To explore the specific regulation mechanism of myostatin (MSTN) gene on muscle development of bovine,a total of ten healthy bovine were selected from the same farm,including five MSTN^-/- gene-edited bovine and five wild-type bovine,and extracted the RNA of bovine muscle tissue sample.Collecting the calf-hip muscle samples of two groups,and the transcriptome analysis were sequenced by Illumina high-throughput sequencing technology.Bioinformatics method was used to compare the differentially expressed genes between two groups,and Go and KEGG enrichment analysis were performed.Real-time quantitative PCR was used to verify the transcriptome sequencing data.A total of 18 071 genes were obtained from the gene-edited bovine and wild-type bovine.Under the conditions of log₂|FoldChange|≥ 1.48,a total of 406 significantly differentially expressed genes were screened,in which 347 genes were up-regulated and 59 genes were down-regulated.GO functional analysis results showed that total 915 functional categories were effected after MSTN gene editing (P<0.05).The differentially expressed genes were mainly involved in the binding,regulation of biological process,immune system process and other related functions.KEGG functional enrichment analysis results indicated that differential genes were involved in total of 211 pathways,mainly concentrated in cell adhesion molecule,chemokine,cytokine interaction and other signaling pathways.The differentially expressed genes might be related to cell growth and muscle development were further screened out,including CD14,KIT,CSF1R,FBP1,DUSP4,ULBP21,PRKCB,SPN,CHAD and SRC genes.Real-time quantitative PCR results showed that the expression levels of the differentially expressed genes were basically consistent with the transcriptome expression,indicating the reliability of sequencing results.The results of this study indicated that MSTN gene could mediate the expression of multiple downstream genes after exert its role,effecting the relevant signaling pathways and biological processes.The screened differentially expressed genes in this study could be used as candidate targets for further study of the regulation mechanism of skeletal muscle.

The purpose of this study was to establish porcine aminopeptidase N(pAPN)gene knockout IPI-2I cell lines by CRISPR/Cas9 system,and to further investigate the role and interaction mechanism of pAPN in the process of coronavirus invasion at the cellular level.Two sgRNA vectors (pX330-GFP-g3 and pX330-RFP-g5) targeting the second exon of the pAPN gene were co-transfected into IPI-2I cells.After 48 h of transfection,the GFP and RFP fluorescent was observed in most cells,and the positive cells with the RFP and GFP fluorescent label were sorted by flow cytometry for screening monoclonal cell line.Then genomic DNA was extracted from a small number of monoclonal cells,and the editing site of pAPN gene in monoclonal cell line was identified by PCR and sequenced for obtaining the pAPN gene knockout cell line.Before and after the pAPN gene knockout,the expression of pAPN protein in the IPI-2I cells was also detected by Western blotting.The results showed that most of the IPI-2I cells could simultaneously express GFP and RFP after 48 h of transfection,indicating that pX330-GFP-g3 and pX330-RFP-g5 vectors were transferred into IPI-2I cells.PCR and sequencing results suggested that a total of 48 fragment deletion monoclonal cells were obtained (the knockout efficiency was 15.5%),of which 16 were monoallelic knockout and 32 were biallelic knockout.Among 32 strains of biallelic knockout cells,23 strains of cells were homozygous fragment knockout.Western blotting results revealed that the expression of pAPN protein could not be detected in the homozygous fragment knockout cells.In summary,this study successfully constructed a pAPN gene homozygous fragment knockout IPI-2I cell line using CRISPR/Cas9 system,which laid the foundation for elucidating the mechanism of pAPN in the process of coronavirus invasion and preparing new pig breeds that could be resistant to disease.

The aim of this study was to obtain the coding sequences of long-chain acyl-CoA synthetase 6 (ACSL6) gene in Xinong Saanen dairy goats,and preliminarily explore its effect on lipid metabolism of mammary epithelial cells in dairy goats.Taking the primary mammary epithelial cells of Xinong Saanen dairy goats as experimental materials,ACSL6 gene was amplified and cloned,and tissue expression profiles were analyzed by Real-time quantitative PCR,bioinformatics analysis was carried out based on the sequence of ACSL6 gene by online software,RNA interference technology was applied to interfere the mRNA expression of ACSL6 gene in mammary epithelial cells of dairy goats.The results showed that the length of ACSL6 gene CDS region was 2 169 bp,encoded 722 amino acids.Bioinformatics analysis indicated that ACSL6 protein was an alkaline unstable protein.The results of tissue expression analysis showed that ACSL6 gene was highly expressed in mammary tissue,followed by spleen in dairy goats.The designed synthetic siRNA was transfected into mammary epithelial cells,interference of ACSL6 gene made the mRNA expression of acetyl-CoA carboxylase (ACC),stearyl-CoA dehydrogenase 1 (SCD1),cluster of differentiation 36 (CD36) and sterol regulatory element binding proteins 1 (SREBP1) genes extremely significantly decreased (P<0.01).This results laid the foundation for further study on the effect of ACSL6 gene on lipid metabolism at the protein level and individual level.

This study was aimed to clone BMP2 gene of Guangxi Bama Mini pigs and conduct bioinformatics analysis such as structure and function prediction,and analyze the difference in BMP2 gene expression between Guangxi Bama Mini pigs and Landrace pigs.The genomic DNA of Guangxi Bama Mini pigs was used as template and single nucleotide polymorphism (SNPs) in the exon region of BMP2 gene were identified by sequencing.Then,using variety of bioinformatics software to carry out a series of analysis on the promoter region,coding region and 3'UTR region of BMP2 gene in Guangxi Bama Mini pigs.The basic physical and chemical properties,phosphorylation site,O-glycosylation site,transmembrane region,hydrophilicity and hydrophobicity,protein modification structure,and secondary and tertiary structure of BMP2 protein in Guangxi Bama Mini pigs were predicted and analyzed.The miRNA binding sites and related pathways were analyzed,and the tissue expression profiles of Guangxi Bama Mini pigs and Landrace pigs were drawn.The results showed that the CDS region of BMP2 gene in Guangxi Bama Mini pigs was 1 188 bp in length,encoding 395 amino acids.Compared with pig reference genome BMP2 gene of Guangxi Bama Mini pigs had synonymous mutations in G1663A and G1897C,three missense mutations in C1896T (Pro→Leu),T1971C (Gly→Ser) and G2000A (Leu→Ser),and 13 miRNA binding regions in 3'UTR region,including miR-142-5p and miR-129-5p.The homology of Guangxi Bama Mini pig with Sus scrofa,Bos taurus,Bubalus bubals,Capra hircus,Ovis aries,Equus cabalus,Canis lupus dingo,Canis lupus familiaris and Macaca mulatta was 99.92%,91.75%,91.84%,91.92%,91.92%,90.50%,89.43%,90.91% and 90.91%,respectively.The results of phylogenetic tree showed that Guangxi Bama Mini pigs had the highest similarity with Sus scrofa,had a relatively close genetic distance with Bos taurus,and had a relatively distant genetic relationship with Macaca mulatta.The molecular weight of BMP2 protein was 44.55 ku,and the theoretical isoelectric point was 8.37.It was predicted that there were 12 O-glycosylation sites and 42 phosphorylation sites.The prediction analysis showed that BMP2 protein was hydrophilic and was an extracellular protein.Tissue expression profile showed that the expression of BMP2 gene in growth plate cartilage of Guangxi Bama Mini pigs was significantly lower than Landrace pigs (P<0.05).This study provided a theoretical basis for further exploring the function of BMP2 gene in Guangxi Bama Mini pigs,and also provided an important reference for the utilization of Guangxi Bama Mini pigs breed resources.

To explore the role of Ras homolog gene family,member A (Rhoa) and cyclooxygenase 2 (Ptgs2) molecules in the immune escape of Brucella,Rhoa and Ptgs2 genes of RAW264.7 cells were amplified and cloned to construct eukaryotic expression vectors and predict bioinformatics functions.The primers were designed according to Rhoa and Ptgs2 genes CDS sequences published in GenBank (accession No.:JN971019.1 and NM_011198).The total RNA of RAW264.7 cells was extracted and reverse transcribed into cDNA.The purified Rhoa and Ptgs2 fragments were respectively connected to the linearized pcDNA3.1 plasmid,and the recombinant plasmid was sequenced and identified by double enzyme digestion,and then transfected with Lipofectamine^TM 2000 to 293T cells,the expression of Rhoa and Ptgs2 genes was verified by Real-time quantitative PCR and Western blotting.Bioinformatics software was used to predict Rhoa and Ptgs2 genes.The results showed that the eukaryotic expression plasmids of Rhoa and Ptgs2 genes in RAW264.7 cells were successfully constructed,and was expressed at the transcription level by Real-time quantitative PCR detection.Western blotting results showed that Ptgs2 protein had an obvious band at 70 ku,while Rhoa protein had no band.Bioinformatics analysis results showed that the nucleotide sequences of Rhoa and Ptgs2 genes had high similarity and were relatively conserved among different species.RhoA had no signal peptide and was an unstable protein,while Ptgs2 had a signal peptide at 17-18 amino acids position and was a stable protein.Rhoa and Ptgs2 proteins had 12 and 53 potential phosphorylation sites,respectively,and the secondary and tertiary structures were mainly random coil.In this study,the eukaryotic expression vectors of Rhoa and Ptgs2 genes in RAW264.7 cells were successfully constructed,both were expressed at the transcription level,and their biological functions were analyzed,which provided a tool for the follow-up study of Rhoa and Ptgs2 genes in RAW264.7 cells in the immune mechanism of Brucella.

In order to explore the relationship between ovocalyxin-32 (OCX-32) gene overexpression and Ca²⁺ concentration and lipid indexes of chicken uterine epithelial cells.The Changshun Green-eggshell chicken uterine epithelial cells were isolated and cultured by collagenase Ⅳ method.The expression vector pcDNA3.1(+)+OCX-32+Flag was constructed by homologous recombination method and transfected into uterine epithelial cells.The overexpression of OCX-32 gene,the concentration of Ca²⁺ and the indexes of lipid were determined and the correlation was analyzed.The results showed that the concentration of total cholesterol (TCHO) in the experimental group was significantly higher than that in the control group (P<0.05),and the concentration of Ca²⁺ in the control group was significantly higher than that in the experimental group (P<0.05).The OCX-32 gene overexpression was negatively correlated with Ca²⁺ concentration (P<0.05),and significant positive correlation with triglyceride (TG) concentration (P<0.05),and extremely significant negative correlated with TCHO concentration (P<0.01).Meanwhile,TCHO concentration was extremely significant negative correlated with TG concentration (P<0.01).The results revealed that overexpression of OCX-32 gene could regulate Ca²⁺ concentration and lipid synthesis and transport in chicken uterine epithelial cells.The results of this study provided experimental basis and data support for further elucidating the regulatory mechanism of lipid and calcium secretion in chicken uterine epithelial cells.

In order to explore the regulatory mechanism of miR-150 in ovaries of sheep,the target gene prediction and analysis was performed with bioinformatic method.Mature miR-150 sequences were obtained from miRBase database for sequence alignment and conservative analysis.The Ensemble database was used to search the miR-150 precursor sequence,and the online software PROMO was used to predict the transcription factor binding site of miR-150.The target genes of intersection were predicted by TargetScan and miRwalk,and the Go function and KEGG pathway enrichment analysis of the predicted target genes were performed by DAVID and KOBAS online software.The functional protein interactions was analyzed with Networkanayst online tools.YM500V2 software was used to analyze the differential expression levels of miR-150 target genes in different tissues and diseases.The results showed that the miR-150 mature sequences between species were highly conservative.There were many transcription factor binding sites such as p53,ID1 and FOXO4 in the promoter region of miR-150.GO function enrichment analysis results showed that the predicted target genes of miR-150 mainly enriched in the positive regulation of RNA polymerase Ⅱ promoter transcription for biological process,zinc ions binding,DNA binding and transcription of DNA template for molecular functions,nuclear,nuclear and cytoplasmic and perinuclear area of cytoplasm for cellular components.KEGG pathway enrichment analysis results showed that target genes were significantly enriched in cancer pathways,proteoglycan in cancer,human papillomavirus infection,breast cancer,regulatory stem cell pluripotency and other signaling pathways.miR-150 target gene interaction analysis results revealed that target genes PIK3R1,PIK3CB and CTNNB1 had more targeted relationships with other proteins,and involved in estrogen signaling pathway,cell senescence,phosphatidylinositol signaling system,TGF-beta and other pathways.The expression of miR-150 in different tissues and diseases was analyzed by YM500V2 online software,it was found that the expression level of miR-150 was the highest in blood,relatively high in ovary,pancreas,lymph and skin,and very low or no expression level in brain and adrenal cortex.These results would provide theoretical reference for investigating the function and regulatory mechanism of miR-150,and further laid the foundation for the regulatory mechanism of miR-150 in the ovarian development of sheep.

This study was to clone and analyze the fat mass and obesityassociated protein (FTO) gene of Nubian goat,and construct its eukaryotic expression vector.The tissue of longissimus dorsi muscle of Nubian goat was taken as the experimental material,and the coding region of FTO gene was amplified by RT-PCR.After sequencing and identification,the cloned sequence was analyzed by bioinformatics with the corresponding analysis software,and then the FTO gene fragment was linked with pMD19-T vector and transformed into Escherichia coli DH5α competent cells.The pMD19-T-FTO vector was constructed,and the recombinant plasmid with correct sequencing was digested with double enzymes.The pEGFP-N1 vector was connected to construct the pEGFP-N1-FTO eukaryotic expression vector.The pEGFP-N1-FTO eukaryotic expression vector was then transfected into 3T3-L1 cells and cultured for 48 h.The results showed that the encoding region of Nubian goat FTO gene was successfully cloned.The sequence length was 1 518 bp,encoding 505 amino acids,and the molecular weight was 57 142.24 u.The homology of FTO gene of Nubian goat was 98.7%,96.4%,98.3%,87.2%,64.3% and 81.7% with that of Capra,Bos Taurus,Ovis aries,Sus scrofa,Gallus gallus and Mus musculus published on NCBI,respectively.The phylogenetic tree analysis of this gene showed that Nubian goat had the closest genetic distance to Capra and the furthest genetic distance to Gallus gallus.It was highly conserved in different species.The secondary and tertiary structures of Nubian goat FTO protein were mainly alpha helix and random coil.The constructed eukaryotic expression vector pEGFP-N1-FTO was transfected into 3T3-L1 cells 48 h later,and the expression of green fluorescence was observed under the microscope,indicating that the eukaryotic expression vector of FTO gene was successfully constructed.In this study,eukaryotic expression vector of Nubian goat FTO gene was constructed and successfully expressed in 3T3-L1 cells,which laid a foundation for future research on the correlation between FTO gene and goat fat metabolism.

As the main active form of vitamin D,1,25(OH)₂D₃ affects adipogenesis of humans and animals,but its role in proliferation and differentiation of porcine adipocytes is unclear.To explore the effects of 1,25(OH)₂D₃ on proliferation and differentiation of porcine preadipocytes,the cells isolated from subcutaneous adipose tissue of 3-5 days piglets were cultured and treated with 0,0.1,1,10,100 and 1 000 nmol/L concentration of 1,25(OH)₂D₃,respectively.The proliferation of preadipocytes cultured for 0,1,2,4,6,8 and 10 d was detected by MTT method.The differentiation and expression levels of PPARγ and FAS mRNA for the preadipocytes at 0,1,2,4,6,8 and 10 d after inducing-differentiation were determined by oil red O staining and Real-time quantitative PCR,respectively.The results showed that 0.1 and 1 nmol/L 1,25(OH)₂D₃ significantly promoted proliferation of porcine preadipocytes (P<0.05),but the concentration of 10-100 nmol/L inhibited the cell proliferation (P<0.05).At the concentration of 0.1 and 1 nmol/L,1,25(OH)₂D₃ markedly inhibited the differentiation of porcine preadipocytes and reduced mRNA expression of PPARγ and FAS (P<0.05).On the contrary,10 and 100 nmol/L 1,25(OH)₂D₃ significantly enhanced differentiation of porcine preadipocytes and increased mRNA expression of PPARγ and FAS (P<0.05).When the concentration was 1 000 nmol/L,it might be toxic to the cells.These results suggested that low concentration of 1,25(OH)₂D₃ could promote the proliferation of porcine preadipocytes,but inhibit differentiation through down-regulating PPARγ expression.High concentration of 1,25(OH)₂D₃ could inhibit the proliferation of the preadipocytes and promote differentiation by up-regulating PPARγ expression.And so,1,25(OH)₂D₃ affected proliferation and differentiation of porcine preadipocytes in a biphasic manner.

The purpose of this study was to investigate the effects of peroxisome proliferators activated receptors (PPARγ) agonist (ciglitazone) and insulin on lipid metabolism and its regulatory mechanism in preadipocytes of Yanbian Yellow cattle.The experiments were divided into control group (CON,5% FBS),insulin treatment group (I,5% FBS+10 mg/L insulin),cycloglitazone treatment group (C,5% FBS+10 mg/L insulin+10 mg/L cycloglitazone) and insulin+cycloglitazone treatment group (IC,5% FBS+10 mg/L insulin+10 mg/L cycloglitazone).Each group was treated for 144 h,and transcriptome sequencing was performed on the preadipocytes by RNA-Seq technology,and the differentially expressed genes were analyzed by GO functional annotation and KEGG enrichment.Analysis results of differentially expressed genes(DEGs) in each treatment group showed that:Compared with control group,the number of DEGs in IC group was 1 764,276 and 569 more than that in I and C groups,respectively.There were 371 DEGs in each treatment group.The number of up-regulated genes in IC group was 974,140 and 249 more than that in I and C groups,respectively,while the number of down-regulated genes in IC group was 790,I and C groups was 654 and 470,respectively.The GO analysis of the DEGs showed that the DEGs in each treatment group were involved in the regulation of cellular processes,metabolic processes and biological processes.In terms of molecular function,it mainly included molecular function regulator,transport activity and transcriptional regulation activity.In terms of cell composition,most of the DEGs were enriched into organelles,membranes and extracellular regions.KEGG pathway analysis showed that the DEGs were mainly concentrated in FoxO signaling pathway,PPAR signaling pathway,TGF signaling pathway,p53 signaling pathway,etc.In conclusion,the regulation mechanism of preadipocyte differentiation of Yanbian Yellow cattle treated with insulin and cycloglazidone alone was different from that treated with cycloglazidone together,and the promotion effect of the two treatments on differentiation was more obvious.The results of this study provided a basis for studying the function and molecular mechanism of cycloglitazone and insulin in regulating fat production.

The purpose of the experiment was to investigate the relationship between the growth rate of Altay sheep and the fat deposition by studying the content of main blood biochemical indicators and the expression level of the Leptin gene in the tail fat of Altay sheep at different growth rates.300 Altay rams with similar birth times were selected and fed normally to 6 months of age.During this process,the daily weight gain was recorded.Then the sheep whose daily weight gain was top 10% was defined as the fast growth group (the group with higher body weight,HBW),and those whose daily weight gain was bottom 10% was devided into the slow growth group (the group with lower body weight,LBW).From the fast-growing group,12 sheep were randomly selected and divided into growth inhibition group (low-energy diets,HBW-75%,n=6) and control group (standard diet,HBW-100%,n=6).In the slow growth group,12 sheep were randomly selected into the growth promotion group (high-energy diets,LBW-125%,n=6) and the control group (standard diet,LBW-100%,n=6).After 7 d of prefeeding,the sheep were fed for another 30 d,at the end of feeding,the sheep were weighted and the blood were collected,and tail fat was collected after slaughter.The levels of TG,CK,TSH,T3,T4,S.S,GH,INS,PG,Leptin in blood,the expression of Leptin mRNA and protein,and the morphological changes of tail fat cells were measured.The weight results showed that the initial weight and final weight of the HBW-100% and LBW-100% groups were significantly different (P<0.05),HBW-75% and HBW-100% groups had no significant difference in initial body weight and final body weight (P>0.05);LBW-100% and LBW-125% groups had significant difference in initial body weight (P<0.05),but no significant difference in final body weight (P>0.05).The T4,S.S,Leptin,TSH and T3 content in the LBW-100% group were significantly lower than those in the HBW-100% group (P<0.05).The T3 content in the LBW-125% group was significantly higher than that in the LBW-100% group (P<0.05).The content of GH,PG and Leptin in the HBW-75% group were significantly lower than that in the HBW-100% group (P<0.05).The tail fat cells in the LBW-125% group were larger than that in the LBW-100% group,and the HBW-75% group was smaller than the HBW-100% group (P<0.05).The detection results of Leptin mRNA and protein expression levels showed that there was no significant difference between the Leptin mRNA expression levels in the HBW-100% group and the LBW-100% group (P>0.05),and the Leptin protein expression levels were significantly higher than that in the LBW-100% group (P<0.01).The expression of Leptin mRNA and protein in the LBW-125% group were significantly higher than that in the LBW-100% group (P<0.05).The expression of Leptin mRNA in the HBW-75% group was significantly lower than that in the HBW-100% group (P<0.01),and the expression of Leptin protein was significantly lower than that in the HBW-100% group (P<0.05).In summary,in the fast-growing group and the slow-growing group,the expression of Leptin had a certain connection with body weight and body fat.In the slow-growing group,body weight and fat were increased significantly after promoting growth,and the expression of Leptin showed an upward trend.In the fast-growing group,after inhibiting growth,the body weight and fat were decreased,and the expression of Leptin showed a downward trend.The change of growth rate would affect the expression of Leptin,thereby changing the fat deposition.

This experiment was designed to investigate the effect of β-carotene on milk composition and correlated gut microbiota,to reveal the correlation between gut flora and milk composition.A total of 48 hybrid sows with similar parity were selected and randomly divided into 3 groups (each with 16 replicates) and fed with a diet containing 0,30 and 90 mg/kg β-carotene,respectively.The experiment peroid was from the 90th d of gestation to the 14th day after delivery.Colostrum and fecal samples were collected on the day of delivery,and normal milk samples were collected on the 14th day of lactation.Immunoglobulins (Ig) and other commponents were determined in colostrum and normal milk,while fecal microbiota was characterized by 16S rRNA sequencing.The results showed that dietary supplementation of β-carotene had a tendency to increase the IgM level (P=0.173),but decrease IgG (P=0.155) in sow colostrum.Supplementation of 30 mg/kg β-carotene significantly increased the lactoprotein and urea nitrogen content (P<0.05),but reduced the lactose content (P<0.05) in ordinary milk,while 90 mg/kg β-carotene significantly reduced the total solids content in ordinary milk (P<0.05).Analysis of the fecal microbiota revealed that β-carotene up-regulated the relative abundance of microbial genera such as Eubacterium brachy group,Ruminococcaceae UCG009 and Ruminococcaceae UCG014,but down-regulated the relative abundance of Candidatus Soleaferrea,Coprococcus 3,Lachnospiraceae NK4B4 group and other flora.Correlation analysis showed that there had a negative correlation between Coprococcus 3 and the IgM level in colostrum (P<0.05),while Fibrobacter was positively correlated with IgG and IgM in colostrum (P<0.05).Moreover,Lachnospiraceae NK4B4 was positively correlated with the lactoprotein level and total solids content in ordinary milk (P<0.05),while Ruminococcaceae UCG009 had a negative correlation with the IgG level in colostrum (P<0.05).These data indicated that β-carotene may affect the composition of colostrum and ordinary milk by regulating gut microbiota in sows.

This experiment was conducted to study the effects of two whole corn silage ratios and additional sodium bicarbonate supplementation on growth performance,nutrient apparent digestibility,serum biochemical indexes and economic benefits in Jiangyue donkey.40 healthy male donkeys (106.02 kg±11.81 kg) aged from 6 to 8 months were randomly divided into 5 groups (n=8 in each group).According to isoenergetic and isonitrogenous design,the proportion of whole corn silage in crude feed was 0 (group Ⅰ),30% (group Ⅱ),60% (group Ⅲ),respectively,and supplemented with additional sodium bicarbonate groups Ⅳ (30% of whole corn silage in crude feed+sodium bicarbonate) and Ⅴ (60% of whole corn silage in crude feed+sodium bicarbonate).The normal addition of sodium bicarbonate was calculated as 0.5% of dry matter of concentrate, and the additional addition was calculated as 0.5% of dry matter of concentrate+1% of dry matter of whole corn silage.The first 12 days were the prefeeding period,and the last 120 days were the trial period.The results showed that with the increase of whole plant corn silage addition,the F/G of groups Ⅰ,Ⅱ and Ⅲ decreased gradually,and the F/G of group Ⅲ was significantly lower than that of groups Ⅰ and Ⅱ (P<0.05).The apparent digestibility of crude fat,neutral detergent fiber and crude ash in group Ⅲ was the highest,which was significantly higher than that in group Ⅱ (P<0.05).The contents of total protein,creatinine and alkaline phosphatase in group Ⅲ were significantly higher than that in group Ⅰ (P<0.05).The glucose content in group Ⅲ was significantly lower than that in group Ⅰ (P<0.01).The total cholesterol content of group Ⅲ was significantly lower than that of groups Ⅰ and Ⅱ (P<0.05).Compared with the same proportion of whole corn silage,the average daily gain of group Ⅱ was significantly higher than that of group Ⅳ (P<0.05),the average daily gain of group Ⅲ was higher than that of group V (P>0.05),the F/G of group Ⅲ was significantly lower than that of group V (P<0.05),and group Ⅱ was lower than that of group Ⅳ (P>0.05).The apparent digestibility of crude protein,crude fat,energy and ash in group Ⅳ was not significantly different from that in group Ⅱ (P>0.05).The apparent digestibility of nutrients in group Ⅴ was not significantly different from that in group Ⅲ (P>0.05).The contents of total cholesterol and the activity of alanine aminotransferase in group Ⅳ were significantly higher than that in group Ⅱ (P<0.05).The weight gain cost per kilogram of donkeys in group Ⅰ-Ⅴ were 16.40,15.10,14.79,17.34 and 16.10 yuan,respectively.The gross profit per donkey in the whole experiment period was 711.27,837.53,871.98,629.46 and 738.16 yuan,respectively.It could be concluded that under the conditions of this experiment,based on the growth performance,apparent digestibility of nutrients and serum biochemical indexes,and comprehensive economic benefit comparison,when 0.28% sodium bicarbonate was added to the basal diet (based on DM),the ratio of total silage corn to 60% of the roughage (26.67% added to the basal diet DM) was the best for feeding Jiangyue donkey,and no additional sodium bicarbonate was needed.

Translucent egg has become a focus of attention in egg production in recent years.In order to explore the variation rule of translucent eggs and its influence on egg quality,500 Jining Bairi chickens and 500 Wenshang Luhua chickens with the same age and feeding environment were selected in this study.60 eggs of each of the two varieties were collected,and the changes of translucent eggs at 0,1,2,3,4,12,24 h and 3,5,7 d were recorded,and the proportion of translucent eggs was calculated.At the same time,200 eggs of each of the two varieties were collected.After being placed for 3 d,30 normal eggs of each variety and 30 fourth-grade translucent egg were selected for egg quality detection.The results showed that the translucent egg rate of Wenshang Luhua chickens was lower than that of Jining Bairi chickens.Translucent eggs began to appear after 2 h of egg production,4-12 h was the high incidence period,12 h-3 d was the aggravation period.During 3-7 d,normal eggs would no longer change to translucent eggs,but low-level translucent eggs would change to high-level translucent eggs.There were no significant differences in shell thickness,eggshell ratio,egg yolk color and harrington units between translucent eggs and normal eggs of two varieties (P>0.05).The albumen height of normal eggs of Jining Bairi chickens was higher than that of translucent eggs (P<0.05),but the difference between translucent eggs and normal eggs of Wenshang Luhua chickens was not significant (P>0.05).In summary,time was an important factor affecting the production of translucent eggs.Different varieties of eggs had different translucent egg production rates,and translucent eggs had a certain effect on egg quality.

The purpose of this study was to study the effects of Cordyceps powder (Cordyceps sinensis culture powder) combined with yeast selenium on laying performance,egg quality,antioxidant function and immune function of laying hens.A total of 450 24-week-old Hy-Line variety Brown were randomly divided into 5 groups with 6 replicates in each group and 15 hens per repeat.The control group was fed with basic diet,and groups Ⅰ-Ⅳ were fed with basic diet supplemented with 0.1 mg/kg yeast selenium and different doses of Cordyceps powder (0,0.2%,0.3% and 0.4%),respectively.The experimental period was 27 weeks.The laying performance,egg quality,immune function and antioxidant function of laying hens were analyzed in the middle period of laying peak (40th week).The results showed that compared with control group,the laying rate and average egg weight of laying hens in group Ⅲ were significantly higher and the ratio of feed to egg was significantly lower (P<0.05);Haugh unit,eggshell color,eggshell strength,yolk color,yolk crude protein content,yolk crude protein and crude fat content were significantly increased (P<0.05);The levels of essential amino acids Leu,Lys,Met,Trp,Phe,Thr,Val and His and non-essential amino acids Ala,Asp,Glu,Pro and Tyr in egg white were also significantly increased (P<0.05);The eggshell color,eggshell strength,Haugh unit and crude protein content of egg white,essential amino acids Ile,Gly,Arg and non-essential amino acids Cys and Ser in egg white were also significantly increased in group Ⅳ (P<0.05),and the selenium content in yolk and egg white were significantly increased in groups Ⅰ-Ⅳ (P<0.05).At the same time,compared with control group,the serum MDA content was significantly reduced (P<0.05),the number of WBCs,the serum IFN-γ and IL-2 levels,and the T-AOC and T-SOD activities were all significantly increased of the laying hens in middle stage of the egg production peak in group Ⅲ (P<0.05).The results indicated that the combination of yeast selenium and appropriate amount of Cordyceps powder could significantly improve the laying performance and egg quality of laying hens in middle of laying peak,enhance immunity and antioxidant capacity,and the effect of 0.3% Cordyceps powder combined with 0.1 mg/kg yeast selenium was the best.

This experiment was conducted to investigate the effects of nano copper,spores of Ganoderma lucidum and soybean isoflavone on growth performance,immune function and antioxidant capacity of Qingyuan Partridge chickens,as to provide reference for the development and application of additives in broiler production.A total of 1 200 1 d chicks were randomly assigned to 4 groups with 10 replicates of 30 birds per replicate.The control group was fed with basal diet (CON),and the birds in treatment groups were supplemented with 2 500 mg/kg nano copper (NC),600 mg/kg spores of Ganoderma lucidum (GLS) and 300 mg/kg soybean isoflavone premix (SI) for 30 d,respectively.At the end of the experiment,plasma and jejunal mucosa were collected to determine the biochemical variables related to antioxidation in plasma and intestinal immune factors.The results showed that,compared with the control group,①the growth performance of chickens was not affected by the three bioactive additives (P>0.05).②GLS increased the thymus ratio (P<0.05).③NC significantly increased the villus to crypt ratio in jejunum.④NC increased activity of glutathione catalase (GSH-Px) in plasma (P<0.05),and decreased jejunal MDA content (P<0.05) significantly.SI significantly reduced both plasmatic and jejunal malondialdehyde (MDA) content (P<0.05),and all three additives increased activity of total superoxide dismutase (T-SOD) in plasma and jejunum (P<0.05) significantly.⑤NC increased immunoglobulin (IgA) content in jejunum (P<0.05),and GLS decreased tumor necrosis factor-α (TNF-α) in plasma and interferon (IFN-γ) in jejunum (P<0.05)significantly.Based on this experiment,NC,GLS and SI did not affect the growth performance of Qingyuan Partridge chickens,while improved the antioxidant capacity.NC increased the villi/crypt ratio,and NC and GLS regulated the secretion of immune factors.Among them,NC had the best effect on antioxidant capacity and GLS had the best effect on immune function.

Proteins represent the second largest component of animal diet.However,the shortage of feed protein ingredients in China has restricted the healthy and sustainable development of animal husbandry severely.As a kind of euryphagous insect,Tenebrio molitor has strong reproductive ability and high output,and also has high contents of crude protein,chitin,unsaturated fatty acids,and vitamins.It can be extracted some biological active substances with the functions of antibacterial,anti-inflammatory and immune regulation.Moreover,it also has many other advantages,such as short growth cycle,easy to raise in commercial and less requirement of land.Therefore,it has the potential to become a new feed protein resource,thus alleviating the current shortage of feed protein raw materials.It is reported that Tenebrio molitor can partially replace conventional protein feed in animal diets,increase growth or production performance of animals,improve the product quality,and improve the body health status.In this review,the author elaborated the nutritional ingredients of Tenebrio molitor and nutrient digestibility of livestock and poultry to Tenebrio molitor,and summarized the functions of Tenebrio molitor for partial replacement of conventional feed protein materials on the basis of literature review:Increasing the growth or production performance of livestock and poultry,improving the slaughter performance and products quality of poultry,regulating the material metabolism,enhancing the antioxidant and immune abilities,and promoting the intestinal health of livestock and poultry.The auther also prospected for the Tenebrio molitor application in animal production,in order to provide a theoretical basis for it using in animal production.

The effects of high-yielding protease yeast on the quality of fermented soybean meal under different conditions were studied in this study.The fermentation time,water content,inoculation amount of bacteria solution and glucose addition were optimized by using protease protease activity as the index.Orthogonal test was designed based on the single factor test results to explore the changes of fermentation on the activities of protease,cellulase,phytase,pectinase and the contenets of crude protein,small molecule polypeptide and pancreatic protein inhibitor in soybean meal.The results showed that the enzymatic activity of soybean meal was the highest when the water content was 50%,the added amount of bacterial liquid was 4% and the glucose addition was 1.5%.Based on these results,orthogonal experiments were designed and found that compared with the control group,all experimental groups exhibited increased protease activity by 100.56%-380.13%,cellulase activity by 2.67%-81.77%,phytase activity by 53.89%-252.81%,pectinase activity by 13.84%-70.83%,small-molecule polypeptide contents by 574.67%-1 981.08%,crude protein contents by 9.24%-16.49%,pancreatic protein inhibitor contents by 7.36%-67.39%.The fermentation of soybean meal by high-protease yeast could significantly improve its nutritional value and reduce its anti-nutritional factors.Soybean meal was fermented by high yield protease yeast.If the soybean meal had the highest hydrolase activity,the fermentation conditions were 1% glucose,2% mixed bacterial fluid and 45% water content fermentation at room temperature for 5 days.When the contents of crude protein,polypeptide,and the contents of trypsin inhibitor detoxification rate were highest,the fermentation conditions were 2% glucose,4% mixed bacterial solution,55% water content and fermentation for 5 days at room temperature.

Lactic acid is an important intermediate metabolite in the rumen of dairy cows.Reasonable regulation of lactic acid metabolic characteristics,full utilization and playing the beneficial functions of lactic acid are of great significance for the healthy production of dairy cows.The synthetic pathway of lactic acid,the main acid-producing bacteria,the metabolic pathway of lactic acid,the main utilizing bacteria,the metabolic mode of lactic acid in the rumen of dairy cattle and the factors affecting its metabolism were reviewed in this paper.The effects of lactic acid-producing bacteria and utilizing bacteria on the regulation of lactic acid metabolism were elaborated in detail.At the same time,the effects of plant extracts on the regulation of lactic acid metabolism and the effects of lactic acid metabolism regulation on the gastrointestinal flora,lactation performance and mastitis of dairy cattle were introduced.It provided a theoretical basis for further understanding the action mechanism of lactic acid on the health of dairy cows and the application of related plant active substances in production practice,and provided new ideas for solving the prevention and treatment of acidosis in dairy cows caused by high-precision diet.

The aim of this study was to investigate the genetic diversity of MAPK10 and MEF2C gene structural variants in mitogen-activated protein kinase (MAPK) signaling pathway and its association with growth traits in Xiang pigs,in order to provide a theoretical basis for breeding improvement of Xiang pigs in Guizhou.The population genetic variation of two structure variation (SV) loci (MAPK10-I₈-sv273 and MEF2C-I₁-sv71) were investigated in 233 Xiang pigs by PCR technology,the growth curve of Xiang pigs was made by Origin software,the polymorphic information content (PIC),heterozygosity (He),homozygosity (Ho) and effective allele number (Ne) were calculated by online software,and the correlation between genotyping results of two SVs and growth traits of Xiang pigs was analyzed by SPSS 26.0 software.The results showed that two SVs were polymorphic in Xiang pig population,there were three genotypes of DD,ID and II,and two alleles of D and I.The allele frequency of D in MAPK10-I₈-sv273 was higher than I,and the genotype frequency of DD was the highest (0.65).The average daily gain,body height and growth rate of the individual with DD genotype were greater than that with II genotype,which indicated that D allele had the genetic effect to improve the growth performance in Xiang pigs.The allele frequency of I in MEF2C-I₁-sv71 was higher than D,and the genotype frequency of II was the highest (0.51).The average daily gain,chest circumference and growth rate of the individuals with II genotype were higher than that with DD genotype,which showed that I allele could improve the growth performance in Xiang pigs.In summary,MAPK10-I₈-sv273 and MEF2C-I₁-sv71 were related to the growth and development of Xiang pigs,and could be used as candidate molecular markers for the selection of growth traits.

The purpose of this study was to study the paternal origin structure and genetic diversity of red deer in China at the molecular level,and to determine the phylogenetic relationships among various groups.The SRY gene sequences of 159 red deers from 11 populations including Tianshan red deer, Aertai red deer, Tahe red deer and Dongbei red deer.They were detected and analyzed by DNA extraction,PCR and direct sequencing.The base composition,nucleotide diversity (Pi),haplotype diversity (Hd) and frequency were calculated to evaluate genetic diversity and construct the haplotype network diagram,and the phylogenetic tree was constructed by neighbor-joining method(NJ) and maximum likelihood method(MJ) with white lipped deer as outgroup to explore the clustering and genetic diversity of red deer.The results showed that the length of the obtained sequence was 1 615 bp,18 SNPs were identified,accounting for 1.11% of the total number of nucleotides.According to the polymorphic sites,14 haplotypes were identified,and the dominant haplotype was Hap-1 which consists of Tianshan red deer, Aertai red deer, Alashan red deer, Tahe red deer, Dongbei red deer, Gansu red deer, North American red deer and King of red deer population accounted for 35.84%.Among them,Alashan red deer, Tahe red deer, Gansu red deer, Chuanzang red deer, North American red deer and King of red deer all had unique haplotypes.The haplotype diversity ranged from 0 to 0.857,and the nucleotide diversity ranged from 0 to 0.00272.The genetic distance between Tahe red deer and Tibet red deer was the largest (0.002406),and that between Alashan red deer and Qinghai red deer was the smallest (0.000124).The phylogenetic tree based on NJ and ML was consistent which showed that there were three branches among the 11 wild red deer populations.Branch S1 included all the red deer populations.Tahe red deer, Gansu red deer, North American red deer and King of red deer King constituted Branch S2,and North American red deer constituted branch S3.The minimum haplotype network diagram was consistent with the phylogenetic tree.The results showed that there were differences in genetic diversity among different red deer populations.There were two paternal types in Tahe red deer, King of red deer and Gansu red deer,three paternal types in North America red deer and only one paternal type in other red deer populations.Hap-1 was in the core position in Hap-lotype group S1,other haplotypes were scattered around Hap-1.Hap-1 was supposed to be the original haplotype in red deer population.

In order to study the effects of different breeds,months,ages and interval of semen collection on semen quality of Canadian boars,and the effects of breeds and age of the first semen collection on semen quality stability,in this study,79 Canadian Large White,Landrace and Duroc boars from a boar station in Jiangxi province were selected as the experimental population.The data of semen collection and semen quality of 3 921 boars from December 2018 to December 2020 were collected.The effects of various factors on semen volume,semen concentration,sperm motility,total sperm number and stability were explored by mixed linear modelandanalysis of variance.The semen volume and total sperm number of Landrace boars were higher than those of Large White and Duroc boars,but the semen concentration of Duroc boars was higher than that of Landrace and Large White boars,and the sperm motility of Duroc boars was the lowest.In different months,the concentration of semen collected in 1 to 3 months was the highest,the motility of semen collected in 4 to 6 months was the highest,the volume of semen collected and the total number of sperm collected in 10 to 12 months were the highest,and the semen dose showed the seasonal variation of more in autumn and winter and less in spring and summer.There were also differences in semen quality traits among boars at different ages.The smaller the age was,the lower the semen volume was.However,the semen concentration was higher and the sperm motility was relatively better.The total sperm number was the highest at the best age of 19 to 24 months.The longer the interval was,the better the semen volume,semen concentration,sperm motility and total sperm number were.When the interval was 7 days,the comprehensive performance was the best,but the longer the interval was,the lower the sperm motility was.The stability of total sperm number in Landrace and Large White boars were significantly better than that in Duroc boars (P<0.05).This results showed that breed,month of sperm collection,age of sperm collection and interval of sperm collection all affected boar semen quality traits.Paying attention to these factors would help boar station to make more perfect production plan and improve the utilization rate in boars.

The aim of this study was to screen the most suitable cryoprotectant for porcine MⅡ oocytes and explore the effect of vitrification on DNA of porcine MⅡ stage oocytes. The oocytes of MⅡ stage were randomly divided into 8 groups. The oocytes of the control group were parthenogenetically activated directly, while the oocytes of the other 7 groups were treated with the most used 7 kinds of cryoprotectant, respectively, and then thawed in the cryoprotectant directly without liquid nitrogen freezing, and parthenogenetically activated after thawing. The suitable cryoprotectant was screened through the statistical results of cleavage rate, blastocyst rate and blastocyst cell number, and porcine MⅡ oocytes were vitrificated using the three selected cryoprotectants. After thawing, porcine MⅡ oocytes were recovered for 2 h, and the normal rate of oocytes morphology and the cleavage rate were acalculated. The ultrastructural changes of porcine MⅡ oocytes after vitrification were observed by transmission electron microscopy. Porcine MⅡ oocytes were randomly divided into control group, cryoprotectant treat group and vitrification group, and the DNA damage of oocytes by vitrification was detected by comet assay. The results showed that compared with control group, the cleavage rate of group 5 and blastocyst rate of group 1 were significantly decreased (P<0.05), but there were no significant differences in the cleavage rate and blastocyst rate of the other groups (P>0.05). The number of blastocyst cells in control group was higher than that of the other groups, but the differences were not significant (P>0.05). The cleavage rate and blastocyst rate in groups 3, 6 and 7 were higher. After vitrification and thawing, the morphological normal rate and cleavage rate of oocytes in group 7 were significantly lower than those in groups 3 and 6 (P<0.05), and the cleavage rate in group 6 was higher than that in group 3. The MⅡ oocytes were obviously shrinkage after being transferred into the pretreatment solution, and quickly dehydrated after being transferred into the freezing solution. After thawing, the zona pellucida of oocytes was broken, and the cytoplasm was shrunk and unevenly distributed. Under transmission electron microscope, the zona pellucida and cell membrane of porcine MⅡ oocytes were damaged after vitrification, microvilli were seriously damaged or even disappeared, cortical granules were arranged under the plasma membrane and decreased in number, morphology of lipid droplets were destroyed and vacuoles were formed, the connection between endoplasmic reticulum and lipid droplets were damaged, mitochondria swelled and cristae were not obvious. Comet assay showed that there were no significant differences in head DNA, tail DNA and Olive tail moment between control group and cryoprotectant-treated group (P>0.05),and there was comet tailing. The head DNA damage, tail DNA damage and Olive tail moment values of vitrification group were significantly higher than those of the cryoprotectant treated group and control group (P<0.05), and there was obvious comet tailing. The results showed that the cryoprotectant with DMSO and EG as main components were suitable for vitrification of porcine MⅡ oocytes, vitrification could damage the ultrastructure and DNA of porcine MⅡ oocytes to some extent, and the damage mechanism needed further study.

The purpose of this study was to select the tetrameric microsatellite markers with high polymorphism,and to establish the paternity testing system of sheep.The Small-tail Han sheep was the main research object in this study,and fifty-three (ATAG)_n repeat microsatellite sites were selected according to the sheep reference genome sequence and a total of 30 microsatellite loci which had stable amplification and high polymorphism information were developed based on genotyping data in Small-tail Han sheep.The genotyping results of 30 loci showed that 253 alleles were obtained from the 30 markers,the average number of alleles was 8.433,the number of alleles was all greater than 5,the polymorphic information content (PIC) varied from 0.566 to 0.898,the calculated observed heterozygosity (Ho) ranged from 0.548 to 0.903 and the expected heterozygosity (He) ranged from 0.631 to 0.921 with a mean of 0.776.The Hardy-Weinberg equilibrium analysis revealed that all 30 sites were in genetic equilibrium.Then,22 of 30 microsatellite loci were selected according to the amplification efficiency of PCR for the calculation of parental exclusion probability,and the combinations were performed by increasing the number of bits from high to low according to the size of the PIC.The results showed that when the genotypes of the two parents were unknown,the cumulative probability of exclusion (CPE) could reach more than 99.99% when the number of marker sites was 15,and the NE-1P varied from 0.321 to 0.663.Further,16 Small-tail Han sheep with genealogical records were tested by using the established paternity test system,the results showed that 4 families with high confidence were identified,and the identification results were in complete agreement with pedigree records.This experiment laid an important foundation for the construction of sheep molecular genealogy,paternity test and the development of sheep breeding.

This experiment was aimed to investigate the regulatory effect of arginine(Arg) on bovine endometrium treated with interferon-tau(IFNT) and its potential molecular mechanism.Bovine endometrial epithelial cells were inoculated into 6-well cell culture plate,different concentrations of IFNT (0,100 ng/mL) were added into the culture medium after the confluence of cells reached 70% to 80%.Cells were harvested to determine the expression of genes related to unfolded protein response (UPR) pathway after 12 h;Different concentrations of Arg (0,0.05,0.1,0.2,0.5,1,2 mmol/L) were subjected to bovine endometrial epithelial cells to detect the proliferation rate for 24 h by CCK8 method,and the optimal concentration of Arg was selected;Bovine endometrial epithelial cells were randomly divided into Arg starvation group,Arg starvation+IFNT treatment group,Arg+IFNT treatment group,100 ng/mL IFNT was added after Arg deprivation or supplementation for 6 h.The expression of genes associated with UPR pathway (BIP,PERK,CHOP,ATF6),apoptosis (Bax,Caspase9) and endometrial receptivity (HOXA10) were detected after IFNT stimulation for 12 h.The results showed that:IFNT stimulation significantly up-regulated the expression of BiP,PERK,CHOP and ATF6 genes implicated in UPR pathway in bovine endometrial epithelial cells.The proliferation rate of bovine endometrial epithelial cells were significantly increased by 0.5 mmol/L Arg (P<0.05);Arg deprivation significantly up-regulated the mRNA level of Bip,PERK,CHOP,ATF6 and BAX genes in IFNT-treated bovine endometrial epithelial cells (P<0.05),and significantly down-regulated the mRNA level of HOXA10 (P<0.05),but had no significant effect on the expression of Caspase9 mRNA (P>0.05).The results indicated that Arg deficiency could lead to endoplasmic reticulum stress in bovine endometrial epithelial cells and influence the establishment of endometrial receptivity,which could provide reference for further revealing the regulation mechanism of Arg on bovine endometrium and formulating nutritional regulation strategies to reduce embryo loss in early pregnancy.

In order to find molecular markers for meat quality of Yili horses,38 Yili horses were used to determine meat quality traits and muscle fiber traits (including water loss rate,cooked meat rate,shear force,muscle fiber cross-sectional area,muscle fiber diameter and muscle fiber density).The polymorphism of myogenin (MyoG) gene exon 1 in Yili horse population was detected by PCR direct sequencing,and the relationship between different genotypes of MyoG gene SNPs and meat quality,muscle fiber traits were analyzed.The results showed that five SNPs were detected in exon 1 of MyoG gene,including SNP1 (g.31187343 A>C),SNP2 (g.31187333 G>A),SNP3 (g.31187132 C>T),SNP4 (g.31187105 C>G) and SNP5 (g.31187099 C>T).The SNP1 was found to be a missense mutation,and the mutation of base A to C occurs,which maked the amino acid mutation from threonine to proline.The other sites were found to be nonsense mutations.SNP3 and SNP4 were found to be moderately polymorphic,while SNP1 and SNP2 were low polymorphic,the four SNPs were in Hardy-Weinberg equilibrium.Association analysis showed that:SNP1 and SNP4 genotypes in exon 1 of MyoG gene were significantly associated with water loss rate,cooked meat rate,muscle fiber cross-sectional area,muscle fiber diameter and muscle fiber density (P<0.05).SNP3 genotypes were significantly associated with cooked meat rate,muscle fiber cross-sectional area and muscle fiber density (P<0.05).There was no significant correlation between SNP2 genotypes and each index (P>0.05).In conclusion,five SNPs were detected in exon 1 of MyoG gene in Yili horse,among which SNP1 (g.31187343 A>C),SNP3 (g.31187132 C>T) and SNP4 (g.31187105 C>G) had significant effects on meat quality and muscle fiber traits.These SNPs might become molecular markers of meat quality in Yili horse.

Copy number variation (CNV) has many forms of variation structure,which plays an important role in the study of variety diversity,biological evolution and disease correlation,and has the characteristics of large fragment length and wide coverage.With the development of molecular biology and the maturity of DNA sequencing technology,the study of genetic variation has been deepened to the DNA molecular level.Polymorphic markers have gradually become the trend and mainstream of animal breeding research in livestock and poultry breeding.As CNV has more significant effects on gene regulation and expression,there are more and more studies on CNV in important livestock and poultry.At present,a large number of gene sequence variations related to important economic traits of livestock and poultry have been detected,and many studies have shown that CNV is related to the important economic characteristics of animals and the occurrence of diseases.By referring to the relevant research reports at home and abroad,the author briefly introduced the related research background,concept and mutation mechanism of CNV,and summarizes the effects of CNV on economic traits,reproductive traits and disease regulation of cattle,sheep,pig and chickens.in order to reveal the genetic mechanism of adaptability and the genetic basis of phenotypic traits through the genomic research on these important livestock and poultry,and to develop corresponding molecular genetic markers.To provide a theoretical basis for marker-assisted breeding of livestock and poultry.

The purpose of this study was to investigate the effects of melatonin (MT) on the pregnancy rate and serum reproductive hormones in Holstein dairy cows.A total of 150 first mating Holstein dairy cows with natural estrus were determined by pedometer method.Among them,70 cows were injected with 30 mg melatonin subcutaneously in the neck,and artificial insemination was performed 12 h later.170 Holstein dairy cows were selected for the first time of postpartum mating for the treatment of simultaneous ovulation and timing insemination.Among them,90 Holstein dairy cows were injected with gonadotropin-releasing hormone (GnRH) and 30 mg melatonin subcutaneously and intramuscularly at the same time.Artificial insemination was carried out 16 h later.153 Holstein dairy cows were selected for subcutaneous injection of melatonin.Pregnancy examination was carried out 20~35 d after insemination.The number of cows in the first mating and second mating and the number of calves were recorded in detail.25 Holstein dairy cows with simultaneous ovulation and timing insemination were selected,8 h after subcutaneous injection of melatonin,the contents of melatonin,follicle stimulating hormone (FSH),luteinizing hormone (LH) and estradiol (E₂) in the serum were detected by radioimmunoassay,and the content of progesterone (P₄) in the serum of pregnant cows at 35 d was detected.The results showed that compared with control group of natural estrus and synchronous ovulation,the pregnancy rate and calving rate of Holstein dairy cow of the corresponding subcutaneous injection of melatonin group were significantly increased (P<0.05),the double calving rate of the subcutaneous injection of melatonin group was significantly increased (P<0.05),the pregnancy rate and calving rate of the first mating were significantly increased (P<0.05),and the pregnancy rate and calving rate of the second mating were not significantly different (P>0.05).The results of serum hormone showed that compared with control group,the levels of melatonin,LH and E₂ in the serum of the subcutaneous injection melatonin group were significantly increased (P<0.05),the content of P₄ in the pregnant cows with subcutaneous injection melatonin was significantly increased after 35 d of pregnancy examination (P<0.05).The results showed that subcutaneous injection of melatonin could improve the pregnancy rate,calving rate and serum LH,E₂ and P₄ contents of Holstein dairy cows,indicating that subcutaneous injection of melatonin could promote oocyte maturation and ovulation,and improve the pregnancy rate of breeding.

This experiment was conducted to explore the association between the polymorphism of lipoprteinlipase (LPL) gene and meat quality traits in Yanbian Yellow cattle.In this study,longissimus dorsi muscle of 80 healthy Yanbian Yellow cattle aged 30 months were used as experimental materials.The SNP of LPL gene was detected by PCR sequencing and HRM,respectively.Genetic effects of the experimental population was analyzed and correlated with the meat quality data of Yanbian Yellow cattle.The PCR sequencing results showed that there were two polymorphic loci (g.6215 A>G and g.18341 C>T) of LPL gene in Yanbian Yellow cattle.g.6215 A>G of LPL gene showed two genotypes of AA and AG,and the dominant allele was A;g.18341 C>T showed two genotypes of CC and CT,C was the dominant allele.The detection results of two loci in the reference showed that g.355427 T>A showed two genotypes of AA and AT,A was the dominant allele,and c.322 G>A,showed no genotyping.All of SNPs showed moderate polymorphism (0.25<PIC<0.5) and were in Hardy-Weinberg equilibrium.The results of HRM genotyping showed that there were three SNPs in genotyping,which were correlated with meat quality traits of Yanbian Yellow cattle.g.6215 A>G was significantly correlated with carcass weight,fat content,protein content and water content of Yanbian Yellow cattle,which were:carcass weight,fat content of AG genotype were significantly higher than AA genotype (P<0.05),and protein content,water content of AA genotype were significantly higher than AG genotype (P<0.05).g.18341 C>T was significantly correlated with pre-slaughter weight,carcass weight and backfat thickness of Yanbian Yellow cattle,which showed that CT genotype was significantly higher than CC genotype (P<0.05).In g.355427 T>A,pre-slaughter weight,backfat thickness and fat content of AT genotype were higher than AA genotype (P<0.05).To sum up,g.6215 A>G,g.18341 C>T and g.355427 T>A had an effect on the meat quality of Yanbian Yellow cattle,and LPL gene could be used as the dominant candidate gene and effective molecular marker for breeding and improvement of Yanbian Yellow cattle breeds.

Exosomes are vesicles widely existed in the intercellular substance of various cells.Exosomes can be produced by various kinds of cells under normal conditions or under stress conditions such as extracellular stimulation.The action mode and functions of exosomes derived from different cell-types are different.Exosomes can transmit bioactive molecules to participate in the process of body immunity and cell differentiation by binding membrane proteins to target cell membrane proteins,binding membrane protein fragments to receptors on cell membrane,and direct fusion between membrane and target cell membrane.Exosomes with the characteristics of natural origin,good barrier crossing capability and excellent biocompatibility have attracted much attention in many fields.The morphological characteristics,formation process,mechanism of action,isolation and purification,biological characteristics,function and application of exosomes were introduced and the application of exosomes applied to livestock and poultry research in recent years were summarized in this paper.Exosomes can not only be used in animal genetics and breeding,but also be used as tools or carriers to study multiple biochemical pathways in animals.This review can provide references for the research and application of exosomes.

The purpose of this study was to explore the effect of meningitis-causing Enterococcus faecalis on the function of blood-brain barrier in vitro,and find a stable method to construct the model of blood-brain barrier injury in vitro.One week old ICR mice were used to isolate primary brain microvascular endothelial cells (BMEC),and the BMEC specific coagulation factor Ⅷ(Factor Ⅷ) was used for fluorescent labeling to identify the isolated cells and determine the isolation rate.Primary astrocytes were isolated from ICR mice aged 1-3 days and labeled with glial fibrillary acidic protein (GFAP) to identify the isolated cells and determine the isolation rate.The primary cells were passaged to the third generation,and the blood-brain barrier models of BMEC monolayer and BMEC co-cultured with astrocytes were constructed by Transwell two-dimensional pore.Meningitis-causing Enterococcus faecalis,non meningitis-causing Enterococcus faecalis and E.coli DH5α competent cells were co-cultured with the blood-brain barrier model in vitro to evaluate the penetration of meningitis-causing Enterococcus faecalis to the blood-brain barrier model in vitro.The effect of meningitis-causing Enterococcus faecalis on blood-brain barrier model in vitro was evaluated by 4 h penetration test,sodium fluorescein penetration test and detection of matrix metalloproteinase 2 (MMP-2) expression.The results showed that BMEC and astrocytes were successfully isolated,and the purity were above 90% and 95%,respectively.The penetration test showed that only meningitis-causing Enterococcus faecalis could cross the blood-brain barrier model in vitro.4 h penetration test and fluorescein sodium penetration test showed that the co-culture model was superior to the monolayer culture model of BMEC.Compared with other groups,the penetration of fluorescein sodium was higher and the expression of MMP-2 was significantly increased in the co-culture group.In conclusion,the co-culture model of BMEC and astrocytes was better than BMEC monolayer culture model,meningitis-causing Enterococcus faecalis could cross the blood-brain barrier model in vitro and mediate the blood-brain barrier damage,which provided a theoretical basis for revealing the mechanism of the blood-brain barrier damage induced by meningitis-causing Enterococcus faecalis.

To understand the genetic variation of Canine distemper virus (CDV) in Shanghai,the full-length genome of CDV SH202003 strain was sequenced by RT-PCR with 11 pairs of over lapping primers.PCR fragments were obtained and sequenced repeatedly to ensure the mostly possible sequences.The sequenced 11 fragments were spliced to obtain the full-length genomes sequences of CDV SH202003 strain.Alignment analysis of the full-length genome and H gene sequences of SH202003 strain and other CDV sequences published in GenBank was analyzed with Lasergene 7.0 and Mega 6.0 software,and the phylogenetic tree was constructed.The results showed that total length of SH202003 strain was 15 690 bp,encoding 6 structural proteins (N,P,M,F,H and L).The gene interval sequences were CUA between H and L,and CAA between L and 5' trailer,respectively.The SH202003 strain had 98.6% nucleotide sequence similarity and 96.6% amino acid similarity with Hebei strain,and had 92.2% to 94.3% nucleotide sequence similarity and 82.7% to 87.0% amino acid similarity with vaccine strains.Phylogenetic tree analysis showed that SH202003 strain was in the same branch with prevalent virulent strains and distant from the vaccine strains.H gene had 98.7% nucleotide sequence similarity and 99.5% amino acid similarity with Hebei strain,and was distant from the vaccine strains such as Snyder Hill,CDV3,Convac and Onderstepoort.SH202003 strain belonged to Asia-1 subtype (Asia-1 virulent strain).SH202003 strain contained 9 potential N-glycosylation sites,which was consistent with Hebei strain.The results indicated that SH202003 strain was virulent and belonged to Asia-1 type.H gene sequences were relatively conservative and had 9 potential N-glycosylation sites.However,SH202003 strain had many mutations in the full-length genome and low homology with the vaccine strains,which might be the main reason for canine distemper in immunized dogs.

To determine whether there was an interaction between Transmissible gastroenteritis virus (TGEV) nonstructural protein 2 (Nsp2) and the host cell protein 26S proteasome non-ATPase regulatory subunit 11 (PSMD11),RT-PCR was used to amplify the porcine PSMD11 gene and eukaryotic expression vector pCMV-Myc-PSMD11 was constructed,which was verified by sequencing and double enzyme digestion.And then pCMV-Myc-PSMD11 was transfected into intestinal epithelial cell lines J2 (IPEC-J2),Western blotting and indirect immunofluorescence assay (IFA) were used to detect whether pCMV-Myc-PSMD11 could be expressed in IPEC-J2 cells.Then,the co-immunoprecipitation (Co-IP) experiment was used to determine the interaction between TGEV Nsp2 and PSMD11 of the host cell,and the co-localization of TGEV Nsp2 and PSMD11 in the host cell was observed by laser confocal microscope.The results showed that porcine PSMD11 gene was successfully amplified,with a size of 1 474 bp,and the gene sequence was completely consistent with the standard sequence.The constructed eukaryotic expression vector pCMV-Myc-PSMD11 could successfully express PSMD11 protein in IPEC-J2 cells.The results of Co-IP indicated that there was an interaction between PSMD11 and Nsp2.The results of the colocalization experiment showed that the interaction between PSMD11 and Nsp2 occured in the cytoplasm,and the expression position of PSMD11 in the cell was not changed due to the expression of Nsp2.The results of this study provided new clues for further study of the important role of TGEV Nsp2 in the process of virus infection.

In order to explore a new way to control Salmonella infection,phages with high titer and wide lysis spectrum were isolated in this experiment.Firstly,spot test and efficiency of plating (EOP) test were used to isolate and purify Salmonella phage and determine its lysis titer and lysis spectrum.Then the morphological characteristics of one of the phages with high titer and wide lysis spectrum was further observed by electron microscope,and detected the multiplicity of infection(MOI),one-step growth curve,along with temperature,pH and UV sensitivity.The results showed that the lysis rate of the bacteriophage to different host bacteria was different with lysis titer from 10⁹ to 10¹² PFU/mL.The lysis rate of 32 Salmonella strains detected was 59.4%.It was observed that the phage had a long tail phage with a head diameter of about 70 nm and a tail length of about 140 nm under transmission electron microscope.The best MOI was 0.01 when measured by host of Salmonella Typhimurium ATCC 14028,and one-step growth curve showed that the latent period was 20 min,the burst period was 100 min,and the burst size was 113 PFU/cell.The titer was stable at 10¹⁰ PFU/mL at 40-60 ℃,the titer was above 10⁶ PFU/mL at pH 5.0-13.0,and the titer was still 10⁶ PFU/mL after UV irradiation for 50 min.Therefore,the phage had the characteristics of high effective lysis,wide lysis spectrum,good stability to temperature,pH,and UV,which was in line with the relevant characteristics of phage preparations,and had the potential to be developed as a backup strain for phage preparations.

Pigeon salmonellosis is a common and frequent disease caused by Salmonella in pigeon industry.It mainly damages young pigeons and causes serious economic losses in pigeon industry. Salmonella causing the disease is a facultative intracellular parasite,it can survive and reproduce in the host cells and evade the body's immune defense function,which makes it more difficult to purify the disease.At present,the use of antibiotics to control pigeon salmonellosis is the most commonly used effective measure in pigeon breeding.However,the extensive use and even abuse of antibiotics lead to the continuous production of drug-resistant Salmonella strains and drug residues,thus endangering food and public health safety.Nowadays,it has become the mainstream of animal disease prevention and control research to seek alternative antibiotics to reduce or replace the use of antibiotics.Due to the late start of pigeon breeding,few researches on pigeon salmonellosis and its prevention and control measures are done.In this paper,the research progress about salmonellosis of pigeons,the pathogenic mechanism of Salmonella and the prevention and control measures of antibiotic substitutes for Salmonella in recent years were reviewed,in order to provide reference for the related research of pigeon salmonellosis.

The purpose of the experiment was to screen the effective ingredients of Wujin granules in the treatment of lamb dysentery through the mining function of network pharmacology,and explain its potential targets and possible pathways.According to the Traditional Chinese Medicine System Pharmacology Database and Analysis Platform (TCMSP),the main effective compounds of each single medicine of Wujin granules were analyzed,and at the same time their corresponding targets were searched.Compounds without targets were analyzed again by the Swiss Target Prediction database for their targets,it was predicted that the compound with factual target and its corresponding target protein were finally obtained.Cytoscape 3.7.1 software was used to construct the network diagram of the effective ingredient-target protein,the selected target protein was uploaded to the String online database to construct Wujin granules acting on the protein-protein interaction (PPI) map of lamb (Ovis aries) dysentery,and finally uploaded the core targets in the PPI network to the DAVID database for GO function and KEGG pathway enrichment analysis,and at the same time performed gene function cluster analysis.The results showed that a total of 22 compounds with de facto targets were screened,including kaempferol,stigmasterol,methyl arachidonic acid,quercetin,ellagic acid,and so on,and a total of 129 compounds corresponding to target proteins were deduplicated.The PPI network diagram showed that the number of nodes was 74,the number of edges was 287,the average node degree was 7.76,and a total of 67 core target proteins,including INSR,NR3C1,ESR1,PLG,PGR,and so on.GO function enrichment analysis showed that 54 items were related to biological process (BP) (FDR<0.05),11 items were related to cell component (CC) (FDR<0.05),22 items were related to molecular function (MF) (FDR<0.05).GO function enrichment gene items with more count included transcription,plasma membrane,zinc ion binding,and so on,KEGG pathway enrichment results involved 50 genes,and 43 regulatory pathways involved in gene coordination,neuroactive ligand-receptor interaction,pathways in cancer,complement and coagulation cascades,and so on,involved more genes.Gene function classification results showed that of 64 cores among the target genes,12 genes had clustering results.They were divided into two groups with different functions,namely group 1 (enrichment score 6.11,involving 7 core target genes) and group 2 (enrichment score 2.84,involving 5 core target genes).In conclusion,Wujin granules might act on a variety of different targets through a variety of compounds,and participate in the regulation of multiple pathways to achieve the therapeutic mechanism of lamb dysentery.

The purpose of this study was to determine the serotype distribution,virulence gene carrying and drug resistance of Streptococcus agalactiae (S.agalactiae) isolated from tilapia in Guangxi,China,and laid a foundation for the prevention and control of S.agalactiae.47 strains of S.agalactiae were isolated from tilapia farms in Liuzhou,Qinzhou,Nanning and Beihai of Guangxi in 2018-2019,the serotype,distribution of virulence gene and drug resistance were detected and analyzed.The results of serotype test showed that 47 clinical isolates of S.agalactiae strains were Ⅰa serotype.The results of virulence gene detection showed that all clinical isolates of S.agalactiae carried cylE,sodA and gapC genes.The scpB gene was only detected in human reference strain 2603V/R,but not detected in all isolates of piscine S.agalactiae.The results of drug sensitivity test showed that the drug resistance rate of all the piscine isolates to sulfamethoxazole,neomycin,gentamicin and kanamycin was more than 90%,the drug sensitivity of the 47 piscineisolates to of loxacin,levofloxacin,ampicillin,amoxicillin,cefaclor,ceftriaxone,cefoperazone,cefradine,terramycin and doxycycline was 100%.All piscine strains had multiple drug resistance,and 93.62% of the 47 strains were resistanted to more than 5 kinds of drugs.Among all the piscine strains,the isolates resistanted to 9 drugs were accounted for 19.15% and isolated from Liuzhou.The results showed that the serotype of S.agalactiae isolated from tilapia in Guangxi was Ⅰa serotype.All 47 piscine isolates of S.agalactiae carried multiple virulence genes,and the phenomenon of multiple drug resistance was serious.

The purpose of this study was to screen out the monomer inhibitors of Staphylococcus aureus transcriptional regulator MgrA from the small molecule library of traditional Chinese medicine,and explore its effect on the main virulence factors of Staphylococcus aureus and its therapeutic effect on Staphylococcus aureus-induced pneumonia in mice.Fluorescence anisotropy analysis method was used to screen inhibitors,Real-time PCR,hemolysis test and fibrinogen adhesion test were used to investigate the transcriptional expression of related virulence genes regulated by MrgA and the inhibitory effect on hemolysis and adhesion.The thermal stable migration experiment was used to preliminarily explore its inhibitory mechanism.Finally,a mouse pneumonia model was established to evaluate the therapeutic effect of Tectorigenin on Staphylococcus aureus-induced pneumonia in mice.The results showed that Tectorigenin,as an inhibitor of MgrA,could significantly inhibit the activity of MgrA at a low concentration (IC₅₀=20.35 μg/mL) that did not affect bacterial growth (P<0.05).Real-time PCR results proved that Tectorigenin significantly reduced the transcription levels of fnba gene (P<0.05), extremely significantly reduced the transcription levels of hla and RNAⅢ genes (P<0.01),and at the same time extremely significantly increased the transcription levels of ebh, srap and spa genes (P<0.01).Hemolysis experiments showed that Tectorigenin could extremely significantly inhibit the hemolysis of USA300 strain at a concentration of 8 μg/mL (P<0.01).Fibrinogen adhesion experiments confirmed that Tectorigenin extremely significantly inhibited the adhesion activity of USA300 strain at 32 μg/mL (P<0.01).Thermal shift assay revealed that Tectorigenin inhibited its activity by binding to MgrA.The results of mouse pneumonia experiments showed that Tectorigenin could extremely significantly improve the survival rate of mice infected with Staphylococcus aureus (P<0.01),reduce the amount of bacteria in their lungs (P<0.01), and reduce the pathological damage and inflammation of lung tissue in mouse.The above results indicated that Tectorigenin could treat mouse pneumonia caused by Staphylococcus aureus by inhibiting the activity of the transcriptional regulator MgrA.Tectorigenin could be used as a lead compound for the development of treatment of Staphylococcus aureus infection,and provided a theoretical basis for the development of drugs targeting MgrA.

The purpose of this experiment was to study the in vitro inflammatory damage of bovine endometrial epithelial cells caused by Escherichia coli (E.coli),and explore the optimal concentration and duration time of E.coli to induce inflammatory reactions and its mechanism.Firstly,different concentrations of E.coli (5×10⁴,5×10⁵,1×10⁶,2.5×10⁶,5×10⁶ CFU/mL) were used to infect the cells for 3,6 and 9 h,then detected the effect of E.coli on cell viability by observing the cells morphology with inverted microscope morphology and measured the D_{450 nm} with CCK-8 method;Secondly,different concentrations of E.coli (5×10⁴,5×10⁵ CFU/mL) were used to infect the cells for 3,6 and 9 h,then detected the secretion levels of IL-1β,IL-6,IL-8 and TNF-α in the cell supernatant with ELISA method.Finally,different concentrations of E.coli (5×10⁴,5×10⁵ CFU/mL) were used to infect the cells for 6 and 9 h,then the phosphorylation level of IκBα and p65 protein were detected by Western blotting.The results showed that compared with control group,the cell viability of 1×10⁶,2.5×10⁶ and 5×10⁶ CFU/mL E.coli groups was extremely significantly reduced after 9 h of E.coli infection (P<0.01),while the cell viability of 5×10⁵ CFU/mL E.coli group was significantly decreased (P<0.05).The levels of IL-1β,IL-6,IL-8 and TNF-α of 5×10⁵ CFU/mL E.coli group were extremely significantly increassed after 9 h infection (P<0.01).Moreover,the levels of IκBα and p65 phosphorylation protein were significantly higher than those in both 5×10⁴ CFU/mL and control groups (P<0.01).The levels of IL-6,IκBα and p65 phosphorylation protein in 5×10⁵ CFU/mL E.coli group were extremely significantly increased after 6 h infection (P<0.01),while IκBα and p65 phosphorylation protein in the 5×10⁴ CFU/mL E.coli group were significantly increased after 6 h infection (P<0.05).In conclusion,bovine endometrial epithelial cells inflammatory response could be induced by E.coli and the optimal action time was 6 h when E.coli infection concentration was 5×10⁵ CFU/mL,and 9 h when E.coli infection concentration was 5×10⁴ CFU/mL.

The purpose of this experiment was to evaluate the germicidal efficacy of chlorine dioxide disinfectant developed by our laboratory for dairy teat disinfectant,and to preliminarily study the germicidal mechanism of chlorine dioxide.The germicidal efficacy of chlorine dioxide dairy teat disinfectant was evaluated by quantitative sterilization test.The ultrastructural changes of Staphylococcus aureus after chlorine dioxide treatment were observed by transmission electron microscope (TEM).The changes of cell membrane permeability of Staphylococcus aureus after chlorine dioxide treatment were determined by flow cytometry.The results of quantitative germicidal test showed that when the content of chlorine dioxide in the disinfectant was higher than 1.2 mg/L,the logarithm value of killing Streptococcus agalactiae and Pseudomonas aeruginosa was higher than 3.00,that was,the germicidal rate was higher than 99.9%.When the chlorine dioxide content was higher than 0.6 mg/L,the logarithm of killing Staphylococcus aureus,Escherichia coli and Candida albicans was higher than 3.00,that was,the killing rate was higher than 99.9%.When the content of chlorine dioxide was 0.3 mg/L,it could still effectively kill Staphylococcus epidermidis and Streptococcus dyslactiae.When the chlorine dioxide content was higher than 28 mg/L,the bactericidal rate of Bacillus subtilis could reach 100%.The results of TEM showed that the cell membrane of Staphylococcus aureus treated with 100 mg/L chlorine dioxide for 5 min showed slight shrinkage,and there was a slight gap between the cell wall and the cell membrane,and most of the cells still maintained their original morphology.After treated for 15 min,the morphology of Staphylococcus aureus changed obviously,the cell membrane collapsed obviously,the cell wall completely separated from the cell membrane,and the cytoplasm also agglutinated.Flow cytometry detection results showed that the membrane permeability of Staphylococcus aureus was changed after treated with chlorine dioxide,and the change of membrane permeability was the most obvious after treated with 50 mg/L chlorine dioxide for 15 min.Combined with the results of laboratory evaluation of germicidal efficacy,it was found that the change of cell membrane permeability was only of the causes of killing bacteria.The killing mechanism of chlorine dioxide on bacteria was more complex,and it still needed to be further explored.

The aim of the present study was to explore the effect and mechanism of cat adipose mesenchymal stem cells (AD-MSCs) and adipose mesenchymal stem cells conditioned medium (AD-MSCs-CM) on the apoptosis of crandell rees feline kidney (CRFK) damaged by gentamicin.This experiment was divided into 4 groups,which were control group,gentamicin (GM) group,AD-MSCs group and AD-MSCs -CM group.Type Ⅰ collagenase digestion method was used to separate cat AD-MSCs,and identified by flow cytometry identification.CRFK was treated with GM complete medium at concentrations of 0,2,4 and 8 mmol/L,and the cell activity was detected by CCK-8 assay at 12,24 and 48 h to screen the optimal concentration for model making.The damaged CRFK was co-cultured with cat AD-MSCs and AD-MSCs-CM,respectively.After 24,48 and 72 h,the proliferation activity of the cells was detected by CCK-8 assay,and the apoptosis rate was detected by Annexin V flow tometry 24 h later.Finally,the expression levels of cyclin D1 (CCND1),proliferative nuclear antigen (PCNA),Bax and Bcl-2 genes were detected by Real-time quantitative PCR.The results showed that in vitro cultured cat AD-MSCs presented typical long spindle shape,with high expression of MSCs surface markers CD44,CD90 and CD105,but no expression of leukocyte surface marker CD45.The results suggested that AD-MSCs and AD-MSCs-CM enhanced the proliferative activity and decreased the apoptosis rate of damaged CRFK cells by promoting the expression of proliferation gene CCND1,PCNA and anti-apoptotic gene Bcl-2 of GM-damaged CRFK cells and reducing the expression of pro-apoptotic gene Bax.In this experiment,cat AD-MSCs were successfully isolated,and it was confirmed that Ad-MSCs and its conditional medium could promote the proliferation and migration of GM-induced CRFK in vitro,which provided a basis for the treatment of cat AD-MSCs for acute kidney injury.

The aim of this study was to investigate whether Sofosbuvir,a small molecule drug,could inhibit the replication of Bovine viral diarrhea virus (BVDV).The effects of Sofosbuvir on the proliferation of bovine kidney cells (MDBK) at different time and concentrations were determined by MTT method.The optimal concentration of Sofosbuvir which could effectively inhibit BVDV replication was screened by immunofluorescence staining test.The effects of Sofosbuvir on BVDV replication were detected by Real-time PCR,CPE and TCID₅₀.The results showed that Sofosbuvir treated MDBK cells for 24 and 48 h extremely significantly inhibited the proliferation of cells (P<0.01).When Sofosbuvir was 800 μmol/L,the content of double stranded RNA (dsRNA) in MDBK cells infected with BVDV was extremely significantly lower than that in the control group (P<0.01).Real-time PCR showed that compared with DMSO treatment group,the expression of BVDV 5'UTR mRNA in Sofosbuvir treated MDBK cells was extremely significantly decreased at 24 and 48 h after infection (P<0.01).The pathological changes of MDBK cells infected with Sofosbuvir were significantly reduced.Sofosbuvir treatment for 24 h extremely significantly reduced the formation and release of virus particles in the offspring of MDBK cells infected with BVDV (P<0.01).In conclusion,Sofosbuvir could effectively inhibit BVDV replication in vitro.

To understand the proportion of antimicrobial resistance and antimicrobial resistance genes of Escherichia coli from different links of the live poultry production chain in the family farm supply mode,in this study,132 samples were collected from one farmers' market and 33 family farms in Yantan town,Zigong city.Methods of bacteria isolation and purification,morphological examination,specific PCR detection,phylogenetic group detection,drug sensitivity test and antimicrobial resistance genes detection were used for the analysis of bacterial characteristics.The results showed that 83 isolates were E.coli strains which were black and metallic lustre colonies in EMB,Gram-negative bacteria under microscope and positive in specific primer test.Phylogenetic group detection results showed that 83 strains were concentrated in Group A (53.02%) and Group B1 (20.48%).The results of drug resistance test showed that 83 strains were multi-drug resistant bacteria,among which proportion drug resistance of colstin and tetracycline were 100% and proportion drug resistance of ampicillin,cefuroxime,aztreonam and fluorobenzene were over 90%.The results of antimicrobial resistance genes detection showed that antimicrobial resistance genes such as mcr-1, mcr-3, bla_TEM, bla_SHV, bla_{CTX-M-group 1}, bla_{CTX-M-group 9}, qnrS, aac(6')-Ⅰb-cr, tetA and tetB were detected and proportion of these genes were lower than proportion of antimicrobial resistance.In addition,the analysis results showed that proportion of antimicrobial resistance and antimicrobial resistance genes of E.coli from family farm was lower than that from farmers' market.This study proved that farmers' market had the aggregation function of antimicrobial resistance bacterial and antimicrobial resistance genes in live poultry production chain.This study provided experiment basis for the detection,diagnosis and treatment of avian Escherichia coli.As the same time,the study provided reliable data to assess transmission risk of antimicrobial resistant in live poultry production chain.

To study the characteristic and composition of swine manure of two treatments during composting using metagenomic technology.Fecal samples were collected from the same pig tank and randomly divided into two treatments after stirring,such as F group which was added to EM bacteria and K group without EM bacteria.Each treatment was made into a conical pile with a height of 1.2 m and a diameter of 2 m at the bottom.The samples were collected at 0,7,14 and 21 days during composting of two treatments and used to extract genomic DNA.Fecal microbial diversity was investigated by metagenomic sequencing technology.The results showed that in total 1 083 607 ORFs were obtained after sequencing,and the bacteria in the swine manure included 147 phyla,118 classes,258 orders,593 families,2 343 genera,and 13 193 species.At phyla level,the relative abundance of the top 10 bacterias include that Proteobacteria,Bacteroidetes,Actinobacteria,Firmicutes,Verrucomicrobia,Chloroflexi,Tenericutes,Planctomycetes,Spirochaetes,and Acidobacteria.The top 35 bacterias of the two treatments at genera level with the relative abundance were classified into 5 phylas,respectively belong to Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria and Verrucomicrobia.The KEGG functional prediction showed that an abundance of functional genes such as those involved in amino acid transport and carbohydrate metabolism pathway.The differences in the structure of two groups showed that the addition of EM bacteria would promote the changes of the flora at the early stage of composting.