 groups was higher than that of control and antibiotic groups (P>0.05).The IgG content in plasma of antibiotic-free combination 1 group was higher than that of antibiotic group (P>0.05),and significantly higher than that of control group (P<0.05).The IgM contents in plasma of other groups except acidulant group were significantly higher than that of control and antibiotic groups (P<0.05).22 to 42 days,BW and ADG of probiotics 1,oligomeric chitosan and antibiotic-free combination 1 and 2 groups were higher than that of antibiotic group,and ADG of oligomeric chitosan group was significantly higher than that of antibiotic group (P<0.05),F/G of probiotics 1,oligomeric chitosan and antibiotic-free combination 1 groups were lower than that of antibiotic group (P>0.05).Different combinations without antibiotic had no significant effects on immune organ index and plasma biochemical parameters (P>0.05).43 to 66 days,different combinations without antibiotic had no significant effects on growth performance and immune organ index (P>0.05).The IgM contents in plasma of probiotics 1,oligomeric chitosan,antibiotic-free combination 1 and 2 groups were higher than that of antibiotic group (P>0.05),and significantly higher than that of control group (P<0.05).The abdominal fat percentage of probiotics 2 and antibiotic-free combination 1 and 2 groups were lower than that of antibiotic group (P>0.05),which of acidulant group was lower than that of control group (P>0.05),and significantly lower than that of antibiotic group (P<0.05).L^*_{45 min} value of meat of probiotics 2 and antibiotic-free combination 1 and 2 groups were lower than that of antibiotic group (P>0.05),which of oligomeric chitosan group was lower than that of control group (P>0.05),and significantly lower than that of antibiotic group (P<0.05).According to the results of growth performance,immune function,carcass traits and meat quality in three stages of this study,it was recommended to add 50 mg/kg oligomeric chitosan,1 000 mg/kg acidulant and 500 mg/kg complex enzymes in the same time to basic diet instead of antibiotic of rapidly growing Yellow-feathered male chicken aged from 1 to 66 days old could improve the growth performance,immune function,carcass performance and meat quality.

The purpose of this study was to investigate the effects of different levels of chromium niacin on the lactation performance,antioxidant performance and microbial diversity of rumen fermentation in buffalo under heat stress.In this experiment,20 healthy mid-lactating Nili-Ravi buffaloes were selected according to their body weight,daily age,gestational number and milk yield.A single factor randomized block design was adopted,and all the buffloes were divided into 4 groups (Ⅰ,Ⅱ,Ⅲ,Ⅳ groups) with 5 replicates for each group.Each bufflo in control group was fed a basic diet.Each buffalo in group Ⅰ,Ⅱ,Ⅲ and Ⅳ was fed with 0,1.5,3.0 and 4.5 mg chromium niacin per day,respectively.The experimental pre-feeding period was 1 week,and the experimental period was 4 weeks.The experiment results showed that:①Under the condition of heat stress,there was no significant difference in dietary supplementation of different levels of chromium niacin on dry matter intake (P>0.05),and the apparent digestibility of ADF and NDF in the test groups increased to different degrees.②There was no significant effect on the rumen pH and volatile fatty acids such as acetic acid,propionic acid and butyric acid.③Dietary addition of 3.0 mg/d chromium niacin significantly increased milk yield (P<0.05).There was no significant difference in milk protein rate,milk fat rate,milk total solid and non-fat solid among the groups (P>0.05).④The levels of CAT,MDA and GSH-Px in buffalo were not significant (P>0.05) among test groups and the control group.Compared with the control group,the concentrations of T3 and T4 in 3.0 mg/d chromium niacin group were significantly higher than those in the control group (P<0.05).⑤Compared with the control group,the content of fatty acids in long chain milk fat of buffalo in the experimental group was increased.⑥At the phylum level,the dominant flora of each group of water buffalo was Firmicutes and Proteobacteria of Bacteroidetes,and the samples showed rich diversity on the whole.The addition of 3.0 mg/d chromium to the diet could improve the lactation performance and the anti-oxidation performance of the buffalo,and the effect was better than that of other groups.

The aim of this study was to compare reproductive performance of sows under different parity in different-scale pig farms.The detailed production data for sows from 106 pig farms with different scale were collected.According to the actual number of sow stocks,the farms were divided into four scales:<1 000,1 000-5 000,5 000-10 000 and ≥10 000 heads.The differences of reproductive indexes related to 1-9 births in different scale sow farms were investigated,such as live birth rate,healthy piglet rate,deformed piglet rate,stillbirth rate,mummification rate,weaned piglet rate,the average litter number of total,live,healthy,dead and weaned of piglets,average birth weight and average litter weight.The results showed that the rates of live,healthy and weaned piglets under the same parity increased gradually with the increase of the feeding scale,while the rates of deformed piglet,stillbirth piglet and mummy showed opposite trends.From the 1st litter to the 7th litter except for the 4th,the rates of live,healthy and weaned piglets in the farms with ≥10 000 heads scale were significantly higher than those in the farms with <1 000 heads scale (P<0.05),but were not significantly different from other two scales (P>0.05).The stillbirth rate of the 1st,2nd,3rd,5th and 7th litters in the sow farms with <10 000 heads scale was significantly higher than that of other three scale farms (P<0.05).The number of total,livehealthy,deformed,stillbirth and mummy piglets and average litter weight at birth decreased gradually with the expansion of the breeding scale of the farms.The average number of total,live,healthy,stillbirth and weaned,and average litter weight of the 2nd and 3rd litters in the sow farms with <1 000 heads scale were significantly higher than those in the other three scale farms (P<0.05).In conclusion,the breeding scale had a greater impact on the productivity of sows under different parity.The sow fertility in the medium- and large-scale sow farms (≥1 000 heads) was lower than that in small-scale sow farms(<1 000 heads),but the body condition and survival rate of piglets in these farms were better than those in small-scale sow farms.

This study was conducted to investigate the effect of cottonseed peptide-rich fermented cottonseed meal (by Bacillus subtilis-1 and Saccharomycopsis fibuligera complex bacterias) on growth performance and immune function of AA broilers.240 one-day-old female broilers were randomly allocated into 4 dietary treatments,with 4 replicates in each treatment and 15 birds per replicate.6% cottonseed meal was added in the diet of control group(CK),and 4%,6% and 8% fermented cottonseed meal rich in cottonseed peptide were added in the diets of experimental groupsⅠ,Ⅱ and Ⅲ,respectively.The experiment was divided into two stages:1 to 21 d and 22 to 42 d.The broilers were weighed and blood samples were collected at the end of each stage,and the growth performance and immune function indexes were measured.The results showed that:①Compared with CK,the ADG of group Ⅱ was extremely significantly increased at 21 d (P<0.01),and it was significantly higher than groupⅠ(P<0.05).The ADFI of group Ⅲ was significantly increased (P<0.05),and it was extremely significantly higher than groupⅡ(P<0.01) at 42 d.②Compared with CK,the thymus index of group Ⅲ was significantly increased (P<0.05),and it was significantly higher than groupⅠ(P<0.05) at 21 d.The bursa of Fabricius index of groupⅡ was extremely significantly increased at 42 d (P<0.01).③ At 21 d,the phagocytic indices of group Ⅲ was significant higher than the other treatment groups (P<0.05),the phagocytic indices of groupⅠand Ⅱ was significantly higher than CK at 42 d (P<0.05).④ At 21 d,the IL-2 and IL-1β contents of group Ⅲ was significantly higher than CK and groupⅠ(P<0.05),and the IL-6 contents of group Ⅲ was extremely significantly higher than CK (P<0.01).At 42 d,compared with CK,the IL-2 contents of all experimental groups was significantly increased (P<0.05),the IL-1β contents of group Ⅲ was extremely significantly increased (P<0.01),while the IL-6 contents of group Ⅲ was extremely significantly decreased (P<0.01).Meanwhile,the TNF-α contents of groupsⅠand Ⅲ was extremely significantly increased (P<0.01).⑤ Compared with CK,IgM and IgA contents of group Ⅲ was extremely significantly increased (P<0.01),and the IgG contents of groupⅠwas extremely significantly higher than the other treatment groups at 21 d (P<0.01).At 42 d,the IgM and IgA contents of group Ⅱ was extremely significantly increased(P<0.01).In conclusion,adding certain proportion of fermented cottonseed meal rich in cottonseed peptide could improve the growth performance and immune function in broilers.

This study was aimed to analyze the effects of different cotton stalk levels on free gosspol content and apparent digestibility in sheep during fattening period.Designed by single factor random test single factor randomized trial,the similar weight of 60 Small-tailed Han sheep were randomly divided into 6 groups,every group had 10 sheep.The diet was prepared for free feeding based on same energy and same nitrogen content.The control group ck1 did not add cotton stalks and cottonseed meal,the control group ck2 added cottonseed meal,and 20% to 50% cotton stalks were added to 20%,30%,40% and 50% groups,respectively,with a total period of 70 days,of which the pre-test period was 10 days and the feeding test was 60 days.On the 50th day of the trial period,the fecal samples were collected for 24 h using the full-fee method.The acclimation period was 5 days,and the samples were collected continuously for 5 days,weighed and recorded for 3 days.On the 55th to 60th day,the daily feeding amount and the remaining material amount of each group of test sheep were recorded,and the average dry matter intake was calculated.The results showed that expect for ck1 group,the 20% group had the lowest free gossypol residue,which was extremely significantly different from ck2,30%,40% and 50% groups (P<0.01).The apparent digestibility of dry matter,CP,EE,Ca,P and Ash increased with the increase in the proportion of cotton stalk.The addition ratio of 20%-50% had no significant effect on fecal output and the apparent digestibility of NDF and ADF (P>0.05).The results suggested that cotton stalk could be used as raw material for fattening sheep production,it was advisable to consider cotton stalk addition at about 20% which based on the data of free gosspol content and apparent digestibility.

The purpose of this experiment was to study the effects of Enterococcus faecalis on production performance,egg quality and intestinal morphology of laying hens during the late laying period.A total of 270 Hy-Line Brown laying hens aged of 70 weeks were randomly divided into 3 groups with 6 replicates per group and 15 hens per replicates.The test was set three groups:Control group (feeding the basic diet),group Ⅰ (basic diet+3.75 mg/kg Enterococcus faecalis),group Ⅱ (basal diet+7.5 mg/kg Enterococcus faecalis).The preliminary test lasted for 7 days and the experiment lasted for 50 days.The results showed that compared with control group,the egg laying rate and egg mass of groups Ⅰ and Ⅱ were extremely significantly increased (P<0.01),and feed-to-egg was extremely significantly reduced (P<0.01).Compared with control group,there was no significant change in eggshell thickness,eggshell strength,egg shape index,protein height,Haugh unit,relative weight of egg yolk,and relative eggshell weight of groups Ⅰ and Ⅱ (P>0.05).Compared with control group,the mucosal thickness of the duodenum and ileum at the late laying period of groups Ⅰ and Ⅱ were extremely significantly increased (P<0.01),and the ileal villus height was significantly increased (P<0.05).In conclusion,the addition of a suitable dose of Enterococcus faecalis in diets could improve production performance and intestinal morphology of laying hens during the late laying period.

In this study,the effects of dietary crude protein (CP) level on the production performance and egg quality of Dawu Golden Phoenix commercial layer were studied to determine the CP requirement.In the experiment,480 31-week-old Dawu Golden Phoenix commercial layers were selected and randomly divided into 4 treatment groups with 12 replicates in each group and 10 layers in each replicate.The CP levels of the 4 treatment groups were 13.5%,14.5%,15.5% and 16.5%,respectively.The pre-test period was 7 d,and the formal period was 63 d.During the experiment,the number of eggs laid by layers,egg weight and unqualified eggs were recorded every day.Feed intake was counted by repetition every 2 weeks,and average daily feed intake (ADFI) and feed-to-egg ratio (F/E) were calculated.At week 6 of the experiment,three eggs were taken for each iteration to determine egg quality.The results showed that:① The CP level in the diet had a significant impact on the daily egg production,average daily feed intake and feed-to-egg ration of laying hens (P<0.05).The average daily feed intake and the daily egg production of laying hens in 16.5% CP group was significantly higher than that in 13.5% and 14.5% CP groups (P<0.05),and the daily egg production increased linearly with the increase of the CP level in the diet (P<0.05).The feed-to-egg ratio in 16.5% CP group was significantly lower than that in 13.5%,14.5% and 15.5% CP groups (P<0.05).②Dietary crude protein level had a significant effect on the proportion of egg shell and yolk color (P<0.05).The proportion of egg shell in the 13.5% CP group was significantly higher than that in the 16.5% CP group (P<0.05),and the proportion of egg shell decreased linearly with the increase of the dietary CP level (P<0.05).The yolk color in 13.5% CP group was significantly higher than that in 16.5% CP group (P<0.05),and the yolk color decreased linearly with the increase of diet CP level (P<0.05).By establishing the regression curve between feed-to-egg ratio and protein level (y＝275x²-86.4x+8.7356),the protein level for the minimum feed-to-egg ratio was 15.7%.Therefore,considering the results of production performance and egg quality,the dietary crude protein level of Dawu Golden Phoenix commercial layer during peak egg production period was recommended to be 15.7%.

In this experiment,concentrate,hay and tannic acid were used as fermentation substrates to investigate the effects of different levels of tannic acid on in vitro fermentation parameters,methane production,the degradation rate of dry matter and crude protein in sheep,in order to screen out the optimal concentration of tannic acid,and as a theoretical basis for untilization of tannic acid-rich roughage resources apply to dietary formulation and reducing methane emission in the process of ruminate farming.Batch culture in vitro was used for this study,the fermentation substrate was concentrate,hay and tannic acid,and the ratio of concentrate to roughage was 3:7.The supplertation level of tannic acid was 0,0.5%,1.0%,1.5%,2.0% and 3.0%,respectively,the fermentation time was set at 3,6,12 and 24 h,three replicates were set for each treatment.The pH,the concentration of ammonia nitrogen (NH₃-N) and volatile fatty acid (VFA),total gas production,methane production and the degradation rate of dry matter and crude protein were determined.The results showed that pH in 1.0% and 2.0% tannic acid groups was significantly higher than that in control group at 3 h (P<0.05),and there was no significant difference between all groups at other time points (P>0.05).The concentration of NH₃-N in 1.0%,2.0% and 3.0% tannic acid groups was significantly lower than that in control group at 3 h (P<0.05),more over the concentration of NH₃-H in each treatment group was significantly lower than that in control group at 6 and 24 h (P<0.05).The concentration of acetic acid,propionic acid,butyric acid and total VFA in 2.0% tannic acid group was significantly lower than that in control group at 6 h (P<0.05),the concentration of propionic acid,butyric acid and total VFA in 1.0%,1.5% and 3.0% tannic acid groups were significantly lower than that in control group at 24 h (P<0.05),the average concentration of propionic acid,butyric acid and total VFA in each tannic acid adding group was significantly lower than that in control group at 3,6,12 and 24 h(P<0.05).There was no significant difference in total gas production among each group at 12 and 24 h (P>0.05).The methane production in 2.0% tannic acid group was significantly lower than that in control,0.5% and 1.0% tannic acid groups at 12 h (P<0.05).The methane production in 2.0% and 3.0% tannic acid groups were significantly lower than that in control group at 24 h (P<0.05).In conclusion,the addition of tannic acid could significantly reduce the concentration of NH₃-N in vitro fermentation and inhibit the production of VFA,the addition of 1.5% and 2.0% tannic acid could significantly reduce the rumen methane production in vitro,the degradation rate of crude protein in the treatment group with tannic acid was significantly lower than that in control group.

The purpose of this experiment was to study the effect of different strains combination fermentation on the nutritional value and anti-nutritional factors of Calliandra calothyrsus leaf powder.Under the same conditions,Lactobacillus reuteri (A),Lactococcuslactis (B) and Bacillus subtilis (C) were used for solid-state fermentation,and the inoculum amount were single strain (2% v/m) and double strains combination (2%/2%),three strains combination (2%/2%/2%).The results showed as follows:①Compared with the unfermented leaf powder (CON),fermentation treatments significantly reduced the contents of ether extract(EE) and tannin in Calliandracalothyrsus leaf powder (P<0.05).The crude protein (CP) content in B,A+B,A+C and A+B+C groups were significantly increased (P<0.05),while the ash content was significantly increased in C,A+B,A+C,B+C and A+B+C groups (P<0.05).Different fermentation treatments had no significant effect on NDF,ADF,Ca,P,carbohydrate and energy (P>0.05).② The content of asparagine in A+B+C group was significantly higher than that in A,B,C,A+B,and A+C groups (P<0.05).The serine content in B+C group was significantly higher than that in CON,A,B,C,and A+B+C groups (P<0.05).The contents of glutamate,alanine,phenylalanine and proline in A+B+C group were significantly higher than those in other groups (P<0.05).The cysteine content in the CON group was significantly higher than the other groups (P<0.05).The contents of methionine,glycine,leucine,isoleucine,lysine,histidine,arginine,total animo acids (TAA),essential amino acids (EAA),nonessential amino acids (NEAA),EAA/TAA,and EAA/NEAA in CON and A+B+C groups were significantly higher than those in other groups (P<0.05).Different fermentation treatments had no significant effect on the contents of threonine,valine and tyrosine in Calliandra calothyrsus leaf powder (P>0.05).③For Calliandra calothyrsus leaf powder(after and before fermentation),EAA/TAA and EAA/NEAA were close to the ideal protein of FAO/WHO,the TEAA,threonine,leucine,and tyrosine+phenylalanine were higher than egg protein or FAO modes.While the content of cysteine+methionine was lower than egg protein and FAO modes after fermentation.In summary,Calliandra calothyrsus leaf powder (after or before fermentation) was a high-quality protein feed,and A+B+C group had the best effect.

In order to study the effect of the amount of antimicrobial peptide on the immunity-related indicators and anti-injury ability of the serum and tissue of hybrid sturgeon.200 hybrid sturgeons(Russian sturgeon Acipenser gueldenstaedti♀×Amur sturgeon Acipenser schrencki Brandt♂) with robust physique and uniform size were selected and randomly divided into 4 groups.The fishes in control group were fed with basal diet,the fishes in experiment groups were fed with composite additive of antimicrobial peptides with level of 400 mg/kg (group Ⅰ)、800 mg/kg (group Ⅱ) and 1 200 mg/kg (group Ⅲ),respectively.The experiment lasted for 30 d at water temperature of (16.0±1.0) ℃.It was found that the contents of albumin (ALB) and total protein(TP) in serum and tissues were increased at first and then decreased with the increase of dietary antimicrobial peptides concentration,and the contents of ALB and TP in groupⅡ significantly higher than the control group (P<0.05).The activities of lactic dehydrogenase (LDH) and glutamic oxalacetic transaminase (GOT) were shown to be decreased at first and then increased with the increase of antimicrobial peptides concentration in serum.In serum and tissues the activity of GOT in groupⅠwas significantly lower than that in other groups (P<0.05),and the activity of GOT and LDH in group Ⅲ were significantly higher than those in other groups (P<0.05).The total cholesterol (TCHO) contents were gradually decreased with the increase of the concentration of antimicrobial peptides in tissues,and the TCHO contents in group Ⅲ were significantly lower in tissues than those in other groups (P<0.05).The activity of lysozyme (LZM) was gradually increased with the increase of the concentration of antimicrobial peptides,and the activity of LZM was significantly higher in groupⅡ and groupⅢ in heart,spleen and small intestine than that in other groups (P<0.05).The addition of antimicrobial peptides leaded to effectively improve the nonspecific immunity and the damage resistant ability of hybrid sturgeon,and antibacterial peptides had the best effect when the dosage was 400-800 mg/kg.

This study was aimed to excavate the genes affecting follicular development with the mRNA transcriptome difference between large and small follicles in two different goats,in order to provide a reference for further elucidating the mechanism of goat follicular development.Synchronization of estrus was carried out on the Chuanzhong Black and Leizhou goats with cloprostenol sodium.The single follicle (large follicle>6 mm,small follicle<3 mm) was isolated from ovaries which were collected from estrus goats mentioned above with the stereomicroscope.The total RNA was extracted from follicle tissues for RNA-seq,the profiles of mRNA expression were calculated with bioinformatics and differentially expressed genes (DEGs) were performed in GO function and KEGG pathway enrichment.The results showed that 4 451 DEmRNAs were obtained from different follicles in Chuanzhong Black goats,and 2 335 DEmRNAs were obtained from Leizhou goats,two goat breeds 1 771 genes.The common (INHBA,INHA,CYP19A1,KITLG,LHCGR and STAR) and unique (IGFBP6,BMP6 and BMPR2) genes related to follicular development were screened out.In addition,the new genes (GADD45B,TC2N and MSMO1) potentially affecting the reproductive performance in two goat breeds were also identified.GO function analysis showed that the DEGs in two goat breeds were mainly related to the transmembrane transport activity.KEGG pathway analysis revealed significant changes in steroidogenesis,cytokine-cytokine receptor interaction and cAMP signaling in both goat breeds.It was also found that the difference of cytochrome P450 metabolism in steroidogenesis when follicle developed could be a potential factor in the high litter size of Chuanzhong Black goats.The results provided a reference for further exploring the gene functions of follicle development and the research in reproductive performance differences of different goat breeds.

This study was aimed to identify the transforming growth factor-β (TGF-β) gene family in River buffalo,detect their expression profiling,and analyze the possible function of TGF-β gene in preimplantation embryo development.According to the reported TGF-β gene in human,TGF-β family members in River buffalo were identified at the whole genome level by BLASTP,the TGF-β gene information in Bos taurus,Capra hircus and Mus musculus were characterized.Phylogenetic tree of TGF-β gene family was constructed to elucidate evolutional relationship among these five species.The expression of TGF-β gene was calculated by Fragment of Per Kilobase of exon model per Million mapped reads (FPKM) based on RNA-seq data at four stages of preimplantation embryo development (2-cell,8-cell,morula and blastocyst).The results showed that there were 32,23,32 and 26 TGF-β gene in River buffalo,Bos taurus,Capra hircus and Mus musculus,respectively.The similar numbers of TGF-β gene among five species and uniform distribution in the phylogenetic tree indicated that the conservation of TGF-β gene distribution and function among different species.In 32 TGF-β gene of River buffalo,21 TGF-β gene did not express (or extremely low express) in preimplantation embryo development,6 TGF-β gene which had been identified as inhibitor,exhibited low expression level.5 TGF-β gene,which were involved in reproduction,showed high expression level,which suggested that only partial of TGF-β gene (5/32) rather than most of them (27/32) were involved in preimplantation embryo development of River buffalo.The results provided the foundation for the study of TGF-β gene family in River buffalo,and provided a theoretical basis for the functional analysis of TGF-β gene family in early embryonic development.

In order to explore the correlation between growth traits and some immune indexes of high-quality chickens,13-week-old Xueshan chickens were taken as the materials,and the roosters and hens were divided into three groups according to their body weight.Roosters:High weight group(≥1 300 g),medium group(1 100~1 300 g) and small weight group(≤1 100 g);Hens:High weight group(≥1 200 g),medium group (1 000~1 200 g) and small weight group (≤1 000 g).The body weight,body size(shin length and shin circumference) and immune indexes(the serum concentrations of IL-1β,α-interferon,IgM and IgG) of the three groups were measured,respectively.The correlation between body weight,body size and immune indexes were analyzed.The results showed that there was a significantly positive correlation between the body weight and shin length and shin circumference (P<0.05) of 13-week-old Xueshan chickens,and the trend was consistent in different sex.All immune indexes showed significant difference between different weight groups (P<0.05),and the trend of rooster and hen was the same.The serum concentrations of α-interferon,IgM and IgG in the high weight group were significantly lower than those in the medium and small weight groups (P<0.05).The concentration of IL-1β in the high weight group was significantly higher than that of the medium and small weight groups (P<0.05).Except for the inflammatory factor IL-1β,body weight was negatively correlated with immune traits in roosters,with a correlation coefficient ranging from -0.271 to -0.248.Body weight in hens was negatively correlated with IgM and α-interferon (P<0.05),the correlation between body size and immune traits was not significant.There was a significant positive correlation between body weight and body size (P<0.05).There was also a significant positive correlation between immune indexes,especially between cellular immunity and humoral immunity.The results showed that the growth traits of Xueshan chicken had a significant effect on immune performance,and there was an antagonistic relationship between body weight and immune indexes.

This study was aimed to clone and analyze the CYP19A1 gene in laying hens by bioinformatics and investigate its expression in different tissues and egg production.The Taihang chicken in Hebei was taken as the research object.The CYP19A1 gene CDS region was cloned and sequenced by designing primers,and its functional structure was predicted by bioinformatics software.The results showed that the chicken CYP19A1 gene contained a 1 509 bp open reading frame (ORF) encoding 502 amino acids.The results of homology alignment and phylogenetic tree analysis indicated that the sequence of the Taihang chicken CYP19A1 gene was close to that of duck (90.7%).The physicochemical properties of CYP19A1 protein were predicted and analyzed.The structure formula of CYP19A1 protein was C₂₆₀₂H₄₁₀₃N₆₇₅O₇₁₉S₃₆ and the molecular mass was 57.51 ku.The theoretical isoelectric point was 5.99,with leucine being the highest abumdance.The CYP19A1 was hydrophilic protein.There was portions that could form alpha helix (58.95%),extended chain (2.18%),beta sheet (3.93%) and random coils (34.93%).The results of tissue expression profiling showed that the gene was expression in all the tissues,with the highest abundance in ovary.The results of Real-time PCR showed that the expression level of CYP19A1 gene in ovary issure of high-yield group was significantly higher than that of low- yield group (P<0.05).The above results provided important research data for studying the laying performance of the Taihang chicken.

The purpose of the experiment was to understand the contamination of the psychrotrophiles in the large-scale cattle farms in the north and south Xinjiang,and to provide scientific basis for the establishment of effective prevention and control measures.Total of 396 samples were collected from 8 sampling points of 6 cattle farms in Wuchang and Kashgar regions under similar conditions of temperature (TM) and relative humidity (HR).GB 4789.2-2016 National Food Safety Standard for Food Microbiological Testing Determination of Total Colonies was used to isolate and culture psychrotrophiles,and BLAST sequence identification was performed.According to the results,276 strains of halophilic bacteria were isolated from Wuchang region and Kashgar region.Through %ID identification,15 dominant psychrophilic bacteria were identified,including Bacillus thuringiensis,Pseudomonas,Bacillus cereus,Bacillus,Acinetobacter,Bacillus circulans,Bacillus licheniformis,Psychrobacter,Streptococcus sanguis,Bacillus megaterium,Arthrobacter,Pseudomonas fluorescens,Streptococcus,Paenibacillus,Exiguobacterium.Compare the differences between the two places,the results showed that the total number of strains isolated in bed soil,feed and air were the most in Wuchang and Kashgar area.The number of chilling bacteria in 7 environmental sites except the environment of the pipeline group in Kashgar area were lower than that in Wuchang area.Among them,the amount of bacteria in the bed soil,milk tank mouth and feed in Kashgar area were extremely significant or significantly lower than that in Wuchang area (P<0.01;P<0.05).The amount of bacteria in the mouth of the pipeline was significantly higher than that in Kashgar (P<0.01).To sum up,under similar conditions of temperature,humidity and illumination,the management level had a great influence on the contamination of psychrophiles in cattle farms in north and south Xinjiang and the corresponding control measures should be established in different regions.This study provided a theoretical basis for exploring the source of raw milk pollution in north and south Xinjiang.

The purpose of this study was to determine the optimal conditions for the rotation culture of CH/GX/750A/2015 strain of porcine epidemic diarrhea virus (PEDV) on Vero cells,and provide technical support for the mass production of high-efficiency vaccine.The virus was cultured on Vero cells in a 10 L spinner flask.The conditions of trypsin concentrations(0,2,4,6,8,10 μg/mL),cell density(40%,60%,80%,100% and 200%),virus inoculation dose (MOI=1,0.1,0.01 and 0.001),virus adsorption time (0,30,60,90,120 and 240 min),virus harvest time (12,24,36,48,60,72 h after virus inoculation),culture temperature after virus inoculation(35,36,37 and 38 ℃) and spinner speed(6,8,10,12,14 and 16 r/h) were optimized.The results showed that the best culture condition of PEDV CH/GX/750A/2015 strain in 10 L transfer flask was to inoculate the virus when the cell was just covered with a single layer,the dose of virus was MOI=0.01,the concentration of trypsin in the culture medium after virus inoculation was 6 μg/mL,without adsorption,the culture temperature after virus inoculation was 37 ℃,the best spinner speed 12 r/h,and the best virus harvest time was 48 h after virus inoculation.After optimization,the virus titer can reach 10^6.50 TCID₅₀/0.1 mL.This study provided a reference for the large-scale culture of PEDV mutant strain,and laid a foundation for the development of high-efficiency vaccine of PEDV mutant strain.

The purpose of this study was to investigate the infection of Piroplasma in yak in all 18 counties (cities) of Ganzi Tibetan autonomous prefecture (Ganzi prefecture).From June to November 2018,a total of 1 381 whole blood samples of clinical healthy yak were collected in all 18 counties of Ganzi prefecture.A nested PCR targeting 18S rRNA was used to detect Piroplasma.Furthermore,a total of 124 positive PCR products were randomly selected to identify parasite species.The results showed that 438 of 1 381 yak blood samples were detected as Piroplasma positive with an average positive rate of 31.72%.Specifically,the 385 of 1 093 yak blood samples were detected as Piroplasma positive in semi-agricultural and semi-pastoral counties,the average positive rate was 35.22%.53 of 128 yak blood samples were detected as Piroplasma positive in pure pastoral counties,the average positive rate was 18.40%.In 124 positive PCR products,the species infecting yak were Theileria sinensis (76),Theileria luwenshuni (25),Theileria orientalis (3),Theileria ovis (2),Babesia bigemina (1) and Theileria buffeli (1),respectively.The infection rate of Theileria sinensis was 61.29%,and the infection rate of Theileria luwenshuni was 20.16%.The results in this study showed that the infection rate of Piroplasma in yak was high in Ganzi prefecture,and was different among regions,the predominant species were Theileria sinensis and Theileria luwenshuni,contributing to the prevention and control of piroplasmosis in yak in Ganzi prefecture.

The purpose of this study was to establish a double antibody sandwich ELISA method for quantitative detection of classical swine fever virus (CSFV) E2 recombinant protein.CSFV WH303 monoclonal antibody was used to coat 96 well enzyme-linked plate,CSFV 1B6 monoclonal antibody was used to connect HRP as enzyme-linked antibody,CSFV-E2 protein expressed and purified by baculovirus was used as standard protein,and a quantitative method of double antibody sandwich ELISA was established.The purity of standard protein was detected by SDS-PAGE and the concentration of standard protein was quantified by BCA protein quantitative method.The standard was diluted to the linear range and draw the standard curve.Chessboard method was used to determine the optimal dilutions of monoclonal antibody,enzyme labeled antibody and standard protein.The repeatability and stability of the method were verified intra-assay and inter-assay.SDS-PAGE result that the purity of CSFV E2 protein standard was more than 95%,and the concentration of CSFV E2 protein standard by BCA protein quantitative method was 1.553 mg/mL.The concentration of the coated monoclonal antibody was 0.1 g/mL,and the optimal dilution of the enzyme labeled antibody was 1:400.The dilution concentration of CSFV E2 protein standard was in the range of 2.43 to 77.65 g/mL,and the correlation coefficient R² was above 0.99.The coefficients of variation of repeatability test of intra-assay and inter-assay were both less than 15%.A double antibody sandwich ELISA method for the quantitative detection of CSFV E2 protein was successfully established.The method had good repeatability and stability,and provided an effective method for the quantitative detection of CSFV E2 protein.

The study aimed to investigate the effect of tetramethylpyrazine(TMP) supplementation on Listeria monocytogenes loads,listeriolysin O (LLO),proinflammatory factors (TNF-α,IL-1β and IFN-γ),immune organ index and natural immunoglobulins of Rex rabbits infected with Listeria monocytogenes.A total of 180 Rex rabbits were randomly assigned to 5 groups with 6 replicates of 6 rabbits.The control group was fed with the basal diet,and the other four groups were fed with the addition of TMP at 0,50,100 and 150 mg/kg of diet,respectively.Each Rex rabbit in the four treatments was fed with 1 mL (10⁷ CFU) of Listeria monocytogenes,and the control group was fed with the culture medium of aseptic strainat first day of feeding trial.The trial lasted for 14 days.The results showed that TMP addition decreased the loads of Listeria monocytogenes in the spleen,liver and lymph nodes (P<0.05).The pathogen loads in caecal content,liver,spleen and lymph nodes linearly decreased (P<0.05).Also,TMP decreased serum LLO levels and proinflammatory factors TNF-α and IL-1β (P<0.05).Linearly decreased for LLO and linearly and quadratically decreased for TNF-α and IL-1β (P<0.05) at 14 d.The indexes of spleen and liver and the serum levels of IgA and IgG inhanced with the increasing doses of TMP,but the spleen index on 7 d post administration linearly decreased (P<0.05).In conclusion,supplemental TMP in the rabbit diet could attenuate Listeria monocytogenes infection by reducing the pathogen loads and virulence,increasing immune organ indexes and natural immunoglobulins.

Chlamydiosis of animals is caused by Chlamydia infection,and the clinical symptoms such as abortion,stillbirth or decreased egg yield usually appear in the infected animals.The disease is widespread in cattle,sheep,pigs,chickens,ducks and other livestock,as well as foxes,deer and other special economic animals,seriously hindering the healthy development of animals.This paper reviews the current prevention and control of animal chlamydiosis,mainly including vaccine immunization and drug treatment.Vaccine immunization is the most economical and effective method to prevent animal chlamydiosis at present,and drug treatment is the best choice for animals infected with Chlamydia.This paper focuses on the characteristics and the latest research progress of inactivated vaccine,attenuated live vaccine,DNA vaccine,subunit vaccine,live vector vaccine and other vaccines.The drug treatments mainly introduced the research progress of antibiotics and traditional Chinese medicine,the prospect of other drug treatment was explored,and the future prevention and control of animal chlamydia disease was prospected.

To investigate the correlation between transposon and integron carrying and multiple drug resistance of Escherichia coli,259 strains of Escherichia coli were isolated from 12 large-scale pig farms by collecting anal swabs.Minimum inhibitory concentration (MIC) values of isolated strains against 7 classes (16 kinds) of antibacterial drugs were measured by microbroth dilution method,and the carrying of transposons and integrons was detected by PCR.The results showed that the tested strains showed different degrees of resistance to 7 classes (16 kinds) of antibacterial drugs.It was highly resistant to spectinomycin,gentamicin and tetracycline,and the drug resistance multiple of MIC₉₀ was 32 times.It was sensitive to imipenem and polymyxin B,and the drug resistance multiple of MIC₉₀ was 2 times,while the resistance of other drugs was between them.The multiple drug resistance up to 7 times,2 times resistant up to 100%.There were 14 types of multidrug resistance spectrum,mainly including β-lactams+aminoglycosides+tetracyclines+amidoalcohols+sulfonamides+fluoroquinolones,β-lactams+aminoglycosides+tetracyclines+amidoalcohols+sulfonamides+fluoroquinolones+peptides,accounting for 56.97%.The detection rate of transposons and integrons was higher.There were 20 types of transposon and integron phenotypes.Among them,the combination types of IScp1+IS26+tnpA+tnp513+intI1 and IS26+tnpA+tnp513+intI1 accounted for the most,48.48% and 26.06%,respectively.The results showed that insertion element IScp1 was significantly correlated with β-lactam drug resistance (P<0.05,P<0.01).The insertion element IS26 was extremely significantly correlated with ampicillin resistance (P<0.01) and significantly correlated with sulfadioxazole resistance (P<0.05).Integron intI1 was extremely significantly correlated with ampicillin resistance (P<0.01),IntI2 was extremely significantly correlated with cefetadime resistance (P<0.01),and significantly correlated with cefetifur,enoxacin and imipenem resistance (P<0.05).The dominant combination type IScp1+IS26+tnpA+tnp513+intI1 was extremely significantly correlated with the drug resistance of compound sulfamethoxazole (P<0.01).The combination type of IS26+tnpA+tnp513+intI1 was extremely significantly correlated with the drug resistance of compound sulfamethoxazole (P<0.05) and was extremely significantly correlated with ceftifur resistance (P<0.01),but it was negatively correlated with gentamicin resistance (P<0.05).The multidrug resistance of the tested strains to β-lactams,fluoroquinolones,sulfonamide and aminoglycosides were significantly or extremely significantly correlated with transposon and integron carrying type.

In order to confirm the cause of death of Tupaia belangeri yaoshanensis in the artificial domestication and breeding experiment center,the pathogenicity and drug resistance of the isolated strain were investigated.Pathogenic bacteria was isolated and obtained by plate drawing,and further morphological observation,culture characteristics analysis,biochemical test and 16S rRNA sequence analysis were carried out on the isolated bacteria.A pathogenic Aeromonas hydrophila was purified and obtained.The lethal dose 50%(LD₅₀) of the isolated strain was determined by pathogenicity test on Kunming mice.At the same time,the sensitivity of Aeromonas hydrophila isolates to common drugs was analyzed using the paper sensitive method,and the drug resistance genes of the isolates were detected by PCR.The results showed that a strain of Aeromonas hydrophila was isolated from the diseased tree shrews and the microscopic examination showed Gram-negative bacteria in a short rod shape.The 16S rRNA sequence of this strain showed that a strain of Aeromonas hydrophila was isolated from the diseased tree shrew,which was 100% homologous to the Aeromonas aeruginosa collected by GenBank SWCY-3.27 isolated from freshwater lake,and had the closest genetic evolution.The LD₅₀ of the isolates to adult Kunming mice was 1×10⁷ CFU,which was more virulent than the model strain ATCC 7966 and similar to the prevalent strains NJ-35 and J-1,etc.in China.The pathogenicity test identified the isolates as highly virulent.The biochemical identification of esculin,arabinose and oxidase was positive,and various biochemical identifications were consistent with the biochemical characteristics of Aeromonas hydrophila.The drug sensitivity test showed that the strain had high sensitivity to 9 drugs such as minocycline,streptomycin and tobramycin,completely resistant to 10 drugs such as tetracycline,erythromycin and penicillin.PCR amplification was used to detect 12 drug resistance genes of 6 major categories in the isolated bacteria,and it was found that there were two antibiotic resistance genes of aminoglycoside (Aph-(3)-lia) and β-lactams (mecA) in the isolated bacteria,which were basically consistent with the drug sensitivity test results.This study indicated that the pathogen causing the death of tree shrew was Aeromonas hydrophila,which could be treated with high-sensitivity drugs,and provided reference for the prevention and treatment of tree shrew diseases caused by Aeromonas hydrophila infection.

To investigate the antimicrobial resistance and the prevalence and tranmission of extended-spectrum β-lactamase CTX-M,a total of 199 samples were collected from geese and the environment from 10 geese farms in Jiangmen and Yangjiang of Guangdong.Isolation and identification of the Escherichia coli (E.coli) were performed by MALDI-TOF-MS.Antibiotic susceptibility test of E.coli strains was characterized using agar dilution method.The bla_CTX-M gene and its genetic environment were detected by PCR analysis among cefotaxime-resistant E.coli isolates.Pulsed-field gel electrophoresis (PFGE),conjugation experiments and plasmid replicon types were conducted to determine the transmission of bla_CTX-M gene.A total of 196 E.coli strains were obtained and antimicrobial susceptibility testing showed that over half of these isolates were resistant to ampicilin,florfenicol,doxycycline and streptomycin.Meanwhile,10% to 25% were third-generation cephalosporins-resistant,and 49 (24.6%) isolates showed resistance to cefotaxime.For comparison,resistance rates of all the tested drugs among E.coli strains from Yangjiang and geese were generally higher than the ones from Jiangmen and environment,respectively,especially for cefotaxime and ceftiofur (P<0.05).Among the cefotaxime-resistant E.coli strains,19 possessed bla_CTX-Mgene,including bla_CTX-M-55(n=17),bla_CTX-M-27(n=1) and bla_CTX-M-65(n=1).All of the bla_CTX-M-carrying strains showed multidrug-resistant phenotype against 5 to 11 kinds of antimicrobial agents.The ISEcp1-bla_CTX-M-55-orf477 structure was found in all of the 17 bla_CTX-M-55-carrying E.coli strains with three different spacer sequences between ISEcp1 and bla_CTX-M-55,while the ISEcp1-bla_CTX-M-27/65-IS903 structure was identified in 2 isolates harboring bla_CTX-M-9G.Ten pulsed-field gel electrophoresis (PFGE) patterns were found in 19 bla_CTX-M-positive E.coli strains,with a dominant profile (47.4%),including isolates from various sources,indicated the clonal transmission of bla_CTX-M-positive isolates in local regions.The bla_CTX-Mgene was successfully transferred among 12 isolates (63.2%) carrying bla_CTX-M,and IncFⅡ plasmid (n=10) was the most prevalent replicon identified among these bla_CTX-M-carrying transconjugants,followed by IncHⅠ2 (n=2).The doxycycline- and florfenicol-resistant phenotypes were co-transferred with bla_CTX-M gene.In conclusion,our study revealed that it was serious for the antimicrobial resistance of E.coli strains in Yangjiang and a relatively high prevalence of bla_CTX-M gene,mainly bla_CTX-M-55 gene,was found.The clonal spread and horizontal transfer via plasmids and ISEcp1 element were major factors contributing to the dissemination of bla_CTX-M gene in the geese farms in Guangdong.It should be further monitored and attached more attention.

In order to investigate the virulence and drug resistance of Klebsiella pneumoniae from Nanning companion animals,the fecal swabs of dogs and cats were collected,and the bacterial characteristics were analyzed by isolation and culture,morphological observation,drug sensitivity test and PCR amplification of 16S rRNA,khe gene,virulence genes and drug resistance genes.The results showed that four of the isolated strains could form a liquid colony on MacConkey medium,and they were short rod-shaped Gram-negative bacteria,suspected to be Klebsiella pneumoniae.The results of 16S rRNA sequencing showed that the homology between the isolated strain and Klebsiella pneumoniae was 99%,and Klebsiella pneumoniae specific gene (khe) was positive.Among them,GXKP-C14 and GXKP-D15 were sensitive to most commonly used antibiotics,GXKP-D1 showed high level of multi-drug resistance,followed by GXKP-D4 which showed ampicillin,piperacillin,cefuroxime,tetracycline,doxycycline,nitrofurantoin and sulfamethoxazole resistance,but sensitive to cefepime,meropenem,imipenem,amikacin,gentamicin,streptomycin and colistin.Some strains carried drug-resistant genes such as tet(A),QnrS,bla_SHV,sul2,mcr-1 and virulence gene WabG.The results of this study provided experimental basis for the detection,diagnosis and treatment of Klebsiella pneumoniae in dogs and cats.At the same time,the detection of colistin resistance gene mcr-1 would provide a reference for the control and rational use of polymyxin resistance.

This study was aimed to explore the antibacterial mechanism of the main components of Chinese traditional medicine Coptidis Rhizoma based on network pharmacology.The effective components and target targets of Coptidis Rhizoma were screened from the TCMSP database,and the corresponding target genes were transformed using the Uniprot database.The active components and target genes were imported into the Cytoscape 3.6.1 software to construct an active component-target network diagram.Combined with the GeneCards database to search the antibacterial related targets of Coptidis Rhizoma,the core target genes were uploaded to the STRING platform to build a protein interaction network.Finally,the DAVID database was used to analyze key targets by GO biological processes and KEGG pathway enrichment.The results of network pharmacology showed that 9 effective components of Coptidis Rhizoma acted on 105 antibacterial targets and participated in 22 biological processes (18 biological processes,3 molecular functions,1 cell composition) and 19 related signal pathways.Among them,the main active ingredients of Coptidis Rhizoma were quercetin,(R)-Canadine,berlambine,berberine and palmatine,etc.Antibacterial targets were IL2,VCAM1,STAT1,NOS2,TGFB1,CASP9,MMP1,NFKBIA and CXCL10,etc.The involved biological processes mainly included inflammatory response,positive regulation of RNA polymerase Ⅱ promoter transcription,response to lipopolysaccharide,negative regulation of apoptotic process,cell adhesion,etc.Mainly act on Toll-like receptors signal pathway,TNF signal pathway,NF-kappa B signaling pathway,etc.Coptidis Rhizoma might through its active ingredients such as quercetin,berberine,palmatine act on other key targets such as IL2,VCAM1 and STAT1 to participate in the Toll-like receptor signaling pathway and TNF signaling pathway to exert antibacterial effect.It fully reflected the antibacterial action mechanism of Coptidis Rhizoma with multiple components,multiple targets and multiple pathways,and provided a theoretical reference for further clarifying the antibacterial mechanism of Coptidis Rhizoma.

The purpose of the experiment was to evaluate the feeding safety of Tribonema by the acute toxicity and subchronic toxicity test on rats.5 000 mg/(kg·BW) of Tribonema powder was given to Wistar rats by gavage for acute toxicity test.The rats were fed with 0,1 250,2 500 and 5 000 mg/kg of Tribonema powder for 90 days.The weight and feed consumption of rats were measured every week.The rats were dissected at 45 and 90 days after feeding.The blood routine and biochemical indexes were detected.The organ coefficient was calculated and the main organs were examined by histopathology.The results showed that there was no death in the rats treated with 5 000 mg/(kg·BW) of Tribonema powder.LD₅₀ was more than 5 000 mg/(kg·BW).According to WHO’s toxicity rating of chemicals,Tribonema powder belonged to the actual non-toxic substances.In the subchronic toxicity test,there was no significant difference in the average feed consumption,body weight growth and organ coefficient of rats fed with high,medium and low doses of Tribonema powder after 90 days compared with the control group (P>0.05).There was no significant difference in blood routine indexes and blood biochemical indexes between low dose group and control group (P>0.05).Besides the blood total cholesterol (CHO) and triglyceride (TG) in the high and middle dose groups were significantly lower than those in control group (P<0.05),there was no significant difference in other blood biochemical indexes (P>0.05),but the CHO and TG in the high and middle dose groups were all in the normal biochemical indexes of rats within the scope.No significant changes were found in gross anatomy and HE staining of main organs.The results of this experiment showed that the Tribonema powder belonged to the actual non-toxic substance,the rats were fed with 5 000 mg/kg for 90 days,and no obvious harmful effect was observed,which was expected to be applied in animal feed.

For the further development and utilization of the drug,wild medicinal plant Dicranostigma leptopodum (Maxim.) Fedde in Pingliang area of Gansu province was sekcted in this experiment as the research object,the essential oil components were extracted by steam extraction,distillation extraction (SDE) and supercritical CO₂ extraction.Chemical composition of essential oil was analyzed by GC-MS,and the antibacterial activity of supercritical CO₂ extracted essential oil was determined against Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa using plate count method.The results showed that the essential oil obtained by steam extraction and distillation extraction was orange yellow and turbid.The essential oil obtained by supercritical CO₂ extraction was light yellow and clear liquid.The essential oil extracted by the 3 methods was all contained 24 common components.The supercritical CO₂ extraction method was the best,and the extraction rate was 2.1220%.The steam extraction method was the lowest,and the extraction rate was 1.7870%.Supercritical CO₂ extracted essential oil was applied to 3 strains of bacteria tested,and the minimal inhibitory concentration (MIC) was determined as Escherichia coli 0.2500 g/mL,Staphylococcus aureus 0.3000 g/mL and Pseudomonas aeruginosa 0.2600 g/mL,respectively.The bactericidal inhibitory effect was significant.The results showed that supercritical CO₂ extraction was the best method to extract the essential oil of Dicranostigma leptopodum (Maxim.) Fedde,which had good antibacterial effect on Escherichia coli,Staphylococcus aureus and Pseudomonas aeruginosa.

Baiqin extract is composed of Phellodendron chinense,Scutellaria baicalensis,Carthamus tinctorius,Codonopsis pilosula,etc.In order to investigate its therapeutic effect against cow endometritis,the main pharmacodynamics was studied by using experimental rabbit model of endometritis.Besides,the clinical efficacy was evaluated according to the 'Guideling principles for the safety and efficacy test of antimicrobial veterinary drugs for the prevention and treatment of clinical endometritis in dairy cattle' which was issued by the Ministry of Agriculture and Rural Affairs of the People's Republic of China.The results of the pharmacodynamic experiments revealed that Baiqin extract could obviously improve the clinical symptoms of experimental endometritis in rabbits as compared to model group.It has been examined that differential leukocyte counting,uterus secretion smear microscopy,chemical examination of urine and uterus secretion were returned to normal,and abnormal histopathological changes of uterus such as hyperemia,inflammatory cells infiltrates and shedding of epithelial cells were partially alleviated in the uterus of rabbits.Clinical efficacy results showed that at 10th day after administration,the curative rates against endometritis in the high- and medium-dose groups of Baiqin extract were extremely significantly higher than that in the low-dose group (P<0.01),and were no significant difference compared with the positive drug group (P>0.05);While there was no significant difference in the total effective rates among all the tested groups (P>0.05).On 21st day post-treatment,the curative rates in the high- and medium-dose groups of Baiqin extract were significantly higher than that in the low-dose group (P<0.05),and were no significant difference compared with the positive drug group (P>0.05);While there was no significant difference in the total effective rates among all the tested groups (P>0.05).These results indicated that Baiqin extract had a good therapeutic effect on rabbit experimental endometritis and cow clinical endometritis,and the therapeutic efficacy of 1.5 or 1.0 g/mL concentration (30 mL at a time) on clinical cases was excellent,and similar with that of oxytetracycline,the therapeutic efficacy of 0.5 g/mL was non-obvious.The study would lay a foundation for further research and development.

With the extending of the livestock and poultry farming,veterinary drugs are widely used to treat diseases or as growth promoters.From 2010 to 2017,the sales of veterinary antibacterial drugs increased from 31 500 to 56 800 t.The irrational application of veterinary drugs,improper handling of wastes in livestock and poultry breeding,and the excretion of unchanged or metabolites of veterinary antibiotics are the main reasons of environmental pollution caused by veterinary drugs.After entering the environment,the migration and transformation of veterinary drugs has caused serious harms,including the emergence of resistant bacteria or antimicrobial-resistant genes,and the exposure of veterinary drugs in plants and animals’ products as well as drinking water,which indirectly affect human health and the ecological toxicity of non-target animals.The paper discussed the processes of environmental exposure,fate and the impacts on the ecological environment of veterinary drugs.Then the importance of environmental risk assessment of veterinary drugs was analyzed.Finally,the potential ways to find measures and solve the environmental exposure risks of veterinary drugs were prospected.This review will help to attract people's attention to environmental risk assessment of veterinary drugs.