To investigate the molecular characteristics of a Getah virus (GETV) strain SZC30 isolated from midge in Yunnan province and its phylogenetic relationship with GETV isolated from other vectors and host animals at home and abroad,the structural genes sequencing of a GETV strain SZC30 isolated from midge in Yunnan province for the first time in 2013 were amplified by 5 pairs of specific primers of GETV RT-PCR reactions.The sequenced cDNA fragments were analyzed by DNAStar software (SeqMan) and spliced to a complete structural genes.The structural genes of SZC30 strain was 3 762 nt in length and encoded capsid protein (C),E1,E2,E3 and 6K proteins with the length of 804,1 317,1 266,192 and 183 nt,and encoding 268,438,422,64 and 61 amino acids,respectively.Phylogenetic analysis based on C,E1 and E2 genes showed that SZC30 strain and other 27 strains of GETV isolated from different regions and hosts from 1955 to 2018 gradually evolved into four distinct evolutionary populations,including groupsⅠ,Ⅱ,Ⅲ and Ⅳ.The SZC30 strain was clustered within group Ⅲ with other GETV isolates from mosquitoes or animals in China,Korea and Japan,with nucleotide homology of more than 98.0%,amino acid homology of more than 98.9%,which indicated more related relationship between those GETV.However,SZC30 strain and other GETV isolates from mosquitoes in Malaysia,Russia were from a different evolutionary branch,and the homology of nucleotides were lower than 97.6%,which indicated that SZC30 strain had a far relationship with those GETV isolates from mosquitoes.Compared with other GETV isolates from mosquitoes in Malaysia (GenBank accession No.:AF339484),Hainan and Yunnan (GenBank accession No.:EU015061 and KY434327),there were 31 amino acid difference sites on C,E1 and E2 proteins of GETV strain SZC30 isolated from midge,but there were no difference with other GETV isolates from mosquitoes in Japan (GenBank accession No.:LC152056) and pigs in China (GenBank accession No.:MG865966 and MG865969),and the homology of amino acids were 100%.There were 2 potential glycosylation sites and 3 transmembrane domains on both of E1 and E2 proteins between SZC30 strain and other 27 strains of GETV.The analysis of antigen epitopes of T cells predicted that there were epitope differences between SZC30 strain and GETV isolated from mosquitoes,pigs,foxes,cattle and horses,and more epitope differences were found in E1 and E2 proteins.These results suggested that GETV isolated from midge had high homology and close phylogenetic relationship with most GETV isolated from other vectors and host animals and had similar similar molecular characteristics such as amino acid difference sites,potential glycosylation sites and transmembrane domains,suggesting that midge might be involved in the spread of local GETV as a potential vector.

In order to understand the genetic characteristics and the correlation of Gosling plague virus (GPV) from Jilin district with isolated strains from Heilongjiang and other provinces of China and abroad,the intestinal tissue of dead goslings from Jilin district in 2018 was detected by PCR.At the same time,the non-structural protein NS1 gene and structural protein VP1 gene of GPV were cloned and sequenced.Sequence analysis were conducted with the corresponding sequence of 16 GPV reference strains of China and abroad.The results showed that the mixed infection of GPV and EDSV was confirmed in dead gosling.The NS1 gene of GPV from Jilin was 1 884 bp in length,encoded 627 amino acids,and the nucleotide homology with the reference strains were 93.8% to 99.8%,and the amino acid homology were 97.1% to 99.7%.The VP1 gene of GPV was 2 199 bp in length,encoded 732 amino acids,and the nucleotide homology with the reference strains were 93.4% to 99.9%,and the amino acid homology were 96.4% to 99.9%.The phylogenetic tree analysis of NS1 and VP1 genes showed that GPV from Jilin and Harbin isolated 98E strain belonged to the same evolutionary branch,and the relationship was the closest,but far away from abroad,Taiwan and Anhui isolated strains.The NS1 and VP1 amino acid sequence of GPV from Jinlin were analyzed with GPV origined different kinds of waterfowls,the results showed that GPV from Jilin had a closer relationship and the higher homology with goose-origin parvovirus.This study provided basic date for clarifying the spatial epidemic regularity of GPV in Northeast China,and provided a reference basis for the diagnosis and treatment of GPV in Northeast China.

The present study was aimed to explore the physicochemical properties and structural characteristics of chlorocebus sabaeus glucose regulatory protein 78 (GRP78) gene,and to elucidate the molecular chaperone effect and regulatory mechanism of GRP78 gene in porcine epidemic diarrhea virus replication.GRP78 gene was amplified by RT-PCR and inserted into pMD20-T Simple vector for cloning and sequencing,and its amino acids sequence,transmembrane structure,glycosylation site,phosphorylation site,and tertiary structure were predicted and analyzed by bioinformatics methods.The GRP78 gene was inserted into pCDNA 3.1,and transfected into Vero-E6 cells.Western blot was employed to confirm the expression of GRP78 in Vero-E6 cells.Bioinformatics analysis results showed that the total length of GRP78 gene was 1 965 bp,encoding 654 amino acids.The molecular weight of GRP78 protein was 72.33 ku.The theoretical isoeletric point was 5.07,and the molecular formula was C₃₁₈₉H₅₁₅₃N₈₆₅O₁₀₁₉S₁₃.The homology of amino acid sequence of GRP78 gene in Chlorocebus sabaeus with Camelus bactrianus, Canis lupus, Felis catus, Gorilla, Myotis lucifugus, Papio anubis, Sus scrofa, Equus caballus were 99.5% to 99.8%.Genetic analysis showed that gorillas and baboons were the closest relatives.The prediction results of transmembrane region and signal peptide showed that the protein had signal peptide but no transmembrane region.The protein had 6 O-glycosylation sites and 28 phosphorylation sites but no N-glycosylation modification sites,which suggested that GRP78 might have binding sites for PKC and PKA specific protein kinases related to kinase phosphorylation,and might be involved in hexose metabolism and monosaccharide metabolism.

Sleep deprivation has negative effects on both animals' and humans' physical structures and functions.It can lead to multiple diseases,even cause death,especially connect to the body's digestion and metabolism.This article mainly summarizes the effects of sleep deprivation on the structure and function of stomach,intestine,liver and other organs,and focuses on the effects of sleep deprivation on the body's oxidative stress level,cytokine concentration and hormone secretion,and why they may cause digestive system disorders.In addition,food intake also affects the state of sleep deprived animals.The effects of sleep deprivation on the digestive system include damage to the digestive tract mucosa and liver cells,decreased gastric motility,dysbiosis of the gut microbiota,abnormal secretion of gastrointestinal hormones and insulin,etc.,which in turn affects energy metabolism of the body,slows down its recovery from damage,and increases the chance of getting malignant diseases.Sleep deprivation,as a stressor,will significantly increase the body's oxidative stress level,increase the metabolic burden of cells,and do harm to a variety of biological macromolecules,especially DNA.The increased release of cytokines such as TNF-α leads to widespread inflammation,hormone secretion is also a part of the damage process.If the damage continues to develop,it will increase the risk of cancer.Practice has also proved that some foods and drugs can affect sleep deprivation,but the mechanism is still unclear.At present,there are still many unknowns in the mechanism research of sleep deprivation on the digestive system and even the whole body,and there are broad research prospects.

The present study was aimed to explore the effect of oleic acid on the adipogenic transdifferentiation of Yanbian bovine skeletal muscle satellite cells.The experiment was set up 1 blank control group (CON) and 3 oleic acid (OA) induction groups:50,100 and 200 μmol/L oleic acid groups (OAL,OAM and OAH).96 h after oleic acid induction,the effect of oleic acid on cells was evaluated by measuring cell size and viability.Oil red O staining method and triglyceride determination were used to verify the formation of lipid droplets,the expressions of related myogenic adipogenic genes were measured by Real-time quantitative PCR to verify the production of adipocytes.The results showed that,compared with the control group,when oleic acid was added,lipid droplets were formed in Yanbian cattle skeletal muscle satellite cells,and the amount of lipid droplet formation and trigbyceride accumulation were dose-dependent with oleic acid.Real-time PCR measurement results showed that the myogenic related factors Pax3 and MyoD were significantly down-regulated (P<0.05),the adipogenic factors C/EBPβ and PPARγ were significantly up-regulated (P<0.05),the fatty acid metabolism-related factor SCD was significantly down-regulated (P<0.05),and the PLIN2 gene was significantly up-regulated (P<0.05).The results of the experiment showed that the induction and treatment of Yanbian bovine skeletal muscle satellite cells with oleic acid could promote the adipogenic transdifferentiation of the cells.

N⁶-methylation modification (N⁶-methyladenosine,m⁶A) is a post-transcriptional modification of eukaryotic mRNA,which is a dynamic and reversible process,catalyzed by methyltransferases,demethylases and binding proteins.It can mediate various biological processes of eukaryotes and participate in the regulation of various cellular gene expression and pathological processes of diseases.With the continuous deepening of people's understanding of RNA modification and the development of high-throughput sequencing technology in recent years,people are increasingly exploring the biological functions of m⁶A methylation modification in cell differentiation,animal growth and development,and disease occurrence.This article introduced the characteristics of m⁶A methylation modification and its related three enzymes,detection technology of m⁶A modification,as well as its biological functions in mRNA regulation,stem cell differentiation,tumorigenesis and metastasis,and a brief overview of m⁶A methylation modification regulating the growth and development of livestock and poultry animals such as pigs and chickens.Finally,the future research direction and development prospects of m⁶A methylation modification were prospected,with a view to the subsequent m⁶A methylation modification in the animal life process,in order to provide references for in-depth research and application in the prevention and treatment of diseases.

This experiment was aimed to isolate sheep skeletal muscle satellite cells(SMSCs),establish an optimal system for isolation,culture and identification of sheep SMSCs in vitro,thereby laying a seed cells for future pertinent researches.The SMSCs were isolated from new-born healthy sheep,disassociated with collagenase Ⅳ and trypsin and purified via differential adhesion method.RT-PCR and immunofluorescence methods were used to identify the expression of SMSCs marker genes paired box 7 (Pax7),Desmin and myogenic regulatory factors 1(MyoD1),and serum withdrawal method was used to induce SMSCs to differentiate into myoblasts.Satellite cells underwent muscle differentiation and formed myotubes.Muscle differentiation marker myosin heavy chain (MHC) in cytoplasm were detected by immunofluorescence.RT-PCR results showed that the amplified bands were consistent with the expected results,indicating that the cells expressed SMSCs marker genes Pax7,Desmin and MyoD1.The cultured cells expressed marker proteins Pax7,Desmin and MyoD1,demonstrating that SMSCs were obtained.Muscle differentiation marker MHC was detected in these cells.From the above results,the isolated and cultured cells were SMSCs,and a suitable in vitro culture system for sheep SMSCs was established,and myogenic differentiation was successfully carried out,which provided experimental materials and technical support for the future research on the mechanism of sheep skeletal muscle growth and development.

The purpose of the experiment was to comprehensively analyze the quality of whole-plant corn silage by principal component analysis and grey correlation,and to provide a theoretical basis for the rational use of whole-plant corn silage.The samples were collected from different large-scale pastures located in Huang-Huai-Hai plain of China (Shandong,Henan,Hebei),the middle and lower reaches of Yangtze River (Anhui),the Northwest area (Shaanxi,Shanxi,Qinghai,Ningxia,Gansu,Xinjiang,Inner Mongolia),the Northeast area (Heilongjiang,Jilin,Liaoning),the South China (Guangxi) and Southwest (Yunnan,Guizhou).Relevant indicators of whole-plant corn silage quality in different regions were detected by near-infrared spectroscopy and further comprehensively analyzed by principal component analysis and grey correlation analysis methods.The results showed that:①The nutrient composition and fermentation quality of whole-plant corn silage in different regions were significantly different (P<0.05).②Principal component analysis extracted 4 principal components,and the cumulative contribution rate reached 82.882%.The 4 principal components respectively reflect the content of crude fiber,organic acid,and neutral detergent fiber degradation rate of whole-plant corn silage,and could be used to measure the comprehensive quality of whole-plant corn silage in different regions.The comprehensive quality of whole-plant corn silage in different regions from high to low was Huang-Huai-Hai plain,Northwest,Northeast,middle and lower reaches of Yangtze River,Southwest and South China.③By analyzing the quality of whole-plant corn silage in different regions and comparing with the ideal quality index by grey correlation analysis,the equal weight correlation degree of corn silage quality in different regions was 0.71308-0.71264.The quality of corn silage in Huang-Huai-Hai plain was better than that in Northwest,followed by the Middle and Lower reaches of Yangtze River,Northeast,South China and Southwest.In summary,the results of principal component analysis and grey correlation analysis were basically consistent.The quality of whole-plant corn silage in Huang-Huai-Hai plain and Northwest regions of China was high,while it was low in South China and Southwest regions.The comprehensive evaluation model of whole-plant corn silage quality established based on the two methods could support each other,and had certain feasibility and scientificity.

The methods of chemical composition analysis,carbohydrate fractions analysis,protein fractions analysis,energy value prediction and in vitro artificial rumen were used to evaluate the nutritional values of potato pulp and sweet potato pulp.5 potato pulp samples were collected from starch factories in Chengde,Zhangjiakou,Jilin,Ulanchabu and Xi' an.7 sweet potato pulp samples were collected from starch factories in Shijiazhuang,Baoding,Tangshan,Jinan and Qinhuangdao.The results showed that EE content (0.80%) of sweet potato pulp was significantly higher than that of potato pulp (0.58%) (P<0.05).The content of CP,NDF,ADF and ADL of potato pulp (6.53%,26.03%,20.77% and 4.38%) were extremely significantly higher than that of sweet potato pulp (4.57%,16.46%,12.66% and 1.51%) (P<0.01).The starch content of potato pulp and sweet potato pulp were both higher,which were 41.05% and 41.71% respectively (P>0.05).The CC content of potato pulp (9.76%) was extremely significantly higher than that of sweet potato pulp (3.67%)(P<0.01).The PA content of potato pulp and sweet potato pulp were the highest in protein fractions,which were 2.77% and 2.41% respectively (P>0.05).The content of PB2 of potato pulp (1.39%) was significantly higher than sweet potato pulp (0.74%)(P<0.05).The content of PB3 and PC of potato pulp (0.56% and 1.01%) were extremely significantly higher than that of sweet potato pulp (0.28% and 0.47%) (P<0.01).TDN,DE,ME,NE_m,NE_L and NE_g of sweet potato pulp (81.58%,15.03,13.31,9.09,8.50 and 6.24 MJ/kg) were extremely significantly higher than potato pulp (75.11%,13.82,12.10,8.08,7.66 and 5.40 MJ/kg)(P<0.01).DMD_{24 h} and DMD_{48 h} of sweet potato pulp (53.78% and 62.48%) were extremely significantly higher than potato pulp (49.34% and 58.66%) (P<0.01).In conclusion,the nutritional values of potato pulp and sweet potato pulp were both higher,and had the potential to become a feed material for beef cattle,and sweet potato pulp was better than potato pulp.

In order to promote the resource utilization of herbal tea residue,the solid-state fermention using Aspergillus niger was adopted in this study.The effects of six single factors,including time,temperature,water content,initial pH,nitrogen source,and carbon source,on the degradation rate of herbal tea residue and product pH after fermentation were investigated.According to the single factor experimental results and the actual situation of large-scale production,4% ammonium sulfate was used as nitrogen source,2% glucose as carbon source,through orthogonal experiments,the fermentation process was optimized with the degradation rate as the index.The antioxidant activity of herbal tea residue before and after fermentation was evaluated by measuring superoxide radical scavenging rate,hydroxyl radical scavenging rate and DPPH radical scavenging rate.It was found that the optimal parameters of fermentation were water content 60%,pH 9.0,31 ℃ for 168 h.Under the optimum conditions,degradation rate of herbal tea residue was 25.23%,products pH was 4.53.As the concentration of water-extract of herbal tea residue before fermentation was 24 mg/mL,the scavenging rates of superoxide radical,hydroxyl radical and DPPH radical were 43.56%,47.06% and 90.71%,respectively.When the water-extract concentration of product was 24 mg/mL,the scavenging rates of superoxide radical,hydroxyl radical and DPPH radical were 30.77%,95.63% and 87.36%,respectively.The results showed that Aspergillus niger was suitable for fermentation of herbal tea residue,and herbal tea residue had good antioxidant activity after fermentation by Aspergillus niger.

Two different kinds of antibiotic-free addictive were added to the growing pigs' diet to explore their effects on growth and intestinal health.Sixty LDY crossbred pigs at the weight of (85.95±3.48) kg were selected and divided into control group,groups 1 and 2 with 5 replicates in each group and 4 pigs in each replicate (half of castrated boars and half of sows).The pigs in control group were fed with basal diet,in group 1 were fed with 0.5% Monascus yeast selenium germanium complex preparation,and in group 2 were fed with 0.5% compound Chinese medicine extract in the basic diet.The results showed that:①There was no significant difference in growth performance among the three groups (P>0.05).②Compared with the control group,the villus height(VH) of jejunum,villus height/crypt depth(V/H) and villus height of ileum were significantly increased in group 1 (P<0.05),villus height of jejunum and ileum in group 2 were significantly increased (P<0.05).Villus height of jejunum and ileum in group 1 were significantly higher than that in group 2 (P<0.05).③Compared with the control group,the Shannon index of cecal flora in groups 1 and 2 were significantly increased (P<0.05),and the Simpson index were significantly decreased (P<0.05).The Shannon index of group 1 was significantly higher than that of group 2 (P<0.05).④At the phylum level,compared with the control group,the abundance of Proteobacteria and Actinobacteria were significantly increased (P<0.05),and the abundance of Firmicutes was significantly decreased (P<0.05) in group 1.The abundance of Firmicutes and Proteobacteria were significantly increased (P<0.05),and the abundance of Bacteroidetes was significantly decreased (P<0.05) in group 2.The abundance of Actinobacteria in group 1 was significantly higher than that of group 2,and the abundance of Firmicutes in group 1 was significantly lower than that of group 2.At the genus level,compared with the control group,the abundance of Streptococcus was significantly increased (P<0.05),and the abundance of Turicibacter,uncultured_bacterium_f_Muribaculaceae and Lactobacillus were significantly decreased (P<0.05) in group 1.The abundance of Ruminococcaceae_UCG-005 and Streptococcus were significantly increased (P<0.05),and the abundance of Turicibacter,Clostridium_sensu_stricto_13 and Lactobacillus were significantly decreased (P<0.05) in group 2.The abundance of Clostridium_sensu_stricto_13 and Streptococcus in group 1 was significantly higher than that of group 2 (P<0.05),the abundance of Uncultured_bacterium_f_Muribaculaceae and Ruminococcaceae_UCG-005 in group 1 were significantly lower than that of group 2 (P<0.05).⑤ In group 1,the expression of Occludin,ZO-1 and Claudin-1 were significantly higher than the control group (P<0.05),and the expression of Occludin and Claudin-1 were significantly higher than group 2 (P<0.05).The above results showed that both the Monascus yeast selenium germanium complex preparation and compound Chinese medicine extract could improve the intestinal health of growing pigs,and the feeding effect of Monascus group was better.

This experiment was conducted to investigate the effects of dietary crude fiber level enhancement on rumen microbial composition and diversity of fattening lambs.In this study,healthy and disease-free male lambs were selected.All lambs with similar weight at the age of 2 months were randomly divided into two groups according to the body weight,38 males in each group were fed with different diets.The diets of control group were composed of concentrate and alfalfa hay,while the diets of the treatment group were composed of whole corn silage and peanut seedling based on control group diet.The period of trial lasted 150 d,including 20 d in the preliminary phase.After the end of the experiment,5 lambs from each group were slaughtered,and rumen contents were collected for the measurement of microbial diversity and structural changes by 16S rDNA high-throughput sequencing technology.The results showed that the bacterial diversity in the rumen of the treated lambs was significantly higher than that of the control group (P<0.05).At the level of phylum,the dominant bacteria in the rumen of the lambs in the treatment group were Bacteroidetes and Firmicutes,while the dominant bacteria in the control group were Bacteroidetes and Proteobacteria.After the addition of fiber,the relative abundance of Firmicutes in the rumen of the fattening lambs was significantly increased (P<0.05),the relative abundance of Proteobacteria was significantly reduced (P<0.05).At the level of genus,195 genera were identified from the rumen bacteria of the control group and the treatment group,and the dominant bacteria in the treatment group were Prevotella_1 and Succiniclasticum,while the dominant bacteria in the control group were Succinivibrionaceae and Prevotella_7.The relative abundance of Succiniclasticum,Treponema_2,Ruminococcaceae and Selenomonas_1 in the rumen of fattening lambs was increased by increasing the dietary fiber level,and the relative abundance of Succinivibrionaceae was significantly reduced (P<0.05).Therefore,the increase of dietary fiber level appropriately had a certain effect on the structure of rumen microbial flora of fattening lambs,which could promote the proliferation of some fiber-degrading bacteria,and was beneficial to rumen fermentation and nutrient digestion and utilization of fattening lambs.

The effects of short-term feeding with Bacillus velezensis M2 isolated from Min pigs on the growth performance,blood biochemistry,immunity,and intestinal flora of SD rats were investigated for providing a reference for the application of Bacillus velezensis in livestock and poultry.A total of 24 healthy SD female rats of 21-day-old,similar weight and SPF grade,were randomly divided into two groups (six repeats per group,and two rats per repeat).Pre-feeding period lasted three days.During the trial period,the rats in control group were fed orally 0.2 mL normal saline per day for 7 days,while those in the experimental group were fed orally 0.2 mL bacterial solution per day for 7 days.At the age of 35- and 45-day-old,one rat in each repeat was randomly selected to be executed and sampled.The results showed that feeding with Bacillus velezensis M2 increased the final weight and daily gain,and decreased daily feed intake and feed-gain ratio (P>0.05).Short-term feeding with Bacillus velezensis M2 significantly reduced plasma triglyceride concentration,plasma IgA and TNF-α level of 35-day-old rats,and plasma IL-6 level of 45-day-old rats (P<0.05),and significantly increased plasma IgM level of 35-day-old rats (P<0.05).Short-term feeding with Bacillus velezensis M2 had no significant effect on intestinal flora of 35-day-old rats (P>0.05),but significantly reduced the proportion of Actinomycetes and Proteobacteria in 45-day-old rats (P<0.05).These results indicated that short-term feeding with the Bacillus velezensis M2 regulated the lipid metabolism,immunity and intestinal flora abundance in SD rats.

This experiment was conducted to study the difference in the taste activity value (TAV) of the full spectrum of free amino acids (FAA) in the muscles of Diqing Tibetan pigs(TP) and its hybrid wild boars (WT).12 TPs and 12 WTs with the same parity,similar birth dates,and weighing about 20 kg were selected and fed with the same feed,and then slaughtered when they reached about 100 kg.The longissimus dorsi (LD) was collected and steamed with boiling water for 30 min.Taking TPs as control,the composition and content of FAA were detected by high performance liquid chromatography-quadruple/linear ion trap mass spectrometry (HPLC-Q-Trap-MS),then the differences in TAV of FAA were calculated and compared.The results showed that compared with TPs,the total umami TAV of FAA in WTs was decreased by 16.27% (P<0.05).The total sweet,sour and bitter TAV of FAA in WTs was decreased by 3.50%,11.68% and 1.50% respectively,but the difference was not significant (P>0.05).The umami and sweetness TAV of glutamine (Gln) in WTs were both decreased by 22.89% (P<0.05),and the sweetness and bitterness TAV of valine (Val) were both increased by 36.32%(P<0.05).There was no significant difference in the umami,sweet,sour and bitter TAV of other amino acids such as aspartic acid,glycine,glutamic acid and histidine between the two groups (P>0.05).In conclusion,crossbreeding with wild boars would reduce the umami TAV of TP,the umami and sweetness TAV of Gln,and increase the sweetness and bitterness TAV of Val,which could provide basis for the development and utilization of TP.

The aim of this study was to analyze the molecular characteristics and the expression level of transmembrane epididymal protein 1 (TEDDM1) gene during follicle development of the yak.In this study,cumulus-oocyte complexes (COCs) were collected from small follicles (φ≤3 mm),medium follicles (φ=5-7 mm) and large follicles (φ≥8 mm) of yak during estrus,and total RNA was extracted and reverse transcription was conducted.The complete sequence of the CDS region of the TEDDM1 gene was sequenced.Bioinformatics soft-wares were used to analyze the molecular characteristics of CDS region sequence structure.Using GAPDH as reference gene,Real-time quantitative PCR was performed to reveal expression level of TEDDM1 in small,medium and large follicles.The results showed that the CDS region of yak TEDDM1 gene was 903 bp,encoding 300 amino acid,which was highly similar to Bos mutus,Bos taurus,Bison,Ovis and Bubalus bubalis,and was conserved in evolution.Yak TEDDM1 protein was a non-secretory unstable hydrophobic protein with seven helix structures,and the whole peptide chain crosses the cell membrane seven times,which was a typical G protein-coupled receptor.Twenty-one potential phosphorylation sites were distributed in the protein,including 1 tyrosine,3 threonine and 17 serine phosphorylation sites,only one N-glycosylation site and no O-glycosylation prediction site.Functional domain analysis showed that unknown functional domain protein family 716 domain existed in TEDDM1 protein,covering multiple transmembrane regions.Result of Real-time quantitative PCR analysis showed that the expression level of TEDDM1 in medium follicles was significantly higher than that in large follicles and small follicles (P<0.05),but there was no significant difference between large follicles and small follicles (P>0.05).The study provided the sequence of yak TEDDM1 gene and its expression characteristics in yak COCs,laid a theoretical foundation for further revealing the regulatory role of the yak TEDDM1 gene in follicular development.

In order to verify and discover molecular markers and major genes of lambing traits,541 samples' SNP genotyping of three SNPs (rs268286450,rs268286391 and rs268244272) on chromosome 28 which were selected from Yunshang Black goat in early research was performed by MassARRAY time flight mass spectrometry,and analyzed the relationship between polymorphism of three SNPs and the lambing number in Yunshang Black goat.The results showed that there were a C→T base mutation at 28 498 787 bp of rs268286450,a A→C base mutation at 30 971 277 bp of rs268286391 and a T→C base mutation at 2 707 491 of rs268244272 on chromosome 28.The polymorphism information content (PIC) of three SNPs were 0.36,0.32 and 0.21,respectively.rs268286450 and rs268286391 were in Hardy-Weinberg disequilibrium (P<0.01),while rs268244272 was in Hardy-Weinberg equilibrium (P>0.05).The locus rs268244272 had three genotypes (CC,TT and TC).The wild genotype was TT and its genotype frequency was 0.75.The genotype frequency of TC was 0.22,which was a mutant genotype.The difference of the least squares mean in lambing number between TC and TT genotypes was extremely significant (P<0.01),but not significantly different from CC genotype (P>0.05).There was no significant difference of the least squares mean in lambing number between TT and CC genotypes (P>0.05).The results of association analysis showed that there was no significant correlation between lambing number of Yunshang Black goat and the polymorphism of rs268286450 and rs268286391 (P>0.05),but a significant association existed between the polymorphism of rs268244272 and lambing number (P<0.05).In conclusion,rs268244272 polymorphism on chromosome 28 of Yunshang Black goat was significantly associated with lambing number,and might be used as a molecular marker of lambing number in breeding of high reproduction straits in Yunshang Black goat.

Nowadays,the donkey industry is shifting from short-term fattening to a new breeding model for sustainable development.Similar to other livestock and poultry breeds,the transformation of the donkey industry is also based on the expansion of good populations and the improvement of breeds' quality,and the breeding of good-quality is a key step to increase the quantity and improve the quality of donkeys.However,one of the main factors in restricting the transformation of donkey industry is low reproduction capacity.Low reproduction capacity in donkey not only impacts the expansion speed of the existing donkey,and widens the gap between the stock and the rising demand for skin,meat and milk,but also prolongs the breeding cycle of new breeds.Studies have shown that assisted breeding techniques play important roles in improving the reproductive performance of female donkeys and the genetic traits of donkeys.Assisted reproduction technologies such as simultaneous estrus,ovulation control,artificial insemination and embryo transplantation can largely improve the reproductive capacity of female donkeys.Meanwhile,a series of molecular breeding technologies including microsatellite labeling,mitochondrial DNA sequencing,single nucleotide polymorphism analysis and whole genome selection can further accelerate the genetic improvement of donkeys.Here,a brief review of the studies on the reproductive physiology of the donkey was presented,and then,systematically summarized the progress of recent research on the roles of assisted breeding technologies (simultaneous estrus,ovulation control,artificial insemination,embryo transplantation,molecular marker assistant breeding and genomics-based selection) in donkeys,and made prospects for future research directions.This paper would provide an important reference for improving the reproductive performance of female donkeys and the effective clues to the donkey molecular breeding.

The purpose of this study was to verify the accuracy of specific SNPs of red deer based on genotyping sequencing (GBS) technology,and provide a reliable basis for the identification of sika deer,red deer,and their hybrid offspring.Thirty specific SNPs of red deer were randomly selected,based on the sequences of 200 bp each before and after the SNPs,Primer Premier 6.0 software was used to design specific primers,and the randomly selected DNA of the validation samples was used as a template for amplification,PCR was performed,and the amplified products were sequenced in one generation.The sequences obtained by sequencing were analyzed by Mega 6.0 software,and the genotypes of each SNP site in different validation populations were observed.The results showed that 28 out of 30 specific loci were consistent with the results of the previous study,and the gene frequency of the G allele at one of SNPs (SNP3) was 0.05 in sika deer,while the gene frequency of the G allele was 1 in red deer.The loci showed significant differences in genotype distribution between red deer and sika deer (P<0.05).The gene frequency of the T allele in the other SNP (SNP5) was 0.15 in sika deer,while the gene frequency of the T allele was 1 in red deer,and the genotype distribution of this SNP in sika deer and red deer was significantly different (P<0.05).So these two loci could still be used as specific SNPs of red deer.The results showed that the specific SNPs of red deer selected by GBS sequencing could be used as molecular markers for identification,and laid a theoretical foundation for the identification of sika deer,red deer and their hybrid offspring.

The aim of this study was to investigate the effect of cloprostenol sodium,antepartum backfat thickness,and constipation on gilts synchronized parturition and reproductive performance following farrowing induction.Furthermore,the optimal scheme of synchronous parturition for primiparous was discussed.A total of 295 gilts were involved in this study.The backfat thickness at point P2 was measured on 110 d of gestation,and gilts were randomly divided into 3 groups:control (CON group:0.9% saline),low-dose of cloprostenol sodium (LPG group:0.1 mg),and high-dose of cloprostenol sodium (HPG group:0.2 mg),farrowing induction were performed on 114 d of gestation via perivulvar injection.The results showed that compared with CON group,the duration was significantly shortened from injection to farrowing both in LPG and HPG gilts (P<0.05),but the difference between the LPG and HPG group was not significant (P>0.05).It was worth noting that,as higher as 95% of gilts started farrowing within 48 hours after injection in LPG group,of which the proportion of daytime farrowing reached up to 75.7%.In addition,the birth interval of gilts in LPG group were significantly shorten than CON group (P<0.05).Meanwhile,it was found that the gilts with backfat thickness in the range of 16-20 mm was significantly shorter the duration from injection to farrowing than that of gilts with backfat thickness ≤15 mm (P<0.05).Moreover,the birth duration and farrowing interval of gilts in the constipation index range from 0-1.0 group were significantly longer than those in the constipation index range 1.1-2.0 and 2.1-3.0 groups (P<0.05).Through regression analysis,it was further found that the backfat thickness of gilts were significantly linearly correlated with the farrowing duration,total born,and number of born alive (P<0.01).Furthermore,constipation index was a significantly linear correlated with farrowing duration,birth interval,total born,and number of born alive (P<0.01).In conclusion,administration of the low-dose cloprostenol sodium for gilts on 114 d of gestation via perivulvar injection was recommended,since it had advantages in shorten the onset of start farrowing without considerable negative effects on gilts and piglets,as well as reducing cost of the active ingredient.In addition,both the backfat thickness and constipation of gilts before farrowing seemed to be factors impact on the effect of synchronized parturition.It was necessary to maintaining moderate (16-20 mm) backfat thickness and avoiding constipation before gilts' farrowing.

The purpose of this study was to investigate the lactation law of Hetian multiparous Red sheep and improve the survival rate of double lambs.In this experiment,6 ewes of Hetian multiparous Red sheep with double lambs were selected as the objects to study the changes of milk yield and milk composition from 1 to 28 days after parturition,and analyze the correlation between breast body size index and milk yield on the first postpartum day.The results showed that the total milk yield of Hetian multiparous Red sheep was 29.87 kg from 1 to 28 days after parturition,and the average daily milk yield was 1.07 kg,the milk production increased fastest at 1-3 d,and reached the peak value of 1.23 kg on the 21st day.The lactation curve equation was y=537t^0.477e^-0.033t.The milk fat,non-fat milk solids,density,lactose,solids,protein indexes of Hetian multiparous Red sheep decreased rapidly at 1-3 d after parturition,and tended to be stable at 7-28 d,and there were significant differences between the 1st day and the 7th,14th,21st and 28th days of the six indicators (P<0.01).The lactation correlation between the 1st day and 14th,21st and 28th days was not significant of Hetian multiparous red sheep ewes after delivery (P>0.05),and the lactation correlation between the 2nd day and 28th day was not significant (P>0.05),others were significantly correlated(P<0.05).There was a significant correlation between lactation and breast width on the 1st,2nd,3rd and 7th days of parturition (P<0.05),and there was no significant correlation between lactation and breast body size on the 14th,21st and 28th days of parturition (P>0.05).The above results provided a theoretical basis for the germplasm characteristics,breeding,feed preparation and early weaning of Hetian multiparous red sheep.

Mammal sperm and oocytes are produced by meiosis,leading to haploid genomes.The gametes are highly specialized.After the sperm-oocytes fusion,the number of chromosomes are restored,and the developmental totipotency is re-obtained.Following that the mitosis is restarted and individual starts to grow.During fertilization,sperm causes the calcium ions (Ca²⁺) rising at a certain frequency,which is called Ca²⁺ oscillation.The Ca²⁺ oscillation drives the activity of downstream proteins to complete the activation of the oocytes.This article mainly discussed the mechanism of initiation,maintenance and function of Ca²⁺ oscillation during fertilization.The duration,amplitude,and frequency of Ca²⁺ oscillation affect the secretion of cortical granules,the resumption of MⅡ arrest,and the recruitment of maternal mRNA.This article also reviewed how Ca²⁺ oscillations activate maternal mRNA transcription and restart mitosis of zygote.Recent years,great progress has been achieved in discovering signaling pathways that drive oocytes activation events,but the molecular mechanisms involved in each individual event have not yet been fully determined.This review discussed the deficiencies and future directions of related reasarch.In the future,the elucidation of the role of Ca²⁺ oscillation will definitely benefit reproductive techniques for human and the livestock industry.

This experiment was conducted to compare the distribution of the fibroblast growth factor 22 (FGF22),fibroblast growth factor receptor 2 (FGFR2),and the related regulatory factors heparan sulfate proteoglycans (HSPG) in normal testis and cryptorchidism of Qingyang Black goat,and explore their roles in testicular development and cryptorchidism.HE staining and special staining were used to observe the histological structure of FGF22,FGFR2 and HSPG,and the localization of FGF22,FGFR2 and HSPG in goat normal testis and cryptorchidism was studied by immunohistochemistry and immunofluorescence combined with morphometry.The results showed that the seminiferous tubules of cryptorchidism were narrower than those of normal testis,the arrangement of spermatogenic cells was disordered,the collagen fibers and reticular fibers in interstitium were increased,and the positive reaction of glycogen was weak.The overall expression density of FGF22 in Leydig,Sertoli cells,peritubular myoid cells and vascular endothelial cells of cryptorchidism was significantly lower than that of normal testis (P<0.05).The expression of HSPG in normal testis was significantly higher than that in cryptorchidism (P<0.05),especially in interstitial tissue.The expression of FGFR2 was significantly increased in cryptorchidism group (P<0.05),and Sertoli cells were the main positive cells.The results showed that the cryptorchidism of Qingyang Black goat was more abnormal than that of normal testis,the interstitial tissue had a tendency of fibrosis,and the content of glycogen decreased.The decreased expression of FGF22 and HSPG should be closely related to the changes of local environment temperature of cryptorchidism.The increased expression of FGFR2 in cryptorchidism group suggested that it might be regulated adaptively by Sertoli cells during cryptorchidism.

The purpose of this study was to investigate the growth promoting effect and the safety of the vaccine in crossbred buffalo growing cattle immunized with somatostatin and corticostatin co-expression DNA vaccine.The identified plasmid of pVGS/2SS-2A-S/CST-asd was electroporated into attenuated Salmonella Choleraesuis C500 to construct the co-expression live vector vaccine of somatostatin and corticostatin.Sixteen 6-12 months old crossbred buffalo growing cattle were randomly divided into low dosages group (TL),middle dosages group (TM),high dosages group(TH) and negative control group(NC).At the initial immunization,immunization was performed at 09:00 and 15:00 twice a day for 3 d,and the same procedure was used to strengthen immunization once after 2 weeks.The experimental group and control group was immunized with co-expression vaccine and PBS respectively,and the effect of daily weight gain and immune response level in different periods after the primary immunization.The immune response results showed that all dose groups induced immune response after the primary immunization,the antibody of somatostatin (SS) in TL group was higher than that in TM and TH groups at 2 weeks after primary immunization,and the positive rate of SS antibody in TM group was the highest,which was 100% and better than that in TL (75%) and TH groups (50%).However,the antibody of cortistatin(CST) after 2 weeks of primary immunization showed a completely opposite trend,high dosages group showed the best performance,and with the decrease of immune dose,the antibody of CST also showed a downward trend.After 7 weeks of primary immunization,antibody of SS and CST and positive rate showed a downward trend,but some buffaloes showed an upward trend.In the aspect of cytokines,it was found that the interleukin 4 (IL-4) level at 2 weeks after the primary immunizationin TL and TM groups was significantly higher than that in control group (P<0.01).At 7 weeks after primary immunization,the IL-4 level in control group was significantly higher than that in TL group (P<0.05),and there was no significant difference with other groups (P>0.05).The level of immune interferon γ (INF-γ) at 2 weeks after the primary immunization in control group was significantly higher than that in TL and TM groups (P<0.05).The INF-γ level at 7 weeks after the primary immunizationin in control group was significantly higher than that in TL group (P<0.05),but there was no significant difference with other groups (P>0.05).The IL-4 level at 2 weeks after the primary immunizationin in antibody positive group was significantly higher than that in negative group (P<0.01),and was significantly lower than that in control group at 7 weeks after the primaryimmunization (P<0.05),but the INF-γ level at 2 and 7 weeks after the primary immunizationinin in the antibody positive group was significantly lower than that in the negative group (P<0.05).The weight gain effect test showed that the average daily gain of each experimental group was significantly higher than that of control group at 2 weeks after the primary immunization (P<0.05),but there was no significant difference among the groups in other periods (P>0.05).The antibody positive group was also significantly higher than the negative group at 2 weeks after the primary immunization (P<0.05).The hormone test results showed that growth hormone (GH) and insulin-like growth factor 1 (IGF-1) levels in the experimental groups were significantly higher than those in control group at 2 weeks after the primary immunization (P<0.01),while the levels of GH and IGF-1 in the low dosages group were significantly lower than those in control group (P<0.05),but there was no significant difference between the other groups (P>0.05),the levels of GH and IGF-1 in the antibody positive group were significantly higher than those in the negative group at 2 weeks after the primary immunization (P<0.01),and were significantly lower than those in control group at 7 weeks after the primary immunization (P<0.01).In the aspect of safety,no target fragment was detected in water,soil and fecal samples collected in each period.The results showed that the co-expression vaccine could induce immune response and promote the average daily gain of growing cattle,and no safety problem was detected.

In order to explore the effect of interferon-inducible transmembrane protein 1 (IFITM1) on the replication of porcine coronaviruses,the highly pathogenic virus strain of Transmissible gastroenteritis virus (TGEV) and its host cell PK15 were selected as the research objects.After normal PK15 cells were inoculated with TGEV,Real-time PCR was used to detect the mRNA expression levels of various interferon-stimulated genes (ISGs) in PK15 cells at different time points post infection.PK15 cell lines that stably expressed and stably interfered with the expression of IFITM1 were constructed through the lentiviral expression system.A shRNA sequence targeted to interfere with porcine IFITM1 and a full-length porcine IFITM1 sequence were inserted into pLKO.1-EGFP-Puro vector and pLVML-Myc-MCS-IRES-Puro vector to construct recombinant plasmids pLKO.1-IFITM1shRNA-EGFP-Puro and pLVML-Myc-IFITM1-IRES-Puro,respectively.The recombinant plasmids and the lentiviral packaging plasmids were co-transfected into 293FT cells to obtain a recombinant lentivirus with the target gene.After the lentivirus infected PK15 cells,it was screened with puromycin to obtain a PK15 cell line that stably expressed and stably interfered with the expression of IFITM1,named PK15-IFITM1 and PK15-IFITM1^-/-,respectively,and Real-time PCR,IFA and Western blotting were used to detect the interference efficiency and expression of IFITM1.TGEV was inoculated with PK15-IFITM1^-/- and PK15-IFITM1,and Real-time PCR was used to determine the copies of TGEV in the cells.The results showed that the mRNA level of some ISGs increased within 48 h after PK15 cells were inoculated with TGEV.The interference efficiency of PK15-IFITM1^-/-cell line was 70%,and the expression of PK15-IFITM1 cell line was successful.In the PK15-IFITM1^-/- cell line,the mRNA level of IFITM1 was significantly down-regulated,which promoted the replication of TGEV.Conversely,in the PK15-IFITM1 cell line,the replication of TGEV was inhibited.However,the expression or absence of IFITM1 did not affect the adsorption of TGEV to PK15.In short,IFITM1 had a significant antiviral effect on TGEV,IFITM1 had no effect on the adsorption of TGEV to PK15 cells,which laid the foundation for subsequent research on the anti-coronavirus mechanism of IFITM1.

This study was aimed to obtain a recombinant Clostridium botulinum neurotoxin type C,and evaluate its virulence and immunogenicity.The fragment GMBPCH_Ccomposed of MBP and C-terminal of the Clostridium botulinum neurotoxin type C heavy chain (CH_C) was optimized and synthesized.Then,GMBPCH_C was cloned into pET28a-(＋),and subsequently transformed into E.coli BL21 (DE3) competent cells,followed with induction at 15 and 37 ℃,respectively.The recombinant protein rMBPCH_C expressed in a soluble form was then purified by Ni-IDA chromatograph.Then,4 rabbits were immunized with 100 μg of rMBPCH_C emulsified with oil adjuvant of ISA 201.According to the method prescribed in Chinese Veterinary Pharmacopoeia (2015),serum samples were collected 21 d after the first immunization and 14 d after last immunization to detect the neutralizing titer against the Clostridium botulinum neurotoxin type C (crude toxin).At the same time,rabbits were challenged with crude toxin 14 days after the second immunization.Results showed that rMBPCH_C was expressed predominantly in an insoluble form induced at 37 ℃ and expressed as soluble form with a rate of 50% induced at 15 ℃.The neutralization titer against crude toxin of sera from 4 rabbits immunized with rMBPCH_C could both reach 1 (sera could neutralize 1 mice MLD crude toxin per 0.1 mL) after the first immunization.After the second immunization,neutralization titer could reach 4-8.Moreover,rabbits in rMBPCH_C immunized group 100%(4/4) rabbits were survived at the dose of 10 rabbit MLD crude toxin challenge,whereas 100%(2/2) rabbits were died in control group.These data suggest that rMBPCH_C was a potential vaccine candidate for the subunit vaccine of Clostridium botulinum type C.

As an important component of innate immune system,neutrophils play a critical role in defense against infection with pathogens.One of the mechanisms by which neutrophils play a defensive role is to capture and destroy pathogens by releasing neutrophil extracellular traps (NETs).However,some pathogens have evolved the escaping mechanisms,and cause a persistent damage in organism.In addition,abnormal NETs can also lead to an organism damage.Recently,NETs has become a focus of research in neutrophil function,and has been widely studied worldwide.Some researchers have found that the reticular structure released from avian heterophiles is similar to that of mammalian NETs,and named it heterophil extracellular traps (HETs).In this review,the authors summarized the mechanisms by which NETs resist pathogen invasion and pathogens escape from NETs,and the pathogenesis caused by interaction between NETs and pathogens.

In order to evaluate of the protective efficacy of Pseudorabies virus (PRV) inactivated vaccine (HN1201-ΔgE) against the challenge of PRV epidemic and classic strains,piglets were immunized with the inactivated vaccine (HN1201-ΔgE) and live vaccine (Bartha-K61) separately and the gB antibody (Ab) was evaluated using the serum samples collected pre-immune and 7,10,14,17,21,24 and 28 days post-immunization.The pigs were challenged with the epidemic strain of HN1201 and the classical strain of Fa at the 28th day post-immunization,respectively.After challenging,the body temperature,gE antibody (7 and 14 days post-challenge),and the virus shedding (0-8 days post-challenge) were tested.The results showed that the production of gB Ab in HN1201-ΔgE immunization group was earlier and higher than that in Bartha-K61 immunization group.Compared with control group,the appearance of gE Ab in all immunization groups postponed and showed a lower rate of virus shedding after challenge.The control group showed typical clinical symptoms and pathological damage after challenge,together with the positive result of antigen detection in IHC test,while the piglets in the two immunization groups had no obvious clinical symptoms,and the IHC test was negative,except part of the organs in the Bartha-K61 immunization group had minor pathological damage.Compared with control group,the pigs in two immunization groups showed a late seroconversion of gE antibody.The gE antibody level of the HN1201-ΔgE immunization group was lower than that of the Bartha-K61 immunization group.The Bartha-K61 immunization group showed virus shedding on the 3rd to 5th day post challenge.While no virus shedding was detected in the HN1201-ΔgE immunization group.Our studies suggested that the inactivated vaccine (HN1201-ΔgE strain) provided complete protection against the challenge of both the PRV epidemic and classical strains.

To provide basis for diagnostic methods of Epidemic hemorrhagic disease virus (EHDV),VP7 protein of EHDV was expressed and polyclonal antibody against the protein was prepared in present study.Recombinant VP7 protein expressed in E.coli was purified and renatured through Ni²⁺-NTA affinity chromatography and dialysis,respectively.Specific polyclonal antibodies against the expressed VP7 protein were obtained by immunized rabbits.The titer and reactogenicity of the prepared antibodies were evaluated through indirect ELISA,Western blotting and indirect immunofluorescence assay (IFA),respectively.The results showed that VP7 recombinant protein with molecular weight at approximately 46 ku was efficiently expressed in E.coli as inclusion body when induced at 37 ℃ with the concentration of IPTG at 0.1 mmol/L.After purification and renaturation,the purity of recombinant VP7 protein was 94.52%,which could be recognized by sera of different EHDV serotypes.The titer of the prepared rabbit polyclonal antibody against EHDV VP7 was 1∶24 000 and the VP7 protein expressed in the EHDV infected cells was detected by Western blotting and IFA using the prepared antibody.In summary,VP7 protein of EHDV was successfully expressed in E.coli and rabbit polyclonal antibody against VP7 protein with robust specificity and reactivity was prepared,which provided basic materials for the development of EHDV diagnostic antigen and the establishment of serological detection method.

The pathogenic Escherichia coli (E.coli) was isolated,identified,typed and analyzed for genetic evolution of 106 healthy adult Holstein cows in Karama,Xinjiang,and the epidemic situation of pathogenic Escherichia coli in cows in the investigation area was preliminarily known.With reference to the USDA detection method,106 stool samples were selectively enriched,and the E.coli was separated,purified and identified using MacConkey and Eosin Methylene Blue agar medium.The PCR method was used to identify 55 strains of E.coli.The isolates were subjected to pathogenic E.coli serotype identification,phylogenetic group and MLST type analysis,clustering and evolution studies were performed using goeBURST and Mega 7.0 biological software,and epidemiological analysis was performed.The results showed that the detection rate of Escherichia coli in fresh feces of 106 healthy Holstein cows was 51.89%.Nine serotypes related to pathogenicity were identified in 55 E.coli isolates,the detection rate was 54.55%.The detection rates of EPEC,EIEC and ETEC were 21.82%,18.81% and 14.55%,respectively.The dominant serotypes were O142∶K86 (B) and O124∶K72 (both 10.91%).The former belonged to EPEC,the latter belonged to EIEC,and the detection rate of EHEC serotype was 0.The phylogenetic group results show that the B1 group of 55 strains of E.coli accounted for the highest proportion,which was 70.91%,followed by group A,accounting for 21.82%,group D,which proportion was small,accounting for 7.27%,and the B2 group was not detected.MLST analysis showed that there were 24 ST types in the isolates,among which the dominant ST type was ST154 (18.18%).The evolutionary tree analysis revealed seven branches of evolution.In Conclusion,the feces of healthy dairy cows in Karamay,Xinjiang had a rich diversity of E.coli.There were not only pathogenic E.coli but also a certain evolutionary relationship between E.coli strains.Therefore,the excrement of healthy adult cows also had potential safety risk,so the detection and monitoring of pathogenic E.coli in cows excrement should be strengthened.

In order to reveal the expression of interferon (IFN) and interferon stimulated gene in DF1 cells infected with Avian reovirus (ARV),infect DF1 cells with ARV virus,observe cytopathic changes,collect cell samples at 0,6,12,24,36,48,72 and 96 h after infection,and extract reverse transcription into cDNA.Real-time PCR assays was used to detect the dynamic changes of interferon IFN-α and IFN-β and 9 common avian interferon-stimulating genes at the transcription level at different time points after infection.The results showed that after ARV infected DF1 cells,DF1 cells appeared typical cytopathic effect.The virus began to proliferate rapidly at 12 h after infection and maintained at a high level from 36-96 h.The expression of IFN-α and IFN-β at the transcriptional level was significantly down-regulated after infection (P<0.05;P<0.01);IFI6,OAS,IFIT5,and ISG12 had similar changes in the expression level of transcription,and all showed significant up-regulation (P<0.05;P<0.01),reaching peak at 96 h after infection.Among them,IFIT5 had the largest up-regulation,and its expression at 96 h after infection was 19.62 times than that at 0 h (P<0.01).While the expression changes of Mx,IFITM3,PKR,Viperin,and ZAP were similar,and all showed significant down-regulation (P<0.05;P<0.01),among them,the downward adjustment of Mx,IFITM3 and Viperin was larger than that of PKR and ZAP.The results showed that interferon and various interferon-stimulating genes showed regular changes at the transcription level after ARV infection in DF1 cells,which was related to virus replication in DF1 cells.The results showed that ARV infection could induce the expression of variety of interferon-stimulating genes,which played an important role in resisting ARV virus invasion,inhibiting ARV virus replication,release,and virus clearance.This study provided a further research direction of ARV pathogenesis and virus-inhibiting mechanism responses to ARV infection.

To study the preparation process of a cationic liposome-embedded squalene adjuvant,and the lecithin,cholesterol and steary lamine were used as raw materials to coat metabolizable lipid squalene with immune function,using the method of ethanol injection combined with probe ultrasound in this experiment.Furthermore,the particle size,stability index (TSI) and Zeta potential were used as evaluation indexes and the physical and chemical properties of cationic liposome-coated squalene adjuvant were analyzed.The results showed that the optimal preparation conditions of cationic liposome-coated squalene adjuvant were lecithin 15 mg/mL,cholesterol 2.0 mg/mL,squalene 8 μL/mL,probe ultrasonic time 15 min and ultrasonic power 100 W.Under the optimum conditions,the adjuvant could be obtained with an average potential of 45 mV,an average particle size of 182 nm,and a viscosity of less than 80 mPa·s,meanwhile,it also had a great significance in further researching as a vaccine adjuvant with small particle size,low viscosity and stable performance.

In order to preliminarily explore the molecular mechanism of the difference in the susceptibility of Tibetan chickens and Recessive White chickens to Eimeria tenella (E. tenella) from the perspective of immunogenetics,1×10⁵ live E. tenella sporulated oocysts were used to infect Tibetan chickens and Recessive White chickens,respectively,which displayed different levels of susceptibility to E.tenella infection.The transcription level of IFN-γ,IL-2,IL-16,TLR3 and TLR15 in spleen,cecum,thymus and bursa of Fabricius were detected by Real-time PCR at 0 d before infection and at the 2nd,4th,6th and 8th day after infection.The results showed that the transcription levels of IFN-γ,IL-2,IL-16,TLR3 and TLR15 in the spleen of Tibetan chickens were up-regulated on the 4th and 8th day after infection,while that of Recessive White chickens were not obviously changed.The transcription level of IFN-γ in the cecum of Tibetan chickens were significantly up-regulated on the 2nd day after infection (P<0.05),TLR3 was significantly up-regulated from the 4th day after infection (P<0.05),and other immune-related genes showed little change.The transcription level of IFN-γ in the cecum of Recessive White chickens were significantly up-regulated on the 8th day after infection (P<0.05),IL-2 was significantly up-regulated from the 2nd day after infection (P<0.05),IL-16 was significantly up-regulated from the 6th day after infection (P<0.05),TLR3 was significantly up-regulated on the 2nd and 8th day after infection (P<0.05),and TLR15 had little change.All immune related genes were up-regulated or down-regulated in thymus and bursa of Fabricius of the two breeds,but the transcription level of TLR3 and TLR15 in bursa of Fabricius of Tibetan chickens showed relatively large changes,while the other immune-related genes showed little changes compared with that before infection.The above results showed that E. tenella infection mainly resulted in significant changes in immune related genes in spleen and cecum of Tibetan chickens and Recessive White chickens,indicating that the genetic background of host would affect the immune response of coccidiosis infection to a certain extent.

The purpose of this study was to use Lactococcus lactis to construct a secretory expression system for bovine interferon-α (BoIFN-α) and analyze its antiviral activity.According to the preference of Lactococcus lactis MG1363 codon usage,the BoIFN-α gene was optimized and synthesized.The target gene and the Lactococcus lactis expression vector plasmid pAMJ399 were digested with SalⅠ and BglⅡ,respectively,and the gel recovered products were ligated and transformed into E.coli TG1 competent cells.The positive plasmid was extracted and electrotransformed into Lactococcus lactis.SDS-PAGE and Western blotting methods were used to analyze and identify the expressed protein in the mature cells.At the same time,the recombinant Lactococcus lactis was cultured for 28 h and then the supernatant was taken and concentrated with PEG20000 about 10 times for in vitro antiviral test.The results showed that the recombinant Lactococcus lactis pAMJ399-BoIFN-α/MG1363 was constructed successfully in this experiment.The supernatant and precipitate of the recombinant bacteria showed a target band of about 20 ku,indicating that the recombinant protein was secreted in both the supernatant and the precipitation.The concentrated rBoIFN-α supernatant acted simultaneously with Vesicular stomatitis virus (VSV).The titer of rBoIFN-α was determined to be 1.02×10⁶ U/L by the MDBK/VSV system by the microcytopathic inhibition method.The Alamer Blue method analysis result showed that the addition of rBoIFN-α had little effect on cell activity.Indirect immunofluorescence method showed that the cells had no green fluorescence after adding rBoIFN-α,indicating that rBoIFN-α had a good antiviral effect.Real-time PCR results further confirmed that rBoIFN-α had a certain antiviral effect.In this experiment,Lactococcus lactis could secrete and express rBoIFN-α was constructed.A series of in vitro antiviral tests proved that rBoIFN-α had good antiviral activity.The test results provided a new way for the development of rBoIFN-α as antiviral drugs,immune enhancers,peptide drugs and clinical applications.

In order to find out the pathogen,pathogenicity and drug resistance of ducklings in a duck farm in Guizhou province,we dissected the ducks suspected of bacterial infection in the duck farm,tissues and organs such as nasal mucosa,heart and liver were inoculated in the culture medium for bacterial isolation and identification,and studied the drug resistance and pathogenicity of the isolate by drug sensitivity test,animal regression test and virulence gene detection.The results showed that the isolated bacteria grew on the blood agar medium for 16 h and showed a smooth and glossy milky white colony with β-hemolysis.The isolated bacteria were Gram-negative short bacilli with blunt round ends and irregular arrangement under the biological microscope,which was consistent with Vibrio cholera.The homology and phylogenetic tree of 16S rDNA gene sequence showed that the homology of the isolate and Vibrio cholerae were as high as 99.6% to 99.7% and were clustered into one branch.The results of drug sensitivity showed that the isolate was resistant to most drugs,including ampicillin,clindamycin,compound trimethoprim,oxacillin,clindamycin and so on,and the isolate was highly sensitive to cefoperazone and ceftriaxone.The results of animal regression test showed that the isolated strain could cause all ducklings to die within 5 days,which indicated that the isolate had strong pathogenicity to ducklings.PCR detection of virulence genes showed that hlyA,ompW and chxA genes of Vibrio cholerae were positive,while the O1 group rfb,O139 group rfb,tcpA and ctxA genes were negative,indicating that the Vibrio cholerae isolated in this study carried pathogenic genes,but did not belong to O1 and O139 serogroups.The results showed that the pathogen was non-O1/O139 Vibrio cholerae,which was highly pathogenic and resistant to many antibiotics.The results provided a reference for the prevention and control of duck Vibrio cholerae in Guizhou province.

The aim of this study was to investigate the effect of pH conditions on amyloid fibrils formation.Hen egg-white lysozyme (HEWL) was used as the model protein in this study.HEWL was prepared in different pH conditions,such as pH 2.0,6.5,7.5 and 8.0.Then HEWL samples were incubated at (57.0±0.1) ℃ for 8 days to aggregate into amyloid fibrils.Morphology was imaged by transmission electron microscopy.Thioflavin T (ThT) and Congo red dye methods were used to detect the growth of HEWL aggregates,respectively.8-anilino-1-naphthalenesulfonic acid (ANS) fluorescence was used to quantify the hydrophobicity of formed HEWL fibrils.Circular dichroism was explored to measure the secondary structural transition and forecast the β-sheet content.The results showed that HEWL at pH 2.0 solution produced large rod-shaped amyloid fibrils under transmission electron microscopy after incubating for 8 days,and ThT fluorescence results showed that the fibrils quantity at pH 2.0 were higher than other pH groups 2.0 groups (P<0.01),and the growth of amyloid fibrils was time-dependent.Furthermore,Congo red detected both the shift of absorption peak from 490 nm to 510 nm and characteristic shoulder peak at 540 nm,hydrophobicity and β-sheet content were significantly increased,with β-sheet content was increased from 6.1% of native HEWL to 37.6% of pH 2.0 HEWL.pH 6.5 group produced very fewer amyloid fibrils only under transmission electron microscopy.pH 7.5 group produced no visible amyloid fibrils,while visible abundant spherical or rod-shaped aggregated proteins appeared under transmission electron microscopy.However,pH 8.0 group produced no fibrils and aggregated proteins for any detected methods.The results suggested that HEWL easily aggregated into amyloid fibrils at pH 2.0 with the highest hydrophobicity and structural conversion,which provided basic data for the mechanisms of fibrillary aggregation of prion disease and other amyloidosis.

In this study,the pharmacokinetic (PK) of ceftiofur hydrochloride injection in healthy pigs and infected pigs with pasteurellosis was studied by lung bronchialperfusion technique.Twelve piglets were randomly averagely divided into healthy and infection groups.The infection group was artificially infected with Pasteurella multocida to establish the disease model.Two groups of pigs were intramuscularly injected with ceftiofur hydrochloride injection according to the cross-experiment design.Blood and bronchoalveolar lavage fluid (BALF) were collected at different time points,and the drug content was detected by HPLC.The main PK parameters in plasma and BALF of healthy pigs were as follows:Drug peak concentration (C_max) were 22.33 and 2.49 μg/mL,respectively,the difference was nearly 9 times;Elimination half-life (T_1/2) were 19.51 and 70.19 h,respectively,the elimination in lung was very slow,which was 3.6 times longer than that in plasma;Area under the concentration-time curve (AUC_0-∞) were 372.05 and 94.59 μg·h/mL,respectively;Apparent distribution volume (Vd/F) were 0.41 and 5.24 L/kg,respectively,ceftiofur showed highly combined with lung.The corresponding PK parameters in infection groups were as follow:C_max were 11.81 and 5.05 μg/mL respectively;T_1/2 were 11.79 and 24.65 h,respectively;AUC_0-∞ were 162.65 and 29.73 μg·h/mL respectively;Vd/F were 0.53 and 4.65 L/kg,respectively.It showed the same characteristics as the healthy group.The results showed that ceftiofur hydrochloride injection had the PK characteristics of rapid absorption,slow elimination and high bioavailability in pigs,and there were significant differences in PK parameters between plasma and BALF.

In order to determine whether there were pathogenic bacteria in cow bedding and to further understand the types,drug resistance and pathogenicity of dominant growth bacteria in bedding,bacterial isolation and culture,Gram staining microscopy,biochemical test identification,16S rDNA sequence analysis and homology comparison,drug sensitivity test,pathogenicity test in mice were carried out.The results showed that the isolated bacteria formed round and smooth white colonies on the nutrient agar medium,while thick and large white colonies were formed on the blood nutrient agar medium.The results of Gam staining microscopy showed that the isolate was Gram-positive,spherical,arranged in clusters of grapes or scattered individually.The results of biochemical tests showed that the reaction of xylose,glucose,maltose,fructose,lactose and nitrate were positive,while melibiose,xylitol,sorbitol,urea and V-P test were negative.The 16S rDNA sequence analysis results showed that the nucleotide sequence of 16S rDNA was 1 298 bp,99.85% to 100% homology with Staphylococcus sciuri.The phylogenetic tree showed that the isolate was clustered Staphylococcus sciuri.The animal experiments showed that the isolated bacterium had strong pathagenicity to mice,the mortality was 60% when inoculated with 0.2 mL 7.9×10⁸ CFU/mL isolated strain in 48 h.Drug resistance analysis showed that the isolate was sensitive to 7 drugs,such as sulfamethoxazole,ampicillin,moderately sensitive to clindamycin,cefotaxime and cefuroxime,resistant to penicillin,erythromycin and lincomycin.This study could provide a reference for identification and prevention of Staphylococcus sciuri.

To understand the sensitivity of Mycoplasma gallisepticum (MG) to commonly used clinical drugs in Fujian White-feathered broilers,in this study,40 chicken throat swabs suspected of suffering from chronic respiratory disease (CRD) were collected from two commercial White-feathered broiler farms and inoculated on appropriate mycoplasma medium for purification.Through the analysis of the biological characteristics of the strains,bacterial morphology observation,PCR sequencing and animal regression test for identification,and the use of minimum inhibitory concentration (MIC) to determine the sensitivity of isolates to commonly used clinical antibacterial drugs.The results showed that two MG strains,named MX and HY,were successfully isolated and identified.Inoculating the isolated strain in the liquid medium of mycoplasma containing phenol red could decompose glucose to produce acid and change the color of the medium from red to yellow.A typical "poached egg" colony was observed in solid culture based on the observation of low power microscope.The colony center of the isolate after Dienes staining was dense dark blue,the edge was light blue,and did not fade after 30 minutes,and had the ability to adsorb red blood cells,with typical MG culture characteristics.The results of animal regression test and pvpA gene sequencing analysis showed that the two MG isolates had strong pathogenicity,which confirmed that they had the closest genetic relationship with R strain.The results of drug sensitivity test showed that MX and HY were highly sensitive to tiamulin,florfenicol and telithromycin,followed by enofloxacin hydrochloride and spectinomycin hydrochloride,but poor to tilmicosin,a special antibiotic for livestock and poultry with similar molecular structure and antibacterial spectrum.This difference might be related to the long-term clinical use of the drugs.The results showed that there was an epidemic of pathogenic MG in commercial White-feathered broilers in Fujian,and tiamulin could be used as the first choice for clinical prevention and treatment of MG.

Mesoporous material is a new type of porous solid material with pore size between 2-50 nm.Because of its large specific surface area,excellent chemical and thermal stability,adjustable pore size and low density,mesoporous material has become a promising drug delivery system (DDSs).However,there are still some challenges in clinical application of mesoporous material-based DDSs.In order to systematically summarize the research progress of mesoporous materials in drug delivery and to develop drug carriers based on mesoporous materials more efficiently,the authors classified mesoporous materials according to their spatial distribution order and chemical composition,which were divided into ordered mesoporous materials and disordered mesoporous materials according to their spatial distribution,and silicon-based mesoporous materials and non-silicon-based mesoporous materials according to their chemical composition.Then,the common synthesis methods were introduced,including sol-gel method,hydrothermal synthesis method,microwave synthesis method,phase transformation method and precipitation method,among which sol-gel method and hydrothermal synthesis method were the most commonly used.At the same time,the research progress of typical and most widely studied mesoporous materials in chemical and biotechnology drug delivery and the effects of the type,pore size,surface functional groups and physical state of mesoporous materials on drug delivery efficiency were also introduced.Finally,the existing problems and future development direction of DDSs based on mesoporous materials were discussed in this paper.