The purpose of this study was to clone and analyze of adenylosuccinatelyase(ADSL) gene in Guangling donkey by bioinformatics, detect its expression in different tissues, and provide theoretical reference for exploring the action mechanism of ADSL gene in inosine monophosphate and flavor formation of Guangling donkey.Homologous primers were designed by Primer Premier 3.0 according to the mRNA sequences of ADSL gene in Equus caballus (accession No.:XM_001917207.5), Bos taurus (accession No.:NM_001102377.2) and Sus scrofa (accession No.:GU249574.1), and other species published in GenBank.The CDS sequence of ADSL gene in Guangling donkey was amplified with RT-PCR and cloned.The encoding sequence of ADSL gene was analyzed, and the expression of ADSL gene in heart, liver, spleen, lungs, kidney, longissimus dorsi muscle and subcutaneous fat of Guangling donkey were detected by Real-time quantitative PCR.The CDS of ADSL gene in Guangling donkey consisted of nucleotides of 1 473 bp, encoding 490 amino acids.It was submitted to NCBI, and obtain the accession No.:MW037837.The nucleotide sequence of ADSL gene showed 99.5%, 90.8%, 92.3%, 90.4%, 90.7%, 90.5% and 86.0% identity with that of Equus caballus, Bos taurus, Camelus bactrianus, Sus scrofa, Ovis aries, Homo sapiens and Mus musculus, respectively.Phylogenetic tree results revealed that Guangling donkey was the most closely related to Equus caballus and the farthest related to Mus musculus.The ADSL protein, with molecular weight of 55.44 ku, isoelectric point of 6.52, and grand average of hydrophobicity of -0.243, was an unstable acidic hydrophilic protein.There were 41 phosphorylation modification sites, 6 glycosylation modification sites, and a coiled helix in ADSL protein, with no signal peptide and transmembrane structure, it was mainly located in the cytoplasmic.The secondary structure of ADSL protein was mainly of 68.98% alpha helix.ADSL gene was expressed in 6 tissues, among which the expression in lung was the highest, which was significantly higher than that in other tissues(P<0.05), followed by heart and liver, and the lowest in spleen, kidney and longissimus dorsi muscle.The experiment results provided a solid theoretical basis for further exploring the role of ADSL gene in the molecular mechanism of inosine monophosphate synthesis and flavor formation in Guangling donkey.

The purpose of this study was to clone and analyze the peroxisome proliferator-activated receptor γ(PPARγ) gene of Yanbian cattle, and explore its expression patterns in different tissues of Yanbian cattle.Primers were designed based on the sequence of bovine PPARγ gene in the GenBank database (accession No.:NM_181024.2).Taking 18-month-old Yanbian cattle as the experimental object, the PPARγ gene of Yanbian cattle was cloned by RT-PCR, and BLAST in NCBI was used to carry out homology analysis and construct a phylogenetic tree with other species.Bioinformatics softwares were used to analyze the nucleotide sequence and the physicochemical properties, hydrophobicity, signal peptides, transmembrane domains, subcellular localization, phosphorylation sites, glycosylation sites, secondary structure and tertiary structure of PPARγ protein.Real-time quantitative PCR technology was used to detect the expression level of PPARγ gene in heart, liver, spleen, lung, kidney, small intestine, subcutaneous fat, muscle of Yanbian cattle.The results showed that the CDS region sequence of Yanbian cattle PPARγ gene was 1 620 bp, the sequence and phylogenetic tree analysis showed that it had 99.9% homology with that of yellow cattle and 82.1% homology with chicken.PPARγ gene encoded a protein of 505 amino acids, and the protein had no signal peptide and transmembrane domain, which was hydrophilic protein and mainly distributed in the nucleus and cytoplasm.The main secondary structure of Yanbian cattle were alpha helix and random coil, and there were 53 potential phosphorylation sites and 24 O-glycosylation sites.Real-time quantitative PCR results showed that the expression of Yanbian cattle PPARγ gene in spleen, subcutaneous fat and liver was significantly higher than that in heart (P<0.05), the expression of it in lung, kidney, muscle and small intestine was significantly lower than that in heart (P<0.05).The above results could provide reference for further study of the function of PPARγ gene.

This study was aimed to carry out the bioinformatics prediction and analysis of alpha-1-acid glycoprotein(α1-AGP) of donkey, and an efficient purification method of α1-AGP from donkey was established.Analysis of donkey by bioinformatics online software.The basic physical, chemical properties and the secondary and tertiary structures of α1-AGP were predicted.According to the results of the analysis, the method of cation chromatography gel filtration chromatography was used to purify donkey α1-AGP, after SDS-PAGE, matrix assisted laser desorption ionization-time offlight mass spectrometry (MALDI-TOF-MS) was used to identify donkey α1-AGP.The results showed that donkey α1-AGP was stable and hydrophilic, composed of 201 amino acids, the theoretical isoelectric point(pI) was 4.99, there were 5 potential N-glycosylation sites, and the average value of hydrophilicity(GRAVY) was -0.386.α-helix and β-sheet appeared orderly in the secondary structure, accounting for 88.56%, the GMQE value and the Z value of QMEAN were used to verify that the model was in a reasonable range.After purification by cation exchange chromatography and gel filtration chromatography, a total of 50 mL protein solution was obtained, and a clear protein strip was obtained after SDS-PAGE.After MALDI-TOF-MS and retrieved from NCBI database, the protein with the highest score was donkey α1-AGP, which was much higher than other proteins, indicating that α1-AGP was the main component in the purified protein.In this study, we proposed a new method for the purification and identification of α1-AGP from donkeys, which had the advantages of high purification rate, simple operation and less samples needed, and laid a theoretical foundation for the subsequent biological research and application of α1-AGP from donkeys.

This study was aimed to identify differentially expressed microRNA(miRNA) (DEM)in the breast muscle between high intramuscular fat (IMF) and low IMF content chickens, and explore the miRNA regulatory mechanisms involved in IMF deposition in chicken.120 Muchuan Black-bone chickens were employed in this study.High-throughput RNA sequencing (RNA-Seq) was performed to identify miRNA in the breast muscle between three high IMF (H group) and low IMF (L group) content chickens.The sequencing data was analyzed to identify DEMs.Six miRNAs were randomly selected for quantitative analysis using Real-time quantitative PCR.The results of IMF contents determination showed that the average IMF level of 120 chickens was 4.08 g per 100 g muscle, and there was extremely significant difference in IMF content between H and L groups (P<0.01).Results of 6 RNA-Seq libraries showed that more than 10 000 000 Clean reads were obtained for each library, and the Clean reads ratio was greater than 93%, more than 50% of small RNA (sRNA) ranged from 21 to 23 nt in length, and the 22 nt sRNA was the most abundant.The average GC content was 48.95%.These results indicated that the sequencing data were reliably produced in our study.The proportion of sRNA annotated as rRNA, tRNA, snRNA, snoRNA and repetitive sequence in the 6 RNA-Seq libraries was 2.74%, 1.85%, 6.21%, 1.58%, 2.82% and 2.81%, respectively.A total of 578 known miRNAs and 500 pre-miRNAs were identified from the 6 chicken sequence libraries, with 23 DEMs were identified between two groups, of which 16 were upregulated and 7 were downregulated.The prediction analysis of miRNA target genes showed that 23 miRNAs which were differentially expressed between two chicken groups targeted a total of 628 genes.GO analysis results showed that a total of 30 significantly enriched GO terms were identified, including 6 cellular components, 13 biological process and 11 molecular function.KEGG pathway analysis showed that the target genes were mainly involved with insulin signaling pathway, galactose metabolism, adipocytokine signaling pathway, glycosphingolipid biosynthesis, glycolysis and gtarch and sucrose metabolism.Real-time quantitative PCR results indicated that the expression of gga-miR-124a-3p and gga-let-7a-5p in H group was significantly higher than that in L group (P<0.01;P<0.05), the expression of gga-miR-19a-3p in H group was significantly lower than that in L group (P<0.01), the expression of gga-miR-6553-3p and gga-miR-128-3p in H group were significantly lower than that in L group (P<0.05), and 5 DEMs expression pattern were consistent with sequencing data.The results showed that gga-miR-124a-3p, gga-let-7a-5p, gga-miR-6553-3p, gga-miR-128-3p and gga-miR-19a-3p were associated with adipogenesis in animal, which might be important for IMF deposition in chicken and would provide a foundation for clarifying the molecular regulatory mechanisms of miRNA involved in IMF deposition in chicken.

In order to obtain the high interference efficiency siRNA of adipocyte differentiation associated protein 2 (PLIN2) gene and the high overexpression efficiency gene overexpression vector of Qinchuan new beef line (hereinafter referred to as "Qinchuan beef cattle"), and provide experimental basis for the follow-up study of PLIN2 gene function in the process of adipocyte differentiation of Qinchuan beef cattle, in this study, si-PLIN2 targeting bovine PLIN2 mRNA sequence was synthesized by siRNA mediated target gene silencing and eukaryotic pcDNA3.1(+) -PLIN2 overexpression vector was constructed.FuGENE^® 6 low toxic transfection reagent was used to prepare transfection complex, transfect Qinchuan beef cattle adipocytes and induce differentiation.Real-time quantitative PCR was used to detect the relative expression of PLIN2 gene in each experimental group and control group, and the interference and overexpression efficiency of the target gene were calculated.Western blotting was used to detect the protein expression and the phenotype was observed by BODIPY staining.The results showed that after transfection of preadipocytes in Qinchuan beef cattle, three groups of si-PLIN2 (si-PLIN2_01, si-PLIN2_02 and si-PLIN2_03) compared with control group (si-PLIN2-NC), the expression of PLIN2 gene decreased (P<0.01), and the interference efficiency was 71%, 54% and 50%, respectively.The protein level of the si-PLIN2_01 and si-PLIN2_03 groups showed a downward trend compared with control group.Compared with control group, the number of lipid droplets in the interference group decreased significantly.Compared with control group, the expression of PLIN2 gene in overexpression group pcDNA3.1(+)-PLIN2 was significantly increased by 35 times (P<0.01), and the number of lipid droplets was significantly increased.In conclusion, the silencing of PLIN2 gene mediated by siRNA or the expression vector mediated by pcDNA3.1(+) in vitro could successfully interfere with or overexpress PLIN2 gene in the process of adipocyte differentiation in Qinchuan beef cattle, and affect the number of lipid droplets in the process of adipocyte differentiation.This study laid the foundation for further exploring the function of PLIN2 in the process of adipocyte differentiation in Qinchuan beef cattle.

The aim of this study was to explore the gene variation, genetic diversity and phylogenetic relationships of Aedes albopictus from Huaihua district, Hunan province.A total of 30 Aedes albopictus from Huaihua district were captured to use as research target.The ribosomal DNA 18S rRNA and cytochrome c oxidase subunit 1(cox1) gene sequences were amplified by PCR method, and then their gene variation, genetic diversity and phylogenetic relationships were analyzed, respectively.The results showed that the 18S rRNA and cox1 gene sequences of all samples was 489 and 1 004 bp, respectively.Based on 18S rRNA gene sequences, the intra-species differences of Aedes albopictus from Huaihua district were 0-2.9%, and the inter-species differences were 28.74%-61.46%.Based on cox1 gene sequences, the intra-species differences of Aedes albopictus from Huaihua district were 0-0.2%, and the inter-species differences were 6.08%-12.65%.The results of phylogenetic tree based on 18S rRNA and cox1 gene sequences showed that all samples of Aedes albopictus from Huaihua district together located in the same clade.The results of this study indicated there were a certain degree of genetic variation and genetic diversity within all samples of Aedes albopictus from Huaihua district, but the phylogenetic relationships were more closer among them, there was lower intra-species differences and higher inter-species differences between 18S rRNA and cox1 gene sequences, and 18S rRNA and cox1 gene could be used as an ideal genetic marker for the identification of Aedes albopictus.

The objective of this study was to clone and express virulence gene BvfA of Brucella (B. abortus) S19, and analyzed the bioinformatics of its expressed protein.In the tests, chromosome 1(accession No.:CCP030751.1)of B.arbortus S19 was downloaded from GenBank.According to the reported position of BvfA gene and molecular weight of BvfA protein in B.arbortus S19, the Open Read Frame (ORF) of BvfA proteins in NCBI's ORF Finder was predicted.The obtained BvfA gene was ligated into prokaryotic expression vector pET-32a(+) to construct the recombinant plasmid pET-32a(+)-BvfA, and expressed in Escherichia coli BL21(Ril).The expressed products were analysed by SDS-PAGE and Western blotting, the structure of BvfA protein was predicted by bioinformatics methods.The results showed that the full-length ORF of BvfA gene was 336 bp, and the expressed fusion BvfA protein was about 11 ku.Bioinformatics analysis showed that BvfA was an unstable hydrophilic protein with no transmembrane structure and its signal peptide region was 1-28 amino acid, BvfA protein mainly located in the extracellular protein (55.6%).There were 12 potential phosphorylation sites, and no O-glycosylation sites.BvfA protein secondary structure was mainly composed of alpha helix and random coil.The results of this study provided some references for the further study of the pathogenic mechanism of BvfA protein in Brucella and search for new therapeutic targets for brucellosis.

The aim of this study was to clone and analyze bioinformatics of the sapA gene of Haemophilus parasuis (Hps), and provide theoretical basis for the development and application of sapA gene deletion vaccine. 71 clinical strains were selected as the research objects, and the positive strains were identified by PCR amplification. The cloned sapA gene fragments were linked with pGEM-T vector and transformed into DH5α competent cells of Escherichia coli. The positive clones were identified by PCR and double enzyme digestion and then sequenced. Bioinformatics softwares were used for nucleotide sequence and amino acid sequence alignment, phylogenetic tree analysis, protein secondary structure and tertiary structure prediction, amino acid hydrophobicity prediction, flexible region prediction, B cell epitope prediction and Jameson-Wolf antigen index prediction analysis. The results showed that sapA gene was successfully amplified in 29 out of 71 clinical isolates. The sequence of sapA gene of 29 Hps strains shared 96.5%-98.8% similarity with the published SH0165 strain. The amino acid sequence of sapA gene of 28 Hps strains shared 98.9%-100% similarity with SH0165 strain except for the H88 strain. The secondary structure of sapA protein contained α-helix, β-turn and random coil, with 13 hydrophilic regions and 38 flexible regions, 23 potential sites on the surface of B cell antigen. The above results indicated that the sapA gene of Hps was a conserved gene with high homology in nucleotide sequence, amino acid sequence, and the sapA protein had strong antigenicity.

This study aimed to investigate developmental characteristics of hair follicles and the distribution of vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) in 2-day-old and 35-day-old Tan sheep's skin.The cytochemical characteristics of the skin were observed under the microscopy by HE, modified Masson, Gomori and PATH staining, while the expression of VEGF and VEGFR2 were investigated by histochemical analyses using immunohistochemical and immunofluorescence, IPP image analysis software was used for quantitative analysis.The results showed that compared with 2-day-old Tan sheep, the hair follicle of 35 days was developed well, the boundary between epidermis and dermis was clearer, and the collagen and elastic fibers increased and forming a grid-like distribution, and the density of hair follicles was decreased (P<0.05).The expression of VEGF and VEGFR2 were strong in epidermis, outer root sheath and sebaceous glands of the skin.The average optical of VEGF and VEGFR2 was higher in 2-day-old than in 35-day-old Tan sheep skin tissues(P<0.05).Therefore, the collagen and elastic fibers increased obviously during the development of Tan sheep hair follicle;The pathway of VEGF and VEGFR2 played a direct regulatory role in hair follicle keratinization.

This study was aimed to detect the expression pattern of signal transduction and activator of transcription 3 (STAT3), clone STAT3 gene, construct eukaryotic expression vector, and explore the regulatory effect of STAT3 gene on the differentiation of muscle stem cells in Guangxi cattle.Muscle tissues of Guangxi cattle aged 6, 12 and 18 months, GM and DM stages muscle stem cells were collected, and RNA was extracted and reverse transcribed into cDNA.The expression pattern of STAT3 gene was detected by Real-time quantitative PCR, and the complete coding region sequence of STAT3 gene was amplified to construct over-expression vector pCD-STAT3, so as to test its effects on the differentiation of cattle muscle stem cells.The results of Real-time quantitative PCR showed that STAT3 gene expressed in the muscle tissues of cattle aged at 6, 12 and 18 months, and the expression level was the highest at the age of 18 months and the lowest at 12 months.The expression levels of STAT3 and myogenic regulatory factor 6 (MYF6) genes in differentiated MSCs were extremely significantly higher than those in proliferative MSCs (P<0.01).The coding region of STAT3 gene in Guangxi cattle was 2 313 bp, which was successfully linked with empty vector pcDNA3.1 to construct the overexpression vector pCD-STAT3.The pCD-STAT3 was transferred into muscle stem cells in Guangxi cattle in vitro, the expression of STAT3 gene and myoblast differentiation marker genes myogenic differentiation 1 (MyoD1) and muscle myosin heavy chain (MyHC) in muscle stem cells were extremely significantly increased (P<0.01), and the expression of myopoietin (MyoG) gene was significantly increased (P<0.05).Moreover, the number and size of myotubes in pCD-STAT3 group were significantly higher than those in pcDNA3.1 group (P<0.05).In this study, the expression pattern of STAT3 in longissimus dorsi muscle of Guangxi cattle was investigated, and overexpression of STAT3 gene could significantly promote the differentiation of bovine muscle stem cells, which laid a foundation for further study on the role of STAT3 gene in muscle growth and development and its molecular regulatory mechanism.

Milk fat is a high quality natural fat which can provide nutrition and energy for human.Among all kinds of dietary fat and oil, it is the easiest to be digested and absorbed.Milk fat is a kind of lipid substance formed by esterification of de novo synthesis or exogenous intake of fatty acid and glycerol in the mammary gland, its content is related to the quality of milk and the processing characteristics of dairy products.During the lactation cycle of dairy cows, the lactation function of mammary gland is affected by many factors, among which a variety of hormones secreted by the endocrine glands have positive regulatory effects on the synthesis of milk fat in bovine mammary epithelial cells.To sum up, the authors introduced the regulatory mechanism of hydrocortisone, prolactin, insulin and growth hormone on milk fat synthesis in bovine mammary epithelial cells, explaining its regulatory effect from the appropriate amount of hormone in milk fat synthesis and the effect of hormone on the morphology of milk fat globule.And from the key enzymes of milk fat synthesis and transcription factors, hormones on the expression of milk fat synthesis-related genes, the mechanism is explained in depth, aiming to provide reference for studying the regulation mechanism of lactation-related hormones on milk fat synthesis in dairy cows' mammary glands.

This experiment was conducted to investigate the effects of three additives on growth performance, antioxidant capacity, meat quality and content of flavoring substances in muscle of Qingyuan Partridge chickens aged 91 to 115 days.A total of 1 200 91-day old chickens were randomly assigned to 4 groups with 10 replicates of 30 birds per replicate.The control group was fed with basal diet(CON), and birds in treatment groups were supplemented with nano copper compound premix(NC), spores of Ganoderma lucidum compound premix(GLS) and soybean isoflavone premix(SI) for 25 days, respectively.At the end of the experiment, samples of plasma, jejunal mucosa and pectoral muscle were collected to determine the antioxidant capacity of blood and jejunum, the meat quality and the content of flavor substances.The results showed that, compared with the control group:①The growth performance and the carcass traits of Qingyuan Partridge chickens were not affected by the three compound additives (P>0.05).②All these three additives significantly reduced L^* value (P<0.05), increased pH (P<0.05) of breast muscle, and GLS reduced the drip loss of breast muscle (P<0.05).③The scores of color and appearance in SI group were increased when steamed (P<0.05), and the scores of juiciness in GLS group were increased when steamed or boiled (P<0.05).④NC and SI increased the contents of inosinic acid and glutamic acid in muscle (P<0.05), and SI increased the content of intramuscular fat (P<0.05).⑤NC increased the activity of glutathione catalase (GSH-Px) in plasma (P<0.05), and SI increased that in jejunum (P<0.05);GLS decreased the MDA content in jejunum (P<0.05).Based on this experiment, the NC, GLS and SI did not affect the growth performance and carcass traits of Qingyuan Partridge chickens at 91 to 115 d.All these additives enhanced the antioxidant capacity of chickens, improved the meat quality, and increased scores of sensory evaluation.NC and SI also affected the contents of flavor substances in meat, had the better effects.

This study was aimed to investigate the effects of concentrates (CF) and pasture feeding (PF) on meat quality, muscle fiber characteristics, microstructure and protein degradation during postmortem aging of biceps femoris in Tan sheep.28 male Tan lambs aged 4 to 5 months were randomly divided into two groups with 14 replicates each.A replication consisted of 1 sheep.Each group was raised for 60 days under CF and PF, respectively.The results showed that the shearing force, hardness and cohesiveness of muttons in PF group were significantly higher than that in CF group (P<0.05), while the springiness of muttons in PF group was significantly lower than that in CF group (P<0.05).The muscle fiber density and quantity proportion of type Ⅱ muscle fiber of muttons in PF group were significantly lower than that in CF group (P<0.05).Meanwhile, the diameter, cross-sectional area and quantity proportion of type Ⅰ muscle fibers in PF group were significantly higher than that in CF group (P<0.05).During postmortem aging, the sarcoplasmic protein solubility in CF group was significantly higher than that in PF group (P<0.05).The myofibrillar protein solubility in two groups gradually increased after reaching the lowest at 24 h postmortem, but there was no significant difference in each time pointing (P>0.05).Myofibrillar fragmentation index significantly increased at 50.97% and 41.94% in CF and PF groups, respectively.The microstructure showed that the myofibril structure of postmortem muscle was loose and cracked, Z-line was degraded and myofibril fragmentation appeared.The damage of myofibril structure in CF group was more serious than that in PF group.The sarcoplasmic protein had less degeneration and slow degradation rate in CF group, while the degradation of myofibrillar protein was more obvious, and the amount of small molecular protein in CF group was higher than that in PF group.In conclusion, the feeding system affected the characteristics of the biceps femoris muscle fibers in Tan sheep, and had an impact on the microstructure and protein degradation during postmortem aging.CF increased the density and reduced the diameter and the cross-sectional area of muscle fibers of biceps femoris, and thus reduced the muscle shearing force.In addition, as the proportion of type Ⅱ muscle fibers increased, CF improved the growth rate of myofibril fragmentation index, sarcoplasmic protein solubility and protein degradation rate postmortem, it accelerated the process of postmortem aging and improved the tenderness of biceps femoris.

The experiment was aimed to study the effects of adding low-level condensed tannins to different nitrogen sources on rumen fermentation parameters and degradation rate of Yanbian Yellow cattle, so as to screen out the nitrogen sources with the best adding ratio and higher utilization rate of condensed tannins, and to provide scientific basis for condensed tannins to be used as functional additives in ruminant.Four Yanbian Yellow cattle (452.3 kg±27.1 kg) were used to provide rumen fluid.6 kinds of feedstuff without condensed tannin were used as nitrogen sources (soybean meal, cottonseed meal, rapeseed meal, Medicago sativa, red clover and Vicia amoena).Different levels of condensed tannin (0 (control group), 0.1%, 0.5% and 0.9%) were added to each nitrogen source diet.There were 24 groups with 3 replicates in each group.The pH, ammonia nitrogen (NH₃-N), volatile fatty acids (VFA), gas production, effective degradation rate of dry matter, crude protein and neutral detergent fiber of Yanbian Yellow cattle in vitro rumen fermentation were determined.The results showed that the pH, NH₃-N and VFA of each experimental group changed within the normal range and could meet the normal metabolism of rumen microorganisms.These indexes of forage 0.5% condensed tannin groups (hereinafter referred to as 0.5% group) and cake 0.9% groups were good, and the gas production was lower than their respective control groups.The effective degradation rates of dry matter in soybean meal 0.9% group, rapeseed meal 0.5% group and red clover 0.1% group were slightly lower than their respective control groups (P>0.05).The effective degradation rate of crude protein in cottonseed meal 0.5% group was lower than that in the control group (P<0.05), and the effective degradation rate of crude protein in 3 forage 0.9% groups were lower than that in control group.Except for the red clover 0.1% group, the effective degradation rate of neutral detergent fiber in the other five nitrogen source condensed tannin addition groups were significantly lower than that of the control group (P<0.05).Comprehensively considered, the cake 0.9% groups and forage 0.5% groups were more conducive to optimizing the rumen fermentation environment, which could reduce the degradation rate of crude proteins in the rumen and effectively protect the rumen proteins.Among them, Vicia amoena and cottonseed meal had better effect.

With the improvement of people's quality of life, the catering industry is developing rapidly, and a large amount of food waste (FW) is generated, which seriously affects food safety and causes environmental pollution.At present, the resource utilization of food waste has received widespread attention, among which feed processing technology can realize the regeneration of resource value, meet the requirements of environmental sustainable development, and has economic feasibility.Food waste is rich in nutrients, containing 14.93% to 94.20% dry matter (DM), 13.53% to 42.90% crude protein (CP), 3.89% to 31.57% ether extract (EE), 0.24% to 16.50% ash (Ash), 2.07% to 30.70% crude fiber (CF), 6.72% to 38.90% neutral detergent fiber (NDF), and 5.29% to 25.20% acid detergent fiber (ADF).The safety of food waste includes physical safety, biosafety and chemical safety.The physical safety of food waste can be guaranteed during the collection and pretreatment process.The biosafety of food waste is mainly that untreated food waste is easy to breed bacteria, such as Salmonella, Escherichia coli, and feeding animals with uncooked food waste can cause aftosa, swine fever, and transmissible spongiform encephalopathy.The chemical safety of food waste is that toxins, pesticides, and heavy metals should meet the standards, and the influence of anti-nutritional factors, nitrites, and detergents should be paid attention to.On the whole, food waste has high nutritional value, which can meet the needs of animal growth to a certain extent.However, the biosafety limits the application and development of food waste as feed.

The purpose of the study was to sepatate and purify an antimicrobial peptides from the metabolites of Paenibacillus polymyxa BLCC1-0402, and to provide a reference for the preparation of antimicrobial peptides and the detection of their products.Centrifugation, membrane filtration with different molecular weight, ultrafiltration and Superdex peptide 10/300GL gel filtration chromatography were used to separate and purify the fermentation supernatant of Bacillus polymyxa.Bacteriostatic test was carried out on the collected liquid in different periods to compare and evaluate the effect of step chromatography.The standard strain of Escherichia coli O78 was used as the indicator, and the antibacterial activity was detected by the agar diffusion method.Tricine-SDS-PAGE was used to detect the molecular weight.The results showed that, the 3-5 ku component protein samples obtained by 5 and 3 ku roll membrane ultrafiltration had strong antibacterial activity.The 3-5 ku fraction was purified by gel filtration chromatography.The purified antibacterial peptide A3 had the strongest antibacterial activity.The Tricine-SDS-PAGE small molecule peptide electrophoresis showed that the antibacterial peptide A3 had reached the electrophoresis purity and the molecular weight was 4 ku.The antibacterial activity test showed that the antimicrobial peptide had antibacterial effect on the standard strain of Escherichia coli O78.At the same time, antimicrobial peptide A3 showed good heat resistance, and its antibacterial activity could be maintained at about 96% when treated at 90-100 ℃ for 15 min.It had a good acid-base stability, and its antibacterial activity remains above 90% at pH 2.0-9.0.After pepsin treatment, the antibacterial activity of antimicrobial peptide A3 decreased by 20%, and trypsin treatment, the antibacterial activity of antimicrobial peptide A3 decreased by 18%.Protease K had almost no effect on the antibacterial activity of antimicrobial peptide A3.The results showed that the isolated antimicrobial peptide A3 was a new antimicrobial peptide with antibacterial activity against Escherichia coli O78, which had a certain development potential, and provided a certain reference for the further study of antimicrobial peptide structure analysis and physicochemical properties analysis.

Woody feed is a new type of unconventional feed, rich in a variety of amino acids, vitamins and organic matter, rich in nutritional value, and most of the woody feed plant have higher crude protein content, which can be used as an excellent protein feed resources, and its effective use can alleviate the severe constraints on the development of animal husbandry.However, due to the existence of anti-nutritional factors, untreated woody feed directly feeds livestock has poor palatability and low digestibility.Silage treatment can effectively reduce the loss of feed nutrients, improve palatability and its nutritional components, and enhance feeding effect, which has become an important treatment method.Based on the domestic and foreign research on the silage of woody feed, this paper focused on the evaluation of nutritional value of different woody feed, selection of silage materials, effects of silage additives (chemical additives, lactic acid bacteria additives, enzyme preparations and nutritional additives) on silage fermentation, the mixed silage technology of woody feed and different raw materials (gramineous forage, different woody feed, agricultural by-products, etc.) and its effect on fermentation, the nutritional characteristics of woody forage after successful silage and the feeding effect of different animals in the various feeding experiment of woody silage were systematically reviewed, according to the research status of woody feed and the importance of resources, suggestions and prospects for its future development were put forward, in order to provide reference for the follow-up development and practical application of woody silage.

In order to investigate the effect of single nucleotide polymorphism (SNP) of TBC1 domain family member 7(TBC1D7) gene on growth traits in Guanling cattle, in this study, blood samples were collected from 116 Guizhou Guanling cattle, and 8 growth traits were determined.All exons of TBC1D gene were amplified by mixed DNA pool, SNP was screened by direct sequencing, and then amplified and sequenced with individual sample DNA as template to verify SNP and used for genotyping.The correlation between different genotypes of TBC1D gene and the growth traits of Guanling cattle was analyzed by single factor variance of SPSS 19.0 software.The results showed that g.12972 T>C, g.12984 A>G, g.20538 C>T, g.20577 T>C and g.24807 G>A mutation sites were found in exons 5, 6 and 8 of TBC1D gene.The results of genotyping showed that there were 3 genotypes in all 5 SNPs.Chi-square (χ²) test showed that 5 SNPs were significantly deviated from Hardy-Weinberg equilibrium state (P<0.05).The analysis of population genetic parameters showed that 5 SNPs had high heterozygosity and moderately polymorphic information content (0.25<PIC<0.5).The correlation analysis results showed that the oblique length, chest circumference and rear hip circumference of GA genotype individuals in g.24807 G>A of TBC1D gene were significantly higher than that of AA genotype individuals (P<0.05).There were 6 haplotypes(H1-H6) and 9 diplotypes(H1H1, H2H2, H2H3, H2H6, H3H3, H3H4, H3H5, H3H6 and H5H5) of 5 SNPs in the experimental population, including dominant haplotypes H3 and H2, dominant diplotypes H3H3, H2H2 and H2H3.The results of diplotype association analysis showed that H3H6 and H5H5 individuals had significant advantages in body length and hip circumference compared with other diplotypes, and H3H6 and H5H5 might be the favorable genotypes.This results showed that g.24807 G>A of TBC1D gene was an important SNP affecting the growth and development of Guanling cattle, which provided a theoretical reference for screening molecular markers that were helpful to the growth and development of Guanling cattle.

In this study, gene expression differences in hypothalamus-pituitary-ovary axis of estrous and anestrous primiparous sows was analyzed and molecular mechanisms for the regulation of sow estrus was explored.6 healthy primiparous sows were divided into 2 groups:The estrous group (3 sows) and anestrous group (3 sows), and then all sows were slaughtered to collect their hypothalamuses, pituitaries and ovaries for RNA-Seq analysis, GO classification, KEGG enrichment analysis and Western blotting analysis of related protein.It was found that 52 432 800, 52 573 730 and 52 209 252 clean reads were obtained from hypothalamuses, pituitaries and ovaries of estrous sows, respectively.And the ratio of these reads to porcine reference genome (Sus scrofa 11.1) were 78.69%, 81.49% and 79.26%, respectively.52 516 724, 52 476 820 and 52 195 962 clean reads were obtained from hypothalamuses, pituitaries and ovaries of anestrous sows, respectively.And the ratio of these reads to porcine reference genome (Sus scrofa 11.1) were 82.38%, 83.05% and 80.20%, respectively.Compared with estrous sows, there were 710 differentially expressed genes in the hypothalamus of anestrous sows, including 392 up-regulated and 318 down-regulated genes.There were 707 differentially expressed genes in the pituitary of anestrous sows, including 283 up-regulated and 424 down-regulated genes.There were 956 differentially expressed genes in the ovary of anestrous sows, including 635 up-regulated and 321 down-regulated genes.36 differentially expressed genes were shared in 3 kinds of tissues.This study found KISS1, GPR54, TAC3, TACR3, CYP17A1, CYP19A1, STAR, GnRHR and ESR1 genes were all significantly down-regulated in anestrous sow (P<0.05;P<0.01), which were related to regulation of sow estrus.Western blotting analysis indicated TAC3, TAC3R, KISS1 and GPR54 proteins were also all significantly down-regulated in anestrous sow.Analysis of GO and KEGG pathway revealed the differently expressed genes were significantly enriched in some signaling pathways related to steroid hormone production and follicular development, such as positive regulation cholesterol esterification, ovarian steriodogenesis, GnRH signaling pathway and FoxO signaling pathway.This study showed that genes related to the regulation of sow estrus were differentially expressed in hypothalamus-pituitary-ovary axis of estrous and anestrous primiparous sows and especially the mRNA and protein expression levels of Kisspeptin/GPR54 and TAC3/TACR3 systems were all significantly down-regulated in hypothalamus of anestrous primiparous sows.It was speculated that impaired expression of Kisspeptin/GPR54 and TAC3/TACR3 systems led to hypothalamic dysfunction, and in turn led to anestrus in primiparous sows.This study provided important support for revealing the molecular regulation mechanism of anestrus in primiparous sows and a important theoretical basis for the use of molecular technology to improve sow anestrus in the future.

Accurate measuring of the inbreeding degree and genetic relationship on yellow Boer goat was an effective way to improve breeding efficiency and genetic progress of Yunnan Yellow goat breeding.The genotyping-by-sequencing (GBS) was used to sequence 37 yellow Boer rams from Yunnan breeding promotion center.High quality and high density SNP variation information was obtained through quality control.Principal component analysis (PCA), calculation of identity by state (IBS), construction of G matrix, calculation of genetic coefficient, NJ cluster analysis and calculation of inbreeding coefficient were carried out by Gmatrix v2, Plink v1.90 and MegaX v10.0 software.The results showed that 88 393 high-quality SNPs were detected on 29 autosomes in 37 yellow Boer rams, and 1 537 long homozygous fragments (ROH) ranging in length from 1 000.582 to 18 400.12 kb with an average length of 2 576.34 kb and 93.15 SNPs were detected.37 yellow Boer rams were divided into 11 families and 3 families had only one ram each.The genetic relationship between family A and family K was the farthest.The average genomic inbreeding coefficient (F_ROH) based on ROH was 0.043 and the F_ROH of 3 yellow Boer rams was more than 0.125 which meant more inbreeding accumulation existing.This study provided a scientific basis for the rational use of yellow Boer goat in the breeding of Yunnan Yellow goat, and also provided a powerful technical means for evaluating the inbreeding level of goats, preventing inbreeding decline, and optimizing the breeding and matching scheme.

The study was aimed to analyze the difference of genome-wide DNA differential methylation in Sujiang pigs with different body weight, as to screen the differentially methylated genes (DMGs) that affect the body weight of Sujiang pigs.In the study, three 180-day-old Sujiang pigs with high body weight and three 180-day-old Sujiang pigs with low body weight were detected by the whole genome bisulfite sequencing (WGBS) method.The degree of genome-wide DNA methylation, differentially methylated regions (DMRs) and DMGs were analyzed.The candidate genes affecting the body weight of Sujiang pig were identified by GO and KEGG pathway enrichment analysis of DMGs.The results showed that average 4.1% cytosine (C) were methylated in the whole genome of Sujiang pigs.Average 98.41% methylcytosines occurred on CG sequences.The DNA methylation level of exon, intron and 3'UTR was higher than that of promoter and 5'UTR at CG sequence context.A total of 1 657 DMRs and 575 DMGs were detected in this study.The distribution of DMRs on chromosome 8 was the most, 88.89% of the DMRs were within the length of 500 bp, and 62.78% of the DMRs were distributed in the distal intergenic.98 DMGs were significantly enriched in 53 GO terms and 29 signal pathways, including negative regulation of TOR signaling, carbohydrate derivative catabolic process, adipocytokine signaling pathway and FoxO signaling pathway.Five candidate genes related to body weight of Sujiang pigs were identified:leptin receptor(LEPR), tumor necrosis factor receptor associated factor 6(TRAF6), myogenic factor 6(MYF6), calcium dependent secretion activator 2(CADPS2) and epidermal growth factor(EGF).In this study, the whole genome DNA methylation map of Sujiang pigs with high and low body weight was drawn by WGBS, which laid a foundation for the further study of the molecular mechanism of body weight difference in Sujiang pigs.

In order to provide basic data for breeding and fine housing of Beijing-You chickens, the pure line of Beijing-You chicken was used here to compare the feather development, growth and reproductive performance of early and late feathering roosters.According to the relative length characteristics of the primary feathers and the coverts, the newborn male chicks were identified and divided into early and late feathering subgroups.For which, the early feathering included K1 (primary feather is at least 5 mm longer than the covert feather) and K2 (primary feather is 2-5 mm longer than the covert feather);the late feathering included M1 (the primary feather length is equal to or slightly longer than (<2 mm) the covert feather), M2 (the primary feather is shorter than the covert feather) and M3 (the primary feather is not growing out).Before 7 days of age, the length of primary feathers and coverts were measured every other day, and every other week from 7 to 42 days.Body weight was measured every week from 1 to 8 weeks, and every other week from 9 to 18 weeks.The overall plumage condition was observed at 10 weeks of age.Determination of general semen quality, sperm kinetic parameters, and fertilization and hatching traits of early and late feathering roosters was performed at 47 weeks of age.The results showed that the early and late feathering accounted for 11.60% and 88.40% of Beijing-You male chickens, respectively.The majority of late feathering hens were M2 type, and there were a few M1 and M3 type.There was no difference in body weight between late and early feathering male Beijing-You chickens during the brooding age (1-18 week)(P>0.05).Besides the fact the primary feathers and the coverts of late feathering chickens grew slowly than the early ones, only 44% of the late feathering chicks showed intact plumage of back and the thigh area, and this percentage was the highest (over 90%) in the equal-length type late feathering chickens, and low (17%) in the no-primary feathers type.The early and late feathering roosters did not show difference in general semen quality traits, while the sperm linearity was higher (P<0.05)and the straight-line velocity was inclined to be higher (P=0.06) in late feathering.The fertility of early feathering male Beijing-You chickens was higher than the late feathering ones (P<0.05).Although not significant, the hatchability of setting eggs and hatchability of fertilized eggs was apt to higher than the late feathering ones (P>0.05).The results of the present study showed that feather development and reproductive performance of Beijing-You chickens could be affected by feathering phenotype.It is necessary to further study the cause of the late feather development and identification method.Furthermore, the selection breeding for semen quality traits should be enhanced in the late feathering roosters of Beijing-You chickens.

This study was aimed to investigate the association of the polymorphism of oligoadenylate synthase 1 (OAS1) gene with reproductive traits in Songliao Black pigs.130 Songliao Black pigs were selected as the research objects, the SNPs of OAS1 gene exons 1-8 were found by Sanger direct sequencing, and the correlation between SNP of OAS1 gene and reproductive traits of Songliao Black pigs was analyzed using SPSS 19.0 software.The results showed that 33 mutations were detected in exons 2, 3 and 6 of OAS1 gene in Songliao Black pigs.There was a SNP at 110 bp of OAS1 gene exon 2(G110C), which contained three genotypes of GG, GC and CC.There was a SNP at 176 bp of OAS1 gene exon 3(G176T), which contained three genotypes of CC, CT and TT.There was a SNP at 145 bp of OAS1 gene exon 6(C145T), which contained three genotypes of CC, CA and AA.There was a SNP at 166 bp of OAS1 gene exon 6(G166A), which contained three genotypes of GG, GA and AA.There was a SNP site at 206 bp of OAS1 gene exon 6(A206G), which contained three genotypes of AA, AG and GG.The chi-square fitness test showed that G110C of OAS1 gene exon 2 in Songliao Black pigs was in Hardy-Weinberg equilibrium, C176T, C145A, G166A and G206A in exons 3 and 6 deviated from Hardy-Weinberg equilibrium.The results of population genetic parameters analysis showed that the genetic heterozygosity of each SNP was in the middle level andmoderate polymorphism (0.25<PIC<0.5).The correlation analysis results showed that the total number of litter, live litter and weaning piglets of GC genotype in G110C were significantly higher than that of GG genotype (P<0.05).The number of weaned piglets of CT genotype in G176T was significantly higher than that of CC genotype (P<0.05).The total litter number and live number of CC genotype in C145T was significantly higher than that of AA genotype (P<0.05).The number of weaned piglets of GA genotype in G166A was significantly higher than that of GG genotype (P<0.05).The total number of offspring and live litter of GG genotype in A206G were significantly higher than that of AA genotype (P<0.05).The results indicated that there were mutations of OAS1 gene exons, which had significant effect on some reproductive traits in Songliao Black pigs.

The aim of this study was to investigate the genetic diversity of structural variation (SV) of adenylatecyclase 3 (ADCY3) and insulin like growth factor 1 (IGF1) genes in the ovarian steroidogenesis signaling pathway and their correlation with reproductive traits in Xiang pig sows.The SV of ADCY3 and IGF1 genes was studied in 269 Xiang pigs by PCR technique, the genetic heterozygosity (He), genetic purity(Ho), effective allele number(Ne) and polymorphic information content (PIC) were calculated using online software, and the association of different genotypes with reproductive traits such as total litter size, live litter size, newborn weight and number of teats in primiparous and multiparity Xiang pigs were analyzed using One-Way ANOVA and LSD method.The results showed that two SVs ADCY3-I₁-sv506 and IGF1-I₃-sv302 contained plenty of polymorphisms in Xiang pigs population, there were three genotypes of DD, DI and II, and two alleles of D and I.DI genotype (0.569) and D allele (0.601) were dominant in ADCY3-I₁-sv506, II genotype (0.717) and I allele (0.816) were dominant in IGF1-I₃-sv302.ADCY3-I₁-sv506 and IGF1-I₃-sv302 were moderate polymorphism (0.25<PIC<0.5), the heterozygosity was 0.479 and 0.300, respectively.The correlation analysis results showed that the total number born of primiparous and multiparous sows of DD genotype in ADCY3-I₁-sv506 were significantly higher than that of II genotype (P<0.05), the total number born of primipara and multiparous sows and the number born alive of multiparous sows of II genotype in IGF1-I₃-sv302 was significantly higher than that of DI genotype(P<0.05).In conclusion, both of two SVs in of ADCY3 and IGF1 genes had correlation ship with the reproductive performance of Xiang pigs, it could be taken as the candidate molecular markers for breeding of Xiang pigs sows.

The purpose of this study was to investigate the effects of different concentrations of P-coumaric acid (PCA) added to Beltsville thawing solution (BTS) on boar semen quality and antioxidant enzyme activities.The concentrations of 0, 0.03, 0.06, 0.09, 0.12, 0.15 g/L PCA were added into boar semen dilution preserved at room temperature.The sperm kinetic parameters and sperm functional integrity were detected by CASA automatic sperm analyzer and fluorescence probe technology within 1-5 d.The levels of reactive oxygen species (ROS), malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by antioxidant kit on day 1, 3 and 5.The results showed that the sperm motility of the 0.09 g/L PCA group was significantly higher than that of the blank group on the 5th day of semen preservation (P<0.05), the sperm acrosome function integrity, plasma membrane function integrity and mitochondrial function integrity were also significantly higher than blank group (P<0.05).In addition, 0.03, 0.06, 0.09 and 0.12 g/L PCA treatment significantly reduced the levels of ROS and MDA, and increased the activity of SOD (P<0.05).The antioxidant activity of 0.09 g/L PCA, which in comparison with other treatment groups, was won the optimum antioxidant effect.However, when the adding concentration of PCA up to 0.15 g/L, the antioxidant activity of sperm was significantly decreased in comparison with 0.09 g/L (P<0.05).In summary, the addition of 0.09 g/L PCA to BTS could improve the quality of sperm, the best supplementation concentration of PCA was 0.09 g/L.

In recent years, the beef cattle industry in China has been optimized in adjustment, and the number of cows in stock increases continuously.However, the number of breeding beef cows continues to decline.The low conception rate in cows and survival rate of calves have become the bottleneck restricting the development of the beef cattle industry.Ovsynch and timed artificial insemination (Ovsynch-TAI) technology has been widely used in improving the reproduction performance of cows by regulating the estrous cycle through the application of reproductive hormones, promoting the estrus of cows, and raising the estrus detection rate.It is perceived as a major breakthrough in livestock reproduction technique.Notwithstanding, the application of this technique has not been paid extensive attention due to the low intensive degree of beef cattle breeding in China.Different TAI procedures have their own characteristics, and there are many factors affecting the pregnancy rate during its application.How to further optimize this technique is a major problem.Large numbers of research have been done on different concentrations of reproductive hormone, different hormone combination, the interval of hormone injection, as well as hormone replacement.This paper reviewed the principle and main procedures of TAI, and the research progress of this technology in beef cattle, which was expected to establish an effective TAI technique system and provide references for the application in beef cattle industry.

Sertoli cells play a decisive role in maintaining the microenvironment in the process of spermatogenesis.They can promote the process of spermatogenesis through secretion function, blood testis barrier function formed by intercellular connection and phagocytosis, abnormal development of them will lead to different degrees of male reproductive defects.Based on the role of Sertoli cells in the reproductive process of male animals, highly purified Sertoli cells cultured in vitro can be an important cell model to study the two core functions of testis spermatogenesis and sex hormone secretion.In addition, purified Sertoli cells cultured in vitro can also be used as a cell model in toxicology and other newly developing hot fields to provide convenience for assessing and researching on the effects of environmental factors on male reproduction.In this article, the current research progress on the function of Sertoli cell and its commonly methods of in vitro isolation, purification, culture and characterization were systematically summarized to provide references for the use of animal Sertoli cells in the studies of male reproduction.

Muscle participates in a series of life activities such as movement, coordination, and perception of the body.At the same time, livestock and poultry muscles provide the animal protein products for human.Different regulatory mechanisms in the process of myogenesis cause phase differences in muscle development, and the entire muscle development is mainly reflected in the developmental state of two periods (embryonic and post-natal).Long non-coding RNA (lncRNA) is a type of RNA with a length greater than 200 nt that does not have the ability to encode proteins.In recent years, with the rapid development of genomics and molecular biology technology, lncRNA has been found to be widely involved in various stages of muscle development, regulating muscle development with multiple mechanisms of action.In this article, the present finding of lncRNAs that related to muscle development and their mechanisms of action were reviewed, and their important role in different stages of muscle development were explained, thus providing a reference for further study of lncRNAs related to muscle development.

The aim of this study was to construct a recombinant Lactococcus lactis expressing E2 antigen protein of Bovine viral diarrhea virus (BVDV), and lay a foundation for further development of oral live vector vaccine of BVDV.The BVDV E2 gene was amplified and sequenced, the gene sequence was optimized and synthesized according to the codon bias of the Lactococcus lactis strain and inserted into the pNZ8148 expression vectors, the recombinant strain pNZ8148-E2/NZ9000 was constructed by electroporation of competent cells of Lactococcus lactis NZ9000, and the induced with 1 ng/mL Nisin, and E2 expression was analyzed by SDS-PAGE and Western blotting.Healthy calves aged 6-12 months were immunized orally with recombinant Lactobacillus pNZ8148-E2/NZ9000, and blood samples were collected and serum was separated at different time after immunization.The antibody level was detected by indirect ELISA.The results showed that the target fragment of 1 149 bp was amplified by PCR.After optimizing the codon preference of Lactococcus lactis, the GC content changed from 45.28% to 34.30%.The pNZ8148-E2 plasmid was identified as the expected size by enzyme digestion.Approximate 42 ku fusion protein was observed from the cell lysates of pNZ8148-E2/NZ9000 in Western blotting.Furthermore, specific anti-E2 IgG was detected in serum of immunized calves.The results suggested oral immunization with E2-expressing Lactobacillus could induce immune response, and the recombinant Lactobacillus had good immunogenicity.

The purpose of this study was to obtain clinical isolates of Porcine deltacoronavirus (PDCoV) and investigate the biological characteristics and pathogenicity of the coronavirus.The swine testicle (ST) cells were used for PDCoV isolation with the PDCoV positive samples obtained clinically from a pig farm in Henan province.The isolated virus was identified by cytopathic effect (CPE) observation, RT-PCR, indirect immunofluorescence (IFA), electron microscopy, and sequence analysis of spike (S) gene, in addition, the pathogenicity was also evaluated using piglets.The PDCoV in the ST cell was successively passaged for 10 passages and confirmed by RT-PCR.The CPE (cell shrinkage, net pulling, fragmentation, and shedding) could be observed in the ST cells from the 4th passage.The isolated virus were identified by IFA, and typical crown shaped viral particles were observed by negative staining in electron microscope, and then the strain of PDCoV was named HeN10.The S gene of HeN10 had the highest sequence identity with China SD strain (99.8%), within the same branch of domestic prevalent strains phylogenetically, such as CHN-JS-2017 and CHN-HeB1-2017.The results of animal test showed that the virus (oral inoculation at a dose of 5×10^6.3 TCID₅₀/piglet) was highly pathogenic by causing watery diarrhea in 10-day-old piglets (5/5).Overall, the PDCoV HeN10 strain was successfully isolated, it showed high sequence identity with domestic prevalent strains such as SD, and also showed high pathogenicity in piglets, which providing a foundation for the development of diagnostic methods and vaccines.

The aim of this study was to prepare recombinant epitope protein of Streptococcus suis (S.suis) by constructing prokaryotic expression vector pET28a-DCpep-GSE.The transmembrane structures, signal peptides, B cell epitopes, Th cell epitopes and secondary structures of GAPDH, Enolase and SsnA of S.suis were analyzed and predicted by bioinformatics.The amino acid sequence of the recombinant protein (DCpep-GSE protein) was designed and its theoretical isoelectric point, hydrophilicity and other physicochemical properties were analyzed.Recombinant prokaryotic expression vector pET28a-DCpep-GSE was constructed and transformed into E.coli BL21 (DE3).The induced expression conditions were optimized, and the target protein was purified by His nickel column and tested for cytotoxicity and hemolysis.The results showed that the amino acids at sites 1-50 and 1 036-1 059 of SsnA protein were intracellular or transmembrane segments, and the amino acids at sites 1-56 were signal peptides.GAPDH and Enolase had no transmembrane regions or signal peptides.B and Th cell epitopes were located in random coil and β-turn region of secondary structure, showed that epitopes had good antigenicity.DCpep-GSE protein contained 373 amino acids, with molecular weight of 45.3 ku, theoretical isoelectric point (pI) of 4.57, and GRAVY of -0.677, belonged to the category of acidic hydrophilic protein.A 5 369 bp vector band and a 1 131 bp target band were obtained by double digestion of the plasmid.The sequencing results of PCR amplification products were consistent with the designed sequence, and the vector construction was correct.The highest protein expression was induced by 1 mmol/L IPTG at 37 ℃ for 6 h, and the soluble protein was obtained after ultrasonic crushing.The relative proliferation rate of RAW264.7, PK15 and MARC145 cells were 92.3%, 99.5% and 99.7%, respectively, when the protein concentration was 500 μg/mL in experimental group.The cell morphology was the same as that in control group, and the cells grew well.The hemolysis rate of each sample group was less than 5%.In this study, the recombinant protein DCpep-GSE was designed by bioinformatics method, and then DCpep-GSE was successfully expressed and purified by prokaryotic expression system, and its safety was verified.It laid a foundation for the further development of subunit vaccine of S.suis.

In order to understand the low pathogenic Avian influenza virus (LPAIV) epidemic situation in Guangxi border areas from 2013 to 2019, 33 964 poultry swabs samples were collected from large-scale farms and live poultry markets for LPAIV subtype isolation and identification through hemagglutination inhibition test and RT-PCR.The results showed that 3 892 LPAIV strains were isolated, the virus isolation rates from 2013 to 2019 were 17.39%, 11.23%, 13.59%, 9.31%, 9.09%, 9.12% and 15.21%, and the total isolation rate was 11.46%, of which the rate in 2013 was the highest.The isolated LPAIV included 7 subtypes H1, H3, H4, H6, H9, H10 and H11.These virus isolation rates were 0.14%, 4.37%, 0.19%, 4.06%, 2.69%, 0.01% and 0.003%.In the live poultry market, LPAIV subtype H1, H3, H4, H6, H9, H10 and H11 rates were 0.19%, 5.53%, 0.21%, 5.29%, 3.41%, 0.01% and 0.004%, respectively.While in the farms LPAIV subtype H3, H4, H6 and H9 rates were 1.26%, 0.12%, 0.79% and 0.78%, respectively.The virus isolation rate of the farm samples (2.95%) was obviously lower than the live poultry market samples (14.64%).The virus isolation rates of waterfowl (15.10%) and environmental samples (21.67%) were much higher than that from chickens (5.77%).The results showed that the current poultry carried multiple LPAIV subtypes, and waterfowl were high-risk flocks.The live poultry market was seriously polluted, and the market supervision (LPAIV) should be further strengthened, established and implemented more scientific, effective live poultry market operation mechanism.

To establish a more sensitivity, specificity diagnostic method that could be used for the early diagnosis of bovine fascioliasis, Fasciola gigantica excretory-secretory products (FgESP) was chromatographed to screen UV280 chromatographic peak.While UV280 chromatographic peak showing the best detection effect was screened out, the best chromatographic component under corresponding chromatographic peak was further screened as diagnostic antigen.The conditions of coating antigen, antibody dilution, dilution of enzyme-labeled secondary antibody and reaction time were optimized by checkerboard titration test, and the indirect ELISA diagnostic method for detection of bovine fascioliasis was established.Sensitive, specific, clinical tests and 0-14 weeks serum IgG antibody of buffalos experimentally infected Fasciola gigantica were performed on the established indirect ELISA assay.Meanwhile, serum IgG antibody of 160 buffalos were also detected, then result was compared with that of the existing diagnostic antigen (FgESP, the cut-off value was 0.320).The results showed that chromatographic component F22 worked well.The optimal reaction conditions of the indirect ELISA were as follows:The coating antigen of F22 was 0.157 μg/mL, the testing serum dilution and the HRP conjugated anti-bovine IgG dilution were 1:400 and 1:40 000 respectively, the incubation time was 25 min.The established method was used to detect 15 buffalo negative serums, while the cut-off value of D_{450 nm} was 0.408.Comparision of F22 and FgESP indicated that they were comparable in specificity, while F22 showed higher sensitivity.Upon established indirect ELISA, it was found that from the second week anti-F22-IgG was significantly higher than that of anti-FgESP-IgG in the period of 0-14 weeks infected buffalo serum (P<0.01).Therefore, F22 showed better diagnostic effect than FgESP.The positive rate of F22 was 71.25% and FgESP was 63.75% in 160 serum samples detection of buffalos.All above suggested that indirect ELISA based on F22 showed better diagnostic effect than FgESP, which could apply to early diagnosis of fascioliasis in the future.

In order to investigate the molecular genetic characteristics of Pseudorabies virus (PRV) in Southern Henan province, the main PRV virulence genes gB, gE, gC, gD and TK from the tissues of PRV-infected pigs were amplified by PCR and sequenced.The similarity, phylogenetic tree and amino acid variation analysis of major virulence genes between epidemic strains and published reference sequences were performed by MegAlign software.Sequencing results showed that the PRV gB, gE, gC, gD and TK genes from 5 PRV-infected pig tissues were successfully amplified.Amino acid similarity analysis and phylogenetic tree results showed that the 5 PRV epidemic strains in Southern Henan province were in G1 group with the domestic epidemic strains, the amino acid similarity of gB, gE, gC and gD were 98.5%-100%, 97.2%-100%, 97.5%-100% and 98.3%-100%, respectively, relatively distantly related to G2 group (the Bartha strain, Becker strain, and NiA3 strain in Europe and America), the amino acid similarity of gB, gE, gC and gD were 96.4%-97.4%, 94.6%-95.7%, 92.7%-94.0% and 96.3%-99.0%, respectively.Compared with the Bartha-K61 strain (or Becker strain, NiA3 strain, etc.), there were amino acids deletion, insertion and mutation in the 5 epidemic strains, such as ‘PGL’ deletion at position 75-77 and ‘G’ insertion at position 94 in gB amino acids, the ‘D’ insertions at positions 48 and 496 in gE amino acid sequence, the ‘VSGTTGA’ insertions at positions 57-63 and the ‘SPEAG’ mutation to ‘ASTPA’ at positions 65-69 in the gC amino acid, the ‘RP’ or ‘RPRP’ insertion at positions 278-281 in gD amino acid.In addition, there were single amino acid mutations in gB, gE, gC, gD and TK amino acid, respectively.Therefore, the epidemic strains of PRV in Southern Henan province showed the molecular genetic characteristics of the variant strains.The above results confirmed that the 5 PRV epidemic strains were variant strains, which were relatively distantly related to the vaccine strain Bartha-K61.

The aim of the study was to isolate Equine herpesvirus type 8 (EHV-8) from donkeys and analyze genetic characteristics.The lung tissues of diseased donkeys were collected from a donkey farm in Liaocheng area of Shandong province.The infection of EHV-8 was identified by PCR.The lung tissues with positive EHV-8 infection were ground and inoculated into RK-13 cells after repeated freezing and thawing 3 times.The cells were collected when cytopathic effects (CPE) appeared and identified by PCR, indirect immunofluorescence test and transmission electron microscope.The whole genome sequence of ORF70 was amplified by PCR and analyzed by bioinformatics.The results showed that the lung tissues with positive EHV-8 infection were identified by PCR, and typical CPE appeared after inoculating the susceptible cells RK-13.The first three generations of cell cultures were collected, and the ORF70 gene with the expected size was obtained by PCR amplification.The EHV-8 strain was named SDLC66, and the sequence was uploaded to GenBank, and the accession No.was MW816102.Sequencing and sequence alignment showed that the similarity of ORF70 gene between SDLC66 strain and AHV-3 strain (GenBank accession No.:U24184.1) was 99%, and the similarity of ORF70 gene between SDLC66 strain and domestic EHV-8 wh strain (GenBank accession No.:JQ343919.1), foreign EHV-8/IR/2015/40 (GenBank accession No.:MF431614.1) and EHV-8/IR/2003/19 (GenBank accession No.:mf431611.1) reference strain was the highest (99.8%).Phylogenetic tree analysis showed that SDLC66 and EHV-8 (EHV-8 wh, EHV-8/IR/2015/40 and EHV-8/IR/2003/19) were closely related in a small branch.It was closely related to EHV-1 reference strains (Hong Kong/57/1984, United Kingdom/32/1982, Oxford/206/2013), but far from EHV-4 strains (91c1 and TH20p).Indirect immunofluorescence assay showed a red fluorescent signal specifically binding to viral protein.The round virus particles with diameter of about 110 nm were observed by transmission electron microscope, and there was a halo outside the nucleocapsid, and a capsule outside the halo.These data indicated that EHV-8 strain from donkey was isolated, which laid the foundation for the study of pathogenicity and mechanism of EHV-8.

Mycoplasma synoviae (MS) can cause poultry respiratory diseases, avian onfectious synovitis, eggshell apex abnormalities and growth retardation etc, brings huge economic losses to the global poultry industry.MS has no cell wall, only one serotype, isolation and culture requirements are demanding, the genome is smaller than bacteria, it has strong antigen mutation ability, and a variety of membrane proteins are related to immune response and cell adhesion.MS is host-specific and mainly affects young chickens and turkeys.It can be transmitted horizontally and vertically and is distributed worldwide.MS adheres to host cells through membrane proteins, releases toxic products, immune evasion, grabs nutrients etc, resulting in host cell damage or death.MS inactivated vaccine has high safety and protection rate, which can prevent it from colonizing in air sacs and trachea.Live vaccines cannot protect infected chickens, and will induce disease or aggravate symptoms after vaccination.Genetic engineering vaccines are highly safe and easy to mass produce, and will be the future direction of MS vaccine research and development.Macrolides, tetracyclines and other drugs combined vaccines have the best immunization effects, and there are differences in MS resistance in different countries and regions.Based on the problems in the prevention and treatment of MS, the development of new high-efficiency vaccines and antibiotic substitutes that can break through the interference of maternal antibodies and antibiotics will be the focus of future work.The author introduced the pathogenic characteristics, epidemic characteristics, domestic and international epidemic status, laboratory diagnosis methods and pathogenic mechanism of MS, focusing on MS vaccine immunization and drug control, and finally looked forward to the future development trend of the disease control.

In order to screen the suitable method of establishing host infection model of Salmonella Pullorum, the infection model was established by inoculating chicken embryos of different ages with different inoculation methods and different concentration of bacteria solution.90 SPF chicken embryos were randomly divided into 9 groups, 10 in each group, which were divided into A, B, C, D, E, F, G, H, I groups, group A was blank control group, and the other groups were model groups.In the model groups, different concentrations of Salmonella Pullorum C79-3(1×10³, 3×10³, 9×10³, 3×10⁵, 9×10⁵ CFU/mL) were inoculated on SPF chicken embryos of different ages (11 and 14 embryo ages) by air chamber inoculation and eggshell contact inoculation.After hatching, the chicks were reared in different cages according to groups, the clinical symptoms and death of chicks in each group were observed every 12 hours for 7 days.At the end of the observation period, autopsy observation, histopathological examination and isolation and identification of Salmonella Pullorum were performed on the dead and surviving chickens.The results showed that all the chicks in each model group performed obvious symptoms of pullorum and died, and compared with other model groups, group F inoculated with 9×10⁵ CFU/mL of C79-3 bacteria in 11 embryo age eggshell contact infection and H group inoculated with 3×10³ CFU/mL of C79-3 bacteria in 14 embryo age eggshell air chamber infection had higher rate of eggshell emergence, which could reach 80% and 90%, respectively, and the shelled chicks showed obvious characteristics of pullorum dysentery.Autopsy showed liver hemorrhage, cecum enlargement and yolk sac malabsorption.Histopathological sections showed varying degrees of injury to the liver, cecum and heart, with an incidence of 100% and 88.8%, respectively, and the positive rate of Salmonella Pullorum was 70% and 90%.This study showed that the two methods of inoculating 0.1 mL 9×10⁵ CFU/mL C79-3 bacteria in 11 embryo age eggshell contact infection and 0.1 mL 3×10³ CFU/mL C79-3 bacteria in 14 embryo age air chamber infection could be used to establish a stable host infection model of Salmonella Pullorum.

In order to explore the current prevalence and drug resistance of Streptococcus agalactiae isolated from tilapia of Hainan, in this study, 15 strains of Streptococcus agalactiae were isolated from diseased fish in some tilapia farms in Hainan in August 2020.Serotypes, main virulence genes and multiple drug resistance of all isolates were detected.The serotype detection results showed that the serotype of 15 isolated was type Ⅰa, consistent with the reference human strains A909.The results of virulence gene detection showed that scpB gene was only detected in human reference strain Streptococcus agalactiae 2603V/R except all isolates from tilapia.However, the detection rate of major virulence genes, such as cylE, sodA and gapC, was 100%.The drug sensitive test result indicated that the drug resistance rate of 15 isolates to methoxypyrimidine, sulfamisoxazole, cotrimoxazole, streptomycin, neomycin and gentamicin were more than 85%.The sensitive rate of cefcllor, ceftriaxone, cefoperazone, cefradine, kanamycin, erythromycin, rifampicin and clindamycin were more than 80%.No strains were found to be sensitive to methoxymidine, fleroxacin, streptomycin, neomycin and gentamicin.The results of multidrug resistance test showed that 15 isolated strains were resistant to 4 drugs at least, among all the isolates, 86.67% were resistant to more than 6 kinds of antibiotics, and two of them were resistant to 13 kinds of antibiotics.This results provided reference for the application of antimicrobial agents for the treatment of tilapia infection of Streptococcus agalactiaein in Hainan, and also provided new data for the epidemiology and disease control of Streptococcus agalactiae of tilapia in South China.

This experiment was aimed to investigate the prevalence and virulence genes distribution of Salmonella from chicken in Shandong province.Samples were collected from large-scale chicken farms in Shandong province, and tetrathionate broths (TTB) were used as Salmonella selective enrichment broths before bacterial isolation.The isolated strains were purified and cultured, and Gram staining, biochemical tests, colony morphology, PCR amplification with Salmonella and serotype specific primers were carried out.33 kinds virulence genes were detected by PCR to study the virulence genes distribution.The results showed that the isolated strains had grown on LB and MacConkey media with colorless, translucent, smooth and neat edged colonies, and the bacterial morphology were Gram-negative brevibacterium with blunt ends at both ends.25 Salmonella strains were isolated, including 9 Salmonella Enteritis, 8 Salmonella Pullorum strains and 8 Salmonella Typhimurium strains.The detection rates of 20 genes that virulence island (invJ, virK, sopA, mogA, hilA, hisJ, ssaB, ssaQ, ssiD, misL, mgtC, orf319, siiE, siiD, bcfA, pipC, sopB, araB, spoB and avrA genes), enterotoxin gene (stn gene) and fimbriae virulence genes (fimA gene) were 100%.The detection rates of 2 virulence island genes (sipA and sodC genes) and 4 virulence plasmid genes (spvA, spvC, spvD and spvR genes) were 96%.The detection rate of spoE gene was 68%, fliC gene was 36%, gipA and spvB genes were 32%, and sseL gene was 4%.In the 25 Salmonella strains, the numbers of carrying virulence genes were 31(8 strains), 30(1 strain), 29(15 strains) and 24(1 strain), respectively.gipA and spvB genes could be detected only in Salmonella Typhimurium, and the positive rate was 100%.The above results showed that Salmonella Enteritis, Salmonella Pullorum and Salmonella Typhimurium were the main pathogens of salmonellosis in Shandong province.All the chicken Salmonella carried many virulence genes, gipA and spvB genes detected only in Salmonella Typhimurium, it could be used as one of the candidate genes for the identification of Salmonella Typhimurium.

The aim of this study was to clarify the effect of Staphylococcus aureus lipoproteins on immunity of M1 mouse bone marrow-derived macrophages, and provide the theoretical reference for the study of Staphylococcus aureus pathogenicity.In vitro, M1 mouse bone marrow-derived macrophages was infected by WT SA113 and SA113 lgt::ermB strains (SA113Δlgt) of Staphylococcus aureus.The mice were divided into three groups:Blank control group, WT SA113 infection group (MOI:3:1), and SA113Δlgt infection group (MOI:3:1).The tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), chemokines (RANTES) and interleukin-10 (IL-10) in M1 mouse bone marrow-derived macrophages was detected by ELISA method, the expression of Toll-like receptor 2 (TLR2), Toll-like receptor 2(TLR4) and recombinant NLR family, Pyrin domain containing protein 3 (NLRP3) genes were detected by Real-time quantitative PCR, and the effect of lipoproteins on phagocytosis of Staphylococcus aureus by M1 mouse bone marrow-derived macrophages was detected by immunofluorescence.The results showed that compared with control group, both WT SA113 and SA113Δlgt infection could significantly upregulate the secretion of TNF-α, RANTES and IL-10, and WT SA113 infection could significantly increase the expression of TLR2 and NLRP3 genes in M1 mouse bone marrow-derived macrophages polarization (P<0.05), while the expression of TLR4 gene was significantly decreased (P<0.05).Compared with WT SA113 infected group, the secretion of TNF-α, IL-1β, RANTES and IL-10, and the expression of TLR2 (expect for 12 h) and NLRP3 gene in SA113Δlgt infected group were significantly decreased (P<0.05).The immunofluorescence results showed that the phagocytosis effect of M1 macrophage on SA113Δlgt strain was significantly lower than that of WT SA113 strain (P<0.05).In conclusion, the lipoprotein of Staphylococcus aureus induced the production and release of cytokines TNF-α, IL-1β, RANTES and IL-10 mainly through activation of TLR2 and NLRP3 receptors in M1 mouse bone marrow-derived macrophages.

In order to provide scientific basis for the in-depth development of Shuanghuanglian soluble powder(honeysuckle, forsythia, Scutellaria baicalensis, 1:1:2) and the rational selection of medicine for poultry and livestock.In this study, Shuanghuanglian soluble powder produced by production technology of Shuanghuanglian oral liquid and its alcohol precipitates (polysaccharide components) and filtrate (flavonoids components) were used as research materials.The mammals Yak source rotavirus, Newcastle disease virus, yak source pathogenic Escherichia coli, Salmonella, Staphylococcus aureus as the research object, Oxford cup combined with two-fold dilution method was used to investigate the antibacterial activity in vitro.Using yak MA-104 cell line and chicken embryo fibroblast cell line UMNSAH/DF-1 as host cells, Thiazolium blue colorimetry (MTT) and cytopathic detection (CPE) were used to investigate the antiviral activity in vitro.The results showed that the antibacterial effect of the Shuanghuanglian soluble powder on Gram-negative bacteria(Yak Escherichia coli, Yak Salmonella, Escherichia coli quality control strain) was better than that on Gram-positive bacteria (Staphylococcus aureus, Staphylococcus aureus quality control strain from yaks).The flavonoids isolated from the Shuanghuanglian soluble powder might be the main antibacterial component of the Shuanghuanglian soluble powder, while the polysaccharide component had no antibacterial activity.The antibacterial effect of soluble powder flavone components of Shuanghuanglian on Gram-positive bacteria was better than that on Gram-negative bacteria.The minimum nontoxic concentration (TC₀) of Shuanghuanglian soluble powder and its flavonoids and polysaccharides on UMNSAH/DF-1 cells were 2.58, 1.17 and 1.56 μg/mL, respectively.The TC₀ of MA-104 cells were 1.290, 0.585 and 0.780 μg/mL, respectively.They all had good antiviral effect for the Yak rotavirus.The antiviral effect of Shuanghuanglian soluble powder against Newcastle disease virus was better than that of flavonoids and polysaccharides components.The results provided scientific basis for the in-depth development for the Shuanghuanglian soluble powder and rational selection of medicine for livestock and poultry.

The study was carried out to evaluate the safety of Tiandongtanggan powder through the acute toxicity test on mice and subchronic toxicity test in rats.In the acute toxicity test, pre-test (5 000, 1 000, 200, 40 mg/(kg·BW)), formal test (5 000, 2 500, 1 250, 625 mg/(kg·BW)) and maximum dose test (60 g/(kg·BW)) were performed respectively.After administration, the mental state and death of mice were observed for 7 consecutive days, the mice were dissected after the experiment.In the subchronic toxicity test, 80 Wistar rats were randomly divided into 4 groups, including high dose group (10 g/(kg·BW)), middle dose group (5 g/(kg·BW)), low dose group (2.5 g/(kg·BW)) and control group.The administration was continued for 30 days, and the clinical manifestations of the rats were observed during the test.The body weight, food intake and water intake of the rats were recorded, and the weight gain rate was calculated.After 30 days of administration, the blood routine and blood biochemical indexes were detected, the organ indexes were calculated, and histopathological examination was performed on the main organs.The results showed that there was no death of mice and no visible pathological changes in the organs in the pre-test, formal test and maximum dose test of the acute toxicity test.In the subchronic toxicity test, there were no significant differences in the body weight, hematological indexes, blood biochemical indexes, organ coefficient and histopathological examination results of rats in the groups of medium and low dose of Tiandongtanggan powder compared with the control group (P>0.05).The mean erythrocyte volume (MCV) and alanine aminotransferase (ALT) content of male rats in high-dose group were significantly higher than those in male control group (P<0.05).The red blood cell count (RBC), red blood cell distribution width (RDW) and percentage of MONO nuclear cells (MONO%) were significantly lower than those of male control group (P<0.05), but within the normal range, and the pathological tissue blood test results had no significant difference(P>0.05), indicating that 10 g/(kg·BW) Tiandongtanggan powder had certain effects on the hematological indexes and liver and kidney functions of the tested rats.The results showed that LD₅₀>5 000 mg/(kg·BW) of Tiandongtanggan powder in mice.The above results showed that Tiandongtanggan powder in the range of 10 g/(kg·BW) administered intragastrically to rats for 30 days had no obvious toxic and side effects on rats, showing good safety.