【Objective】 The purpose of this study was to compare the diversity,species and relative abundance of microbial community in different intestinal segments of meat ducks by metagenome sequencing,species annotation and functional annotation,so as to explore the spatial heterogeneity of gut microbiota in meat ducks.【Method】 The contents of duodenum,jejunum,ileum and cecum were collected from 6-week-old Beijing ducks and Liancheng White ducks raised in the same environment in turn,and the species and relative abundance of gut microbiota were obtained by metagenome sequencing.Based on the microbial abundance table,wilcoxon rank sum test was used to compare the differences of dominant microorganisms,microbial species,microbial relative abundance,microbial community diversity and microbial functional diversity in different intestinal segments,and t test was used to screen the significantly different bacterial genera in different intestinal segments of Pekin ducks and Liancheng White ducks.【Result】 45 phyla,84 classes,186 orders,418 families,1 400 genera and 3 467 species were identified in this experiment.The dominant phyla of the four intestinal segments were Firmacutes,Bacteroides,Proteobacteria and Actinomycetes,and the distribution of the four phyla in different intestinal segments was different.Bacteroides and Phocaeicola were the dominant genera in the four intestinal segments,and the species and abundance of the dominant genera were different among the groups of different intestinal segments.From duodenum to cecum,the composition,relative abundance and community diversity of microbiota changed significantly (P＜0.05),and the microbial composition of duodenum,jejunum and ileum was highly similar and obviously separated from cecum microbiota.At the same time,the community diversity of cecum microbiota was also significantly higher than that of other intestinal segments (P＜0.01).Compared with duodenum,jejunum and ileum,the metabolic pathways such as bacterial chemotaxis,amino sugar,nucleotide sugar metabolizing fatty acid biosynthesis of cecum microbiota were up-regulated,and 31 different bacteria genera of Beijing ducks and Liancheng White ducks were enriched in cecum,which were higher than that of duodenum,jejunum and ileum.【Conclusion】 The quantity,relative abundance and community diversity of microbiota in cecum of meat ducks were higher than those in duodenum,jejunum and ileum,which provided theoretical basis for analyzing the regulation mechanism of gut microbiota in meat ducks,accurately regulating energy metabolism level and immune function of meat ducks,and provided reference for further study of gut microbiota in meat ducks.

【Objective】 This study was aimed to clone prokineticin receptor 2 (PROKR2) gene,and detect the expression of PROKR2 gene in different tissues during the initiation of puberty in Dolang sheep,so as to provide a basis for exploring the role of PROKR2 gene in the initiation of puberty in sheep.【Method】 Using the hypothalamic cDNA in Dolang sheep after first puberty as a template,PROKR2 gene was amplified by PCR, cloned and sequenced.DNAMAN software was used to splice the sequencing results,MegAlign software was used to compare the similarity among different species and construct a phylogenetic tree,and the physicochemical property and structural function of PROKR2 protein in Dolang sheep were predicted by bioinformatics software.Real-time quantitative PCR was used to detect the expression of PROKR2 gene in hypothalamus,hypophysis,ovary,oviduct and uterus of Dolang sheep in prepuberty,puberty and postpuberty.【Result】 The cloned sequence size of PROKR2 gene was 2 641 bp,including 5'-UTR 143 bp,3'-UTR 1 343 bp and CDS region 1 155 bp,encoding 384 amino acids,and the similarity was 99.83% with the predicted mRNA sequence in sheep on GenBank (accession No.:XM_004014342.5).The phylogenetic tree showed that the genetic distance of the PROKR2 gene in Dolang sheep was the closest to Capra hircus,and the farthest to Gallus gallus.Bioinformatics analysis showed that PROKR2 protein was a hydrophobic and stable basic protein with 7 transmembrane structures,which belonged to the transmembrane protein,and there were 31 phosphorylation sites,which mainly played a role in the plasma membrane.There was an interaction relationship between proteins such as GnRH1,PROK1,ANOS1 and PROKR2 protein in Dolang sheep.The results of Real-time quantitative PCR showed that the expression of PROKR2 gene in each stage of hypothalamus was significantly higher than that of other tissues (P＜0.05),and the expression of PROKR2 gene was the highest in the postpuberty stage,while there was no significant change in hypophysis (P＞0.05).The expression of PROKR2 gene in uterus in prepuberty was significantly higher than that in puberty and postpuberty (P＜0.05).The expression of PROKR2 gene in ovary and oviduct tissues were significantly higher than those in the prepuberty and postpuberty stages (P＜0.05).【Conclusion】 In this study,the CDS region sequence of PROKR2 gene was successfully cloned,and PROKR2 protein was a hydrophobic stable basic protein containing 7 transmembrane structures,which was mainly expressed in hypothalamus,and the general trend in the ovary and oviduct were first rising and then decreasing,which provided a reference for further exploring its regulation in the initiation of early puberty in sheep.

【Objective】 This study was aimed to identify the codon usage characteristic and influencing factors of melanocortin 3 receptor (MC3R) gene in Nyctereutes procyonoides,and understand the relationship between genetic evolution and codon preference among different species,so as to provide a theoretical basis for studying the heterologous high expression of MC3R gene.【Method】 Six parameters of codon preference of MC3R gene CDS sequences in Nyctereutes procyonoides and 15 other species were analyzed using Codon W and EMBOSS softwares,and Neutrality-plot,ENc-plot and PR2-plot were used to explore influence factors of codon bias formation of MC3R gene.The phylogenetic tree was constructed based on the cluster analysis of MC3R gene CDS sequence information,and the heat map was constructed base on relative synonymous codon usage (RSCU) of different species.【Result】 The codon GC and GC3s were both greater than 0.5 of MC3R gene in Nyctereutes procyonoides,and the codon preferred to end with G/C.There were 25 preferred codons (RSCU value ＞1),of which 16 ended in C and 9 ended in G.Neutrality-plot,ENc-plot and PR2-plot analysis results showed that natural selection was the main influencing factor of MC3R gene codon preference formation.The cluster analysis results of gene sequence and RSCU values showed that the Nyctereutes procyonoides belonged to the same cladistic genus as Canis lupus familiaris in phylogenetic relationship,and then clustered with Vulpes vulpes and Vulpes lagopus.But when it came to codon use,Nyctereutes procyonoides were grouped with Homo sapiens,Pan troglodytes and Equus caballus.【Conclusion】 MC3R gene prefered to use codons ending with G/C,and natural selection was the main influencing factor of codon preference.The codon usage bias was species-specific,but codon use patterns were different among closely related species due to other factors.

【Objective】 The study was conducted to establish a chemiluminescent antibody detection method for African swine fever virus (ASFV) with high specificity,high sensitivity,simple operation and high throughput,which could be used for the early monitoring of the prevalence of ASFV.【Method】 In this study,we used carboxylated magnetic beads as a solid-phase carrier to couple ASFV recombinant p72 protein,which was closed with an appropriate amount of bovine serum albumin (BSA),and then added to the test sample for the reaction.Rabbit anti-porcine IgG-AP antibody was used as the enzyme-labeled antibody,and the corresponding substrate was added for the chromatography to optimize the conditions of each reaction, establish the chemiluminescent detection method of the ASFV antibody,and validate the specificity,sensitivity,reproducibility and detection rate of the method.【Result】 The chemiluminescence detection method for ASFV antibody was established,the optimal pH for protein coupling with carboxyl beads was 7.0,the optimal protein coupling concentration was 5 μg/mL,the best effect was achieved by using 10% BSA solution for closure,the final concentration of the reaction beads was 1.00 mg/mL,and the optimal dilution of enzyme-labeled secondary antibody was 1∶40 000.At the same time,the reaction time of this method was 30 min,and the serum dilution was 1∶512.The sensitivity of this method was higher than that of the commercial ASFV antibody ELISA detection kit,and there was no cross-reaction with the antibody-positive standard sera of Classical swine fever virus,Foot-and-mouth disease virus type A,Foot-and-mouth disease virus type O,Porcine reproductive and respiratory syndrome virus,Porcine circovirus type 2,Porcine pseudorabies virus gE,Porcine pseudorabies virus gB.In the reproducibility test,the coefficients of variation ranged from 4.41% to 8.70% in intra-assay and from 2.43% to 8.07% in inter-assay,which were both ＜10%.By testing 120 serum samples and analyzing them in comparison with commercial ASFV antibody ELISA detection kit,the detection rate was 96.7% for commercial ELISA kit and 100% for the chemiluminescent detection method established in this study.【Conclusion】 The ASFV chemiluminescent antibody detection method established in this study could be used to detect the antibody level after ASFV infection and provide a reference for the development of subsequent kits.

【Objective】 In this study,the CDS region sequence of stimulator of interferon genes (STING) gene in White Leghorn chickens was cloned and analyzed by bioinformatics and tissue expression,which laid a foundation for elucidating the role of STING gene in antiviral immune response.【Method】 The CDS region of STING gene in White Leghorn chickens was amplified by PCR and cloned.After sequencing,the similarity of the encoded amino acid sequence of STING gene was compared and the phylogenetic tree was constructed.The physicochemical properties and structural function of STING protein were predicted by bioinformatics,and the expression of STING gene in 14 tissues such as heart and liver of White Leghorn chickens were detected by Real-time quantitative PCR.【Result】 The sequence of the CDS region of STING gene in White Leghorn chickens was 1 140 bp in total length,encoding 379 amino acids.Similarity alignment and phylogenetic tree analysis showed that STING gene in White Leghorn chickens had the highest similarity with Gallus gallus and the closest genetic relationship,and the farthest genetic relationship with Corvus cornix cornix.STING protein in White Leghorn chickens was an acidic,hydrophilic protein with a molecular weight of 42.625 ku,an isoelectric point (pI) of 6.67,an instability coefficient of 69.26, and a fat coefficient of 105.01.STING protein was synthesized mostly on mitochondria and endoplasmic reticulum,contained a transmembrane structure,and no signal peptide.The secondary structure of STING protein included alpha helix (54.62%),extended chain (10.29%),beta turn (3.43%) and random coil (31.66%).Protein interaction analysis showed that there were interactions between STING and NFKB1,DDX41,cGAS,TBK1 and other proteins.The results of Real-time quantitative PCR showed that STING gene was widely expressed in the tissues of White Leghoron chickens,with the highest expression in lung,which was significantly higher than that in other tissues (P＜0.05),and the lowest expression in pectoralis muscle.【Conclusion】 In this study,STING gene in White Leghorn chickens was successfully cloned,the total length of the CDS region was 1 140 bp,encoding 379 amino acids.STING protein in White Leghorn chickens was an acidic,hydrophilic protein with a transmembrane structure.There was the highest expression of STING gene in lung of White Leghoron chickens.The results provided materials for the in-depth study of the function of the protein encoded by STING gene in White Leghorn chickens.

Genome-wide association studies (GWAS) is a method of comparing genotype and phenotype data on a large sample set to identify genetic variations associated with specific traits.With the continuous development of high-throughput sequencing technology,bioinformatics technology,and statistical methods,some genetic variations or small molecular substances with lower frequencies can be detected more accurately and economically.The extension method of GWAS derived from technological progress provides new ideas for precision breeding and genetic improvement of livestock and poultry,including GWAS based on copy number variation (CNV),structural variation (SV),and tandem repeats (TR),and GWAS based on haplotypes,gene expression,and metabolomics.Researchers hope to use different molecular markers to provide more comprehensive and detailed genetic variation information to enhance the interpretability and accuracy of GWAS,or further explain and deepen the results of GWAS by combining other types of data,in order to deeply study the relationship between genetic variation and traits and identify key genes that affect complex traits.The author introduces the application of GWAS based on different molecular markers in livestock and poultry research and discusses its results.The advantages and feasibility of different methods are analyzed,providing more ideas and support for further promoting the application of GWAS in livestock and poultry research,precision breeding,and genetic improvement.

【Objective】 The CDS region of silence information regulator 3 (SIRT3) gene in Hezuo pigs was cloned and bioinformatic analysis was performed,and the expression level in different tissues of Hezuo pigs was explored,laying a foundation for the study of the function of SIRT3 gene.【Method】 Three 3-month-old Hezuo boars were selected to collect heart,testis,lung and other tissues.The CDS region sequence of SIRT3 gene was obtained by PCR amplification and Sanger sequencing,and the phylogenetic tree was constructed by Mega 7.0.Bioinformatic analysis of SIRT3 protein was performed by online software.The tissue expression of SIRT3 gene was detected by Real-time quantitative PCR.【Result】 The CDS region of SIRT3 gene was 774 bp,encoding 257 amino acids.Hezuo pig had the closest relationship with domestic pig,and had a distant relationship with zebrafish.The molecular weight of SIRT3 protein was 28.68 ku,the theoretical isoelectric point was 5.81,and the instability coefficient was 37.92,indicating that SIRT3 protein was a stable hydrophilic protein.SIRT3 protein had no transmembrane region and no signal peptide,but there were 2 low complexity region,which were mainly distributed in the endoplasmic reticulum.The secondary structure of SIRT3 protein was mainly alpha-helix and random coil,and the results predicted by the tertiary structure model were basically consistent with the secondary structure.Real-time quantitative PCR results showed that SIRT3 gene was expressed in different tissues of Hezuo pigs,and the expression level in heart was the highest,which was extremely significantly higher than that in other tissues (P＜0.01),followed by testis,lung and liver tissues.【Conclusion】 The CDS region sequence of SIRT3 gene in Hezuo pigs was successfully cloned,the expression level of SIRT3 gene in different tissues of Hezuo pigs was explored,and the biological function of its encoded protein was preliminatively analyzed,which provided a reference for further study of SIRT3 gene function in Hezuo pigs.

【Objective】 This study was aimed to investigate the damage effect of Brucella virulence factor A (BvfA) in Sertoli cells (TM4 cells),so as to reveal the pathogenic mechanism of Brucella on host.【Method】 RNA-Seq double-end sequencing,Label-free proteomics and LC-MS non-target metabolomics were used to analyze the omics difference of TM4 cells infected with Brucella S19△bvfA and S19 strains.The differentially expressed proteins and differential metabolite pathways were analyzed using KEGG database,and the protein expression were validated by parallel reaction monitoring (PRM).【Result】 There were 400 differentially expressed genes,422 differentially expressed proteins and 271 differential metabolites in TM4 cells infected with S19△bvfA and S19 strains.Multi-omics analysis showed that the common KEGG pathway of differentially expressed genes,proteins and metabolites in TM4 cells infected with S19△bvfA and S19 strains was retrograde endocannabinoid signaling pathway.In this pathway,20 proteins expression was down-regulated in mitochondrial NADH-ubiquinone redoxase Complex Ⅰ of TM4 cells infected with S19△bvfA strain.PRM result showed that the expression trends of Ndufb11,Ndufa9,Ndufv1,Ndufv2,Ndufs8 and Ndufs2 in Complex Ⅰ were consistent with the results of Label-free proteomics.【Conclusion】 BvfA induced variations of transcriptional,protein and metabolic levels in Brucella infected TM4 cells,and BvfA down-regulated the expression of NADH-ubiquinone oxidoreductase complex proteins in mitochondria of Sertoli cells.

【Objective】 The objective of this experiment was to clone the CDS region of myosin light chain 9 (MYL9) gene in Meiren yak and perform bioinformatics analysis,and detect the tissue expression characteristics of MYL9 gene,so as to provide a theoretical basis for exploring the function of this gene.【Method】 Using the cDNA of Meiren yak muscle tissue as template,PCR was used to amplify and clone the CDS region sequence of Meiren yak,similarity comparison with other species and phylogenetic tree construction were carried out.Bioinformatic analysis of MYL9 protein was performed by online software.The expression of MYL9 gene in heart,liver,spleen,lung,kidney,muscle,fat and testis of Meilen yak was detected by Real-time quantitative PCR.【Result】 The CDS region of MYL9 gene in Meiren yak was 516 bp,encoding 171 amino acids.Phylogenetic tree results showed that Meiren yak had the closest genetic relationship with Bos mutus and Bos taurus,and the farthest genetic relationship with Nanorana parkeri.The molecular formula of the protein encoded by the gene was C₈₅₈H₁₃₂₄N₂₃₄O₂₇₄S₁₂,the number of atoms was 2 702,the theoretical isoelectric point was 4.85,and the instability coefficient and the total average hydrophilicity were 37.22 and ―0.772,respectively,which belonged to stable hydrophilic protein.MYL9 protein was a non-secreted protein without signal peptide and transmembrane helix structure.It was mainly localized in mitochondria,nucleus and cytoplasm,and had 17 specific phosphorylation sites. The secondary structure of MYL9 protein was mainly composed of alpha helix,random coil,beta turn and extended chain,and the predicted tertiary structure was consistent with the secondary structure.Real-time quantitative PCR results showed that MYL9 gene was expressed in heart,liver,spleen,lung,kidney,muscle,fat and testis,and the expression level in liver and muscle was significantly higher than that in other tissues (P＜0.05).【Conclusion】 The CDS region of MYL9 gene in Meiren yak was successfully cloned,and the biological function and tissue expression characteristics of MYL9 gene were explored.The results provided a reference for further research on the functional characteristics of MYL9 gene in yak.

【Objective】 The cloned individual of superfine-type Erlangshan cashmere goat was produced by somatic cell cloning technology,which provided a theoretical basis for the application of this technology in the expansion of local high-quality sheep breed resources and the breeding of new varieties.【Method】 A 4-year old Erlangshan cashmere goat with a cashmere fineness of 13.89 μm was selected.The ear tissue was collected and cultured as fibroblasts,which were used as the donor cells.Somatic cell cloning technology was used to expand the propagation of superfine-type Erlangshan cashmere goat.The feasibility of the technique was evaluated by weight,fineness of cashmere and reproductive performance of cloned sheep.【Result】 The stable system of goat oocyte maturation culture in vitro and somatic cell cloning was successfully established.The oocyte maturation rate was 44.8%,and the cleavage rate and blastocyst rate of parthenogenesis was 83.0% and 22.4%,respectively.The cleavage rate and blastocyst rate of the cloned embryos of Erlangshan cashmere goat was 77.8% and 13.7%,respectively.5 cloned sheep were obtained with normal growth and development,and 16 healthy F1 generation individuals were obtained by natural mating with Erlangshan ewes,with normal reproductive ability.The cashmere fineness of cloned individuals was significantly lower than that of control individuals of the same age (P＜0.01).【Conclusion】 Superfine-type Erlangshan cashmere goat individuals was successfully cloned by somatic cell cloning technology,which had normal production and reproductive performance,and retained the characteristics of superfine cashmere,laid a foundation for the expansion of native excellent sheep germplasm resources and the application of new varieties breeding.

【Objective】 The aim of the experiment was to study the effects of adding isoacids to periparturient diets on rumen microbiota,milk yield,milk fat rate and milk protein rate of dairy cows,and to provide a theoretical basis for the rational use of isoacids additives in dairy cows production.【Method】 18 periparturient Chinese Holstein cows with the 3rd lactation and similar production dates were randomly divided into 3 groups (6 replicates per group,1 cow per replicate),the cows in control group (group C) were fed a basal ration,and in experimental groups were added 30 (group A) and 60 mL/d of isoacids (group B),respectively,to the basal diet.The period of pre-feeding was 10 d,and the main test started from 21 d pre-partum to 21 d postpartum.Milk samples were collected from all cows at 10 and 21 d postpartum for determination of milk composition.Rumen fluid was collected from all cows using a rumen catheter before morning feeding at 21 d postpartum for determination of rumen fermentation parameters and microbial diversity.【Result】 ①Milk yield,milk fat rate and milk protein rate of cows in groups A and B were significantly higher than those in group C (P＜0.05).② Acetic acid and total volatile fatty acids (TVFA) were significantly higher in group B than that in group C (P＜0.05).③ The main dominant groups at the level of rumen microbiota of cows in the three groups were Bacteroidota and Firmicutes,and the relative abundance of Bacteroidota was significantly higher in group B than that in groups A and C (P＜0.05),whereas the relative abundance of Firmicutes was significantly lower than that in groups A and C (P＜0.05).The main dominant rumen microbial genera in three groups were Prevotella,Rikenellaceae_RC9_gut_group,Butyrivibrio and F082 unclassified taxa.The relative abundance of Butyrivibrio was significantly lower in groups A and B than that in group C (P＜0.05).④ Milk yield,milk fat rate and milk protein rate were significantly positively correlated with Bacteroidota and Prevotella (R＞0.5,P＜0.05),and significantly negatively correlated with Firmicutes,Spirochaetota and Butyrivibrio (R＜－0.5,P＜0.05).【Conclusion】 Adding isoacids to the ration of periparturient dairy cows could increase the VFA content in rumen and increase milk yield,milk fat rate and milk protein rate,in which the addition of 60 mL/d of isotonic acid was more advantageous for improving the rumen development and production performance of dairy cows.

【Objective】 A high performance liquid chromatography-triple quadrupole mass spectrometry (HPLC-MS/MS) method was developed for the determination of pentachlorophenol (PCP) in swine feed,feed additives and water.【Method】 The samples were extracted with aqueous alkaline methanolic solution,enriched and cleaned up using an anionic solid phase extraction column,reconstituted with 2% formate methanol after nitrogen drying.With 5 mmol/L ammonium acetate solution-methanol as mobile phase,Poroshell 120 EC-C18 (3 mm×100 mm,2.7 μm) column was used to separate PCP.Under the negative ionization,multiple reaction monitoring mode (MRM) was used to determine the content of PCP.Matrix-matched external quantification method was utilized to quantitate PCP.Linear relationship,limits of detection (LOD),limits of quantification (LOQ),recovery,accuracy,precision,matrix effect and other indicators of the established method were investigated.【Result】 The LOD and LOQ were 0.1 and 0.5 μg/kg in livestock feed,feed ingredients and feed additives,while 5 and 12.5 ng/L in farm water,respectively.The linear range of PCP in livestock feed,feed ingredients and feed additives was from 0.5 to 20 μg/kg,and the linear range of farm water was from 12.5 to 500 ng/L.The linear coefficient (R²) was more than 0.999,showing the good linearity.In addition,the recoveries ranged from 69.8% to 119.5% and the relative standard deviation (RSD) were from 1.62% to 9.86%.The matrix effect (ME) was 0.62-1.29.【Conclusion】 The method established in this study with high sensitivity,high accuracy and good robustness was suitable for the screening of PCP in swine formula feed and other farming inputs to ensure the quality and safety of meat products.

【Objective】 The purpose of this study was to explore the effects of high dose of rumen protected fat (RPF) on the growth performance, glucose dynamics and insulin resistance of sheep,so as to provide a scientific basis for the efficient utilization in ruminant nutrition.【Method】 A total of 160 3-months Hulunbuir sheep were selected and divided into 2 groups with 4 pens per group and 20 sheep per pen,which were named HF (23.65% RPF,16.34% corn) and CON (without RPF,36.95% corn) groups.The pre-feeding period of the feeding experiment was 14 days,and the experimental period was 42 days.The feed intake were recorded daily and the body weight of the sheep were measured every 21 days, and the average daily feed intake (ADFI), average daily gain (ADG) anf feed to gain ratio (F/G) were calculated. At the last day of the feeding period,4 sheep were randomly selected from different pens in each group,and they were subjected to intravenous glucose tolerance test (IVGTT),according to 0.25 g/kg BW,50% glucose solution was injected into the jugular vein.The serum from jugular vein blood was collected to measure the concentration of blood glucose and insulin,and the area under curve,maximum concentration,maximum concentration variation,insulin resistance index (HOMA-IR and Matsuda index) were calculated.【Result】 The ADFI and F/G of sheep in the HF group were significantly lower than those in CON group (P＜0.05).During the IVGTT,the serum glucose concentration of sheep in HF group at 2,10,30 min,and area under curve of glucose during the test were significantly lower than those in CON group (P＜0.05).The highest glucose concentration of sheep in HF group was obviously lower than that in CON group (P＜0.05).The insulin concentration at 10 and 30 min,and area under curve of insulin at 60 min in HF group were significantly lower than those in CON group (P＜0.05).The Matsuda index of HF group was obviously higher than that of CON group (P＜0.05).【Conclusion】 Adding high-concentration of RPF in the diet of growing sheep would not affect the growth performance of sheep, while could decrease the serum glucose concertration, and improve insulin sensitivity.

【Objective】 This experiment was to explore the optimal ratio of the mixed silage between distillers grains and navel orange residue by studying the quality and microbial diversity of the mixed silage.【Method】 The crude protein content of distillers grains used in the experiment was 15.05%,and the soluble carbohydrate content of navel orange residue was 24.29% in this study.Seven groups were set up according to a mixed silage proportions (10∶0,9∶1,7∶3,5∶5,3∶7,1∶9,0∶10) of distillers grains and navel orange residue,with 6 repetitions in each group,3 kg for each repetition.The nutritional composition,fermentation quality,and microbial diversity of the silage were measured after 60 days of silage.【Result】 The fermentation quality of the mixed silage of distillers grains and navel orange residue showed that the pH of each group was lower than 4.2.Among them,the pH of the 7∶3 and 5∶5 groups were the lowest and significantly lower than those of the 10∶0 and 0∶10 groups (P＜0.05),and the lactic acid content of the 7∶3 and 5∶5 groups were the highest and also significantly higher than these two groups (P＜0.05).The propionic acid content of the 9∶1 and 7∶3 groups were significantly lower than that of the 0∶10 group (P＜0.05),and the butyric acid of which were not detected.Starting from the ratio of mixed silage reaching 7∶3,the ammonia nitrogen content of silage in each group were significantly lower than that of the 10∶0 group (P＜0.05).The measurement results of microbial abundance showed that the total content of five Lactobacillus,including Lactiplantibacillus,Companilactobacillus,Lactobacillus,Limosilactobacillus,Levilactobacillus,in the 7∶3 and 5∶5 groups were significantly higher than that in the 10∶0 group (P＜0.05).The Bacillus abundance of the 7∶3 group was significantly higher than that of the 0∶10 group (P＜0.05).The Pseudomonas abundance of the 9∶1,7∶3,5∶5 and 3∶7 groups were significantly lower than that of the 0∶10 group (P＜0.05).【Conclusion】 While the mixed silage proportion for silage of distillers grains and navel orange residue was 7∶3,the fermentation quality and microbial diversity of the silage were better,which was the most appropriate proportion for mixed silage.

In recent years,with the attention paid to the function of microorganisms,people’s research on intestinal microbes is also increasing.In the gut of ruminants,there are a large number of microorganisms,which play a very important role in the host’s nutritional metabolism,immune function,etc.,and are one of the key factors affecting body health.Intestinal microbes is affected by diet composition,age,genotype and other factors of ruminants,and dietary composition is the most important factor affecting intestinal microbes.If the diet changes,the crude fiber,protein,carbohydrate and other nutrients will change,and the intestinal microbes will also change.There are beneficial microorganisms in the body of ruminants,which have a positive effect on the animal body,such as Ruminococcus,Micrococcus luteus,Enterococcus bovis,etc.,while some harmful bacteria (such as Clostridium,Zurichbacterium,etc.) destroy the homeostasis of the internal environment of ruminants,reduce the immunity and disease resistance of the body,and are prone to diseases,which seriously affect the health of ruminants.In addition to intestinal microbes affecting the body health of ruminants,probiotics and nutrients also play a regulatory role in the body health of ruminants,and the general measures are to add beneficial probiotics or nutrient diets to the diets of ruminants,which can not only supplement the daily nutrition level,but also prevent the proliferation of harmful microorganisms,so as to ensure the health of ruminants.The authors review the factors affecting the intestinal microbes of ruminants and the effects of probiotics and nutrients on the healthy production of ruminants,aiming to provide a theoretical reference for the reasonable regulation of the intestinal microbes of ruminants and its healthy development,so as to promote the development of animal husbandry.

The lesion rate of woody breast is as high as 20% in fast-growing White feather broiler,it seriously reduces consumers’ desire to buy,and is not conducive to post-processing,resulting in relatively high economic losses.Hypoxia is an important cause of woody breast,and hypoxia inducible factor 1α (HIF-1α) is a key factor in regulating cell function under hypoxia.Ursolic acid has the effects of anti-oxidation,anti-inflammatory and regulating glucose metabolism,and can regulate the expression of HIF-1α through adenylate-activated protein kinase (AMPK) and phosphatidylinositol kinase-3 kinase-protein kinase B (PI3K/Akt) signaling pathways.Therefore,the author reviews the pathogenesis of woody breast caused by hypoxia and how HIF-1α mediated glucose metabolism,oxidative stress and inflammatory responses are regulated by ursolic acid,and further analyzes the potential mechanism of ursolic acid regulating HIF-1α to alleviate the occurrence of woody breast through AMPK,PI3K/Akt and other signaling pathways.

【Objective】 The purpose of this study was to explore the effects of dandelion and Forsythia extracts on production performance,serum antioxidant and immune indexes of dairy cows with subclinical mastitis,in order to provide theoretical basis for the development and application of green feed additives based on plant extract.【Method】 18 multiparous Holstein cows with similar body condition,lactation period,milk production,and the mixed milk sample with somatic cell count (SCC) in milk ＞5×10⁵/mL were selected.According to the principle of similar SCC,milk samples were divided into control group (CON group),dandelion group (DAN group) and Forsythia group (FOR group).The cows in CON group were fed a total mixed diet (TMR),the cows in DAN group were fed TMR＋dandelion extract,and the cows in FOR group were fed TMR＋Forsythia extract.None of the cows received any drug treatment within 1 week before the experiment and the experiment lasted for 45 days.Milk samples were collected every 7 days to determine milk composition and SCC.After the experiment,milk samples were collected to determine the activities of bovine myeloperoxidase (MPO),lactate dehydrogenase (LDH) and N-acetyl-β-D-glucosaminase (NAG).Blood samples were collected and serum antioxidant and immune indexs were measured.【Result】 Compared with the CON group,① Dietary supplementation of dandelion and Forsythia extract had no significant effect on milk production and milk components of dairy cows with subclinical mastitis (P＞0.05).② The LDH activity in milk of DAN and FOR groups was significantly decreased (P＜0.05).NAG activity in DAN group was significantly decreased (P＜0.05).③ The catalase (CAT) activity in the serum of dairy cows with subclinical mastitis in DAN group was significantly increased (P＜0.05),and the total antioxidant capacity (T-AOC) in FOR group was significantly increased (P＜0.05).Both herbal extracts could significantly reduce the serum malondialdehyde (MDA) content of dairy cows with subclinical mastitis (P＜0.05).④ The contents of IgA,IgG and IgM in serum of dairy cows with subclinical mastitis in DAN and FOR groups were significantly increased (P＜0.05),and the IL-1β content in serum was significantly reduced (P＜0.05).【Conclusion】 Dietary supplementation of dandelion and Forsythia extracts could regulate the inflammatory response,enhance the immune and antioxidant capacity of dairy cows with subclinical mastitis,and thus achieve the prevention and control effect of subclinical mastitis in dairy cows.

As the scale of pig farming continues to expand,the sustained growth of meat products is driving the rising demand for animal feed ingredients,especially protein-based feed ingredients.The production of traditional protein feedstocks,including soybean meal and fish meal,requires a lot of natural resources.As a kind of nutrient-rich edible insect,the Hermetia illucens L. larvae is rich in protein,fat and calcium,and the amino acid composition is balanced,rich in glutamate,lysine and valine and other amino acids,which can be used in pig diets instead of protein feed such as soybean meal and fish meal.Hermetia illucens L.larvae show great potential as feed ingredients in pig rearing,which can improve production performance,reduce feed costs,and are environmentally friendly at the same time.The author reviewed the nutritional characteristics of Hermetia illucens L.larvae and its effects on the performance and health of pigs as feed ingredient,so as to provide reference for the application of Hermetia illucens L.as feed materials in pig breeding in the future and provide new ideas for promoting the sustainable development of pig breeding.

Bacillus subtilis is an ideal probiotic species that can form spores with a variety of physiological functions and is non-toxic and has no side effects on animal organisms.Adding Bacillus subtilis to feed can regulate intestinal function,balance microbial flora,enhance immunity,and promote growth.Bacillus subtilis can also be used as a feed fermentation strain.The fermented feed will reduce the content of anti-nutritional factors and increase the content of nutrients,thereby promoting the digestion and absorption of feed nutrients by livestock and poultry.The characteristics of Bacillus subtilis endophytic spores can ensure its functional stability in feed production and livestock and poultry internal environment and its unique advantages as a vaccine expression vector.Therefore,Bacillus subtilis has a very broad application prospect in livestock and poultry production.The authors reviews the biological characteristics,physiological functions,and application of Bacillus subtilis in livestock and poultry production,in order to provide some reference for the application of Bacillus subtilis in livestock and poultry production.

【Objective】 The purpose of this experiment was to evaluate the nutritional value of common fungus bran in Jilin province by in vitro gas production experiment and the ruminal degradation rate at 48 h experiment.【Method】 The single factor experiment design was used,and different fungus bran (Pleurotus citrinopileatus(Y1),Lentinula edodes(X1),Pleurotus ostreatus(P),Auricularia auricula(M), Morchella esculenta(Y2),Hohenbuehelia serotina(Y3),Pleurotus eryngii (X2) and Pleurotus nebrodensis (B)) were used as fermentation substrates.The nutritional value of fungus bran was comprehensively analyzed according to gas production,gas production parameters,fermentation parameters and the ruminal degradation rate of nutrient at 48 h,combined with membership function.【Result】 ①At the end of the 48 h fermentation,the gas production of Lentinula edodes fungus bran was not significantly different from Pleurotus nebrodensis and Pleurotus ostreatus fungus bran (P＞0.05),and was significantly higher than other groups (P＜0.05).②The theoretical maximum gas production of Auricularia auricula and Morchella esculenta fungus bran was significantly lower than that of other fungus bran groups (P＜0.05),the gas production rate of fungus bran was not significantly different among all groups (P＞0.05).③The ammoniacal nitrogen (NH₃-N) contents of Lentinula edodes,Pleurotus citrinopileatus,Pleurotus nebrodensis and Pleurotus ostreatus fungus bran were significantly lower than the other groups (P＜0.05),the total volatile fatty acids (TVFA) and acetic acid contents of Lentinula edodes and Pleurotus ostreatus fungus bran were significantly higher than the other groups (P＜0.05),the propionic acid content of Lentinula edodes fungus bran was not significantly different from that of Pleurotus ostreatus,Hohenbuehelia serotina and Pleurotus eryngii fungus bran (P＞0.05),but was significantly higher than the other groups (P＜0.05).④The ruminal degradation rates at 48 h of dry matter in Lentinula edodes and Pleurotus ostreatus fungus bran were significantly higher than the other groups (P＜0.05),the crude protein ruminal degradation rates at 48 h of Pleurotus ostreatus,Pleurotus eryngii,Pleurotus nebrodensis fungus bran were not significantly different from Lentinula edodes fungus bran (P＞0.05),but were significantly higher than the other groups (P＜0.05).The neutral detergent fiber ruminal degradation rate at 48 h and the acid detergent fiber ruminal degradation rate at 48 h of each fungus bran group was relatively low.⑤According to the analysis of membership function,the comprehensive value of Lentinula edodes fungus bran＞ Pleurotus ostreatus fungus bran＞ Pleurotus nebrodensis fungus bran＞ Pleurotus eryngii fungus bran＞ Pleurotus citrinopileatus fungus bran＞ Hohenbuehelia serotina fungus bran＞ Morchella esculenta fungus bran＞ Auricularia auricula fungus bran.【Conclusion】 There were significant differences in the in vitro gas fermentation effects of various types of fungus bran in Jilin province.Except for the Auricularia auricula and Morchella esculenta fungus bran with low comprehensive scores,other fungus bran had the potential to be used as unconventional feed resources.

【Objective】 The aim of this study was to study the effects of grape pomace on lymphocyte subsets,lymphocyte conversion rate,apoptosis rate and antioxidant activity in peripheral blood of cows.【Method】 Twenty healthy postpartum beef cows with similar parity and body weight were randomly divided into 4 groups with 5 cows in each group.The beef cows in control group were fed a basal diet,and 240,300 and 360 g/d grape dregs were added to the basal diet per head in trial groups I,Ⅱ and Ⅲ,respectively.The pre-trial period lasted for 15 days,and the experimental period lasted for 40 days.The tail vein blood was collected and the lymphocyte subsets,the conversion rate and apoptosis rate of lymphocytes and the antioxidant level of peripheral blood were measured.【Result】 ①On the 20th day of the experiment,the proportion of CD4⁺T cells in peripheral blood of beef cows in all three experimental groups were significantly increased compared to the control group (P＜0.05).On the 40th day of the experiment,the proportion of CD4⁺T cells in groups Ⅱ and Ⅲ were significantly increased compared to the control group (P＜0.05),while the proportion of CD8⁺T cells were significantly reduced compared to the control group (P＜0.05).The CD4⁺/CD8⁺ ratio in all three experimental groups were significantly increased compared to the control group (P＜0.05).②On the 20th and 40th day of the experiment,the peripheral blood lymphocyte transformation rates of cows in groups Ⅱ and Ⅲ were significantly higher than those in the control group (P＜0.01).On the 40th day of the experiment,as the amount of grape residue added increased,the apoptosis rate of peripheral blood lymphocytes showed a linear and extremely significant decrease (P＜0.01).③On the 40th day of the experiment,the serum MDA content of three experimental groups of beef cows were significantly reduced compared to the control group (P＜0.05).The serum T-AOC content of beef cows in experimental groups Ⅰ and Ⅲ were significantly higher than that of the control group (P＜0.01).【Conclusion】 Adding different proportions of grape pomace to the diet of beef cows could improve their immune function.Under the experimental conditions,the appropriate level of grape pomace addition was (300-360) g/d.

【Objective】 The purpose of this experiment was to establish the correlation between boar semen quality and sperm lipid composition,providing support for accurate evaluation of boar semen quality.【Method】 In this study,semen samples were collected from representative breeds of boars,such as Yorkshire pigs and Landrace pigs.The computer-assisted sperm analysis (CASA) system was used to measure semen quality parameters,including sperm density,sperm motility,sperm abnormality rate,and semen volume.The semen samples were classified as two groups based on sperm motility,including normal motility (≥70%,n=11) and low motility (＜70%,n=11).Unbiased lipidomics analysis of the two groups of sperm samples was performed using UPLC-Q-Exactive-Orbitrap/MS multivariate statistical analysis methods,including t tests,partial least squares discriminant analysis (PLS-DA),and correlation analysis,were used to analyze the data of lipidomics.The difference of lipid molecules between the two groups was obtained according to VIP＞1 and P＜0.05.【Result】 A total of 166 differential lipid molecules identified in both positive and negative ion modes,which mainly concentrated in phosphatidylcholine (PC).Lipid molecular species,including diglyceride(DG),sphingomyelin(SM),PC,Zymosterol Ester (ZyE),and simple Glc series (Hex3Cer),showed significant decrease in low motility sperm and were significantly positively correlated with sperm motility (P＜0.05).Triglyceride (TG) and phosphatidylcholine ester (PC(e)) showed significant increase in low motility sperm and were significantly negatively correlated with sperm motility(P＜0.05).【Conclusion】 Lipidomics could accurately distinguish the differences in lipid subpopulations and lipid molecular composition among different motile sperm.The composition and content of lipids such as PC,DG,SM,PC,ZyE,Hex3Cer,TG,and PC(e) could be used as markers to distinguish sperm motility.

【Objective】 The aim of this study was to investigate the polymorphism of glycerol-3-phosphate acyltransferase 3 (GPAT3) gene and its association with growth traits in Jiangquan Black pigs,so as to provide theoretical and technical support for genetic improvement and optimization of the breeding program in Jiangquan Black pigs.【Method】 The ear tissues of 292 Jiangquan Black pigs were collected and subjected to DNA extraction.The GPAT3 gene fragment was amplified using PCR,followed by detection of single nucleotide polymorphism (SNP) and genotyping through Sanger sequencing and MassARRAY^ nucleic acid mass spectrometry.The genetic effects,linkage disequilibrium and haplotype analysis of GPAT3 gene SNP were analyzed using R 3.6.2,HaploView and PHASE softwares.The association between GPAT3 gene SNP and its LD block with growth traits in Jiangquan Black pigs was assessed using SAS 9.2 software.【Result】 A total of 10 SNPs were found in GPAT3 gene and its 5'-regulatory region,of which 4 were low polymorphisms and 6 were moderate polymorphisms.The genotype frequency distribution of 7 SNPs was in Hardy-Weinberg equilibrium.Association analysis found that 9 SNPs were significantly associated with growth traits in Jiangquan Black pigs (P＜0.05),where g.134957929 G＞A,g.134957930 G＞T,g.134958013 G＞A and g.134976983 T＞C had a strong linkage,and the construction of AGGT/AGGT diplotype had significant advantages in chest circumference and body height (P＜0.05),GTGC/GTGC diplotype and GTGC haplotype had significant advantages in body length (P＜0.05).g.135002556 C＞ A,g.135002627 C＞T and g.135002680 C＞T had a strong linkage,and the construction of ATC/ATC diplotype had significant advantages in chest and abdominal circumference (P＜0.05),CCC/ATC diplotype had significant advantage in backfat thickness (P＜0.05),and ATT haplotype had significant advantage in abdominal circumference (P<0.05).【Conclusion】 There were abundant polymorphic loci of GPAT3 gene in Jiangquan Black pigs,9 SNPs and 2 haplotype blocks had significant correlation with the growth traits of chest circumference,abdominal circumference and body height,which could be used as molecular genetic markers for growth traits breeding in Jiangquan Black pigs.

Muscle fibers are the basic units that form into muscle tissue.The muscle fiber types are closely related to meat quality,and commonly altered by external stimuli such as body growth and development,nutrition,and feeding environment.In recent years,studies on the formation and conversion patterns of poultry muscle fiber types have become the hotpot due to the attention given by consumers and breeders to meat quality.In addition,with the development of omics research technology,transcriptomics,proteomics and other technologies have been widely applied in the regulatory mechanism study of poultry muscle fiber types,which has greatly promoted relevant research progress.To fully understand the current research on muscle fiber type transformation is an important theoretical basis for the subsequent research on related meat quality and regulatory mechanism.The author introduces the composition of muscle fiber and the classification methods of different types of poultry muscle fiber.Additionally,the associations between the muscle fiber type and poultry different meat qualities such as color,pH,water binding capacity and tenderness traits are briefly described.This paper also summarizes the effects of growth,breed,environment,and feeding procedures on muscle fiber type,and focuses on the existing advances in molecular regulation studies,aiming to provide basic theoretical support and reference for future research.Finally,the future breeding direction of high-quality broilers is proposed.It is hoped that with the development of modern molecular biotechnology,cutting-edge technologies such as single cell/space omics technology,gene editing technology based on CRISPR/ Cas9 system can be widely used in the field of muscle fiber type research,constantly improve the mechanism of muscle fiber type conversion and establish the molecular breeding method of meat quality traits in poultry.

【Objective】 The aim of this study was to investigate the effects of postmortem aging on the quality of pork,as well as the correlation between gene expression patterns during postmortem aging and pork quality changes,so as to provide a better understanding of the mechanisms underlying the changes in meat quality during the aging process.【Method】 The meat quality of longissimus dorsi muscle in Large White pigs stored at 4 ℃ was determined after 0,1,2,4,6,8 and 10 days of storage.The expression of adenosine monophosphate deaminase 3 (AMPD3),myosin binding protein-C 2 (MYBPC2),myosin light chain 3 (MYL3),solute carrier family 7,member 8 (SLC7A8) and tropomyosin 3 (TPM3) genes in longissimus dorsi muscle were determined by Real-time quantitative PCR,and the correlation with meat quality indexes were analyzed.【Result】 The results showed that the pH of longissimus dorsi muscle in Large White pigs reached a peak value of 6.5 at 2 days,which was significantly lower than 4 and 10 days (P＜0.05).Both cooking loss and drip loss showed an initial increase followed by a decrease trend,while the purge loss reached its highest value of 12.52% at 10 days,which was significantly higher than 0.5-6 days (P＜0.05).The shear force value at 1-10 days were significantly lower than that of 0-1 days (P＜0.05).The thiobarbituric acid (TBARS) value gradually increased with the prolonged aging time,and was significantly higher than other time points at 8-10 days (P＜0.05).Real-time quantitative PCR results revealed that gene transcription abundance in postmortem muscle tissue was still exist.The expression of AMPD3 gene at 6 days of storage was significantly higher than 0,0.5,2 and 10 days (P＜0.05).The expression of MYBPC2,MYL3 and TPM3 genes showed a time-dependent upregulation from 0 to 10 days,the expression of gene at 10 days of storage was significantly higher than 0-2 days (P＜0.05).The expression of SLC7A8 gene peaked at 2 days of storage and was significantly higher than other time points (P＜0.05).Correlation analysis indicated that there was no correlation between the expression of AMPD3 gene and meat quality (P＞0.05).The expression of MYBPC2 gene was significantly positively correlated with TBARS value and purge loss (P＜0.05).The expression of MYL3 gene was extremely significantly or significantly positively correlated with TBARS value,shear force,dripping loss and purge loss (P＜0.01 or P＜0.05),and was extremely significantly negatively correlated with cooking loss (P＜0.01).The expression of SLC7A8 gene was significantly negatively correlated with pH (P＜0.05).The expression of TPM3 gene was significantly or extremely significantly positively correlated with TBARS value,dripping loss and purge loss (P＜0.05 or P＜0.01),and was significantly negatively correlated with cooking loss (P＜0.05).【Conclusion】 This study proved that the changes of gene expression during postmortem pork aging were correlated with the changes of meat quality,the difference of gene expression affected different metabolic types and physiological functions of animals after slaughter,and thus affected meat quality.

The hypoxia environment of the plateau is a major survival challenge for organisms at high altitudes areas.The genetic basis of high altitude adaptation of animals is complex,and relevant studies mainly focus on comparative genome,population genome and evolutionary genome,and positive selection genes are mainly reflected in the hypoxic inducible factor (HIF) regulatory pathway.At present,more and more attention has been paid to the epigenetic mechanism of animal plateau adaptation.Epigenetics can regulate the plasticity and stability of gene expression,and affect cell function and the ability of organisms to adapt to environmental changes.Studies have shown that plateau organisms have experienced long-term hypoxic adaptation,and show adaptive regulation related to epigenetics.DNA methylation and histone modification are important modifications in epigenetics,which are closely related to altitude adaptation and affect cell function by regulating gene expression levels.HIF is a key transcription factor complex in hypoxic environment,and its stability and activity are regulated by epigenetics.DNA methylation and histone modification directly affect the function of HIF signaling pathway,while epigenetic mechanisms such as non-coding RNA and cytochrome P450 (CYP450) enzyme also participate in the regulation of HIF signaling pathway.It is of great significance to deeply understand the relationship between adaptation to high altitude hypoxia environment and epigenetics and the epigenetic mechanism of HIF signaling pathway for revealing the mechanism of adaptive evolution and related diseases.The authors summarized the relationship between high altitude hypoxic environment adaptation and epigenetics,and focused on the mechanism of HIF signaling pathway in epigenetic regulation.

【Objective】 The aim of this study was to investigate the influence of polymorphism in progestogen associated endometrial protein (PAEP) gene on reproductive traits in Kele pigs,with the objective of identifying genetic markers that could enhance their reproductive traits.【Method】 A total of 189 multiparous Kele pigs were used as experimental animals,the single nucleotide polymorphism (SNP) were identified in PAEP gene through PCR product sequencing and sequence alignment.The SHEsis online software was utilized to analyze the population genetic characteristics of SNP.Bioinformatics analysis of SNP was carried out using RNAfold,SOPMA,SWISS-MODEL and SWISS-PdbViewer programs.The SPSS 22.0 software was employed to analyze the correlation between SNP of PAEP gene and reproductive traits in Kele pigs.【Result】 Three SNP sites were identified in PAEP gene of Kele pigs,a missense mutation g.1884992 T＞C in exon 4,resulting in the change of valine (V) at position 135 to alanine (A),as well as mutations in intron 4:g.1885152 G＞C and g.1887834 G＞A.All three SNPs displayed three genotypes.g.1884992 T＞C was low polymorphism (PIC＜0.25),while g.1885152 G＞C and g.1887834 G＞A were moderate polymorphism (0.25＜PIC＜0.5).The genotype distribution of g.1884992 T＞C did not deviate from Hardy-Weinberg equilibrium (P＞0.05),while the genotype distribution of g.1885152 G＞C and g.1887834 G＞A were extremely significantly deviated from Hardy-Weinberg equilibrium (P＜0.01).Linkage analysis revealed that there was complete linkage between g.1885152 G＞C and g.1887834 G＞A,resulting in the generation of 3 haplotypes and 6 diplotypes.g.1884992 T＞C resulted in the changes of the secondary structure in mRNA and protein of PAEP gene.The association analysis showed that g.1884992 T＞C was significantly affected total litter size and total number born in Kele pigs (P＜0.05),while g.1885152 G＞C and g.1887834 G＞A were significantly affected total litter size,total number born,number of weaned and weaning litter weight in Kele pigs (P＜0.05).The total litter size,total number born,number of weaned and weaning litter weight of H3H3 diplotype in Kele pigs were significantly higher than those of other 5 diplotypes (P＜0.05),and the birth weight of H3H3 diplotype in Kele pigs was significantly higher than that of H1H1,H2H2 and H2H3 diplotypes (P＜0.05).The total number born and number of weaned of H1H3 diplotype were significantly higher than those of H1H1,H1H2,H2H2 and H2H3 diplotypes (P＜0.05),and the total number born of H1H3 diplotype in Kele pigs was significantly higher than that of H2H3 diplotype (P＜0.05).【Conclusion】 Three SNPs found in PAEP gene significantly impacted the reproductive traits in Kele pigs.H3H3 was advantageous diplotype,and might serve as a genetic marker for the selection of reproductive traits in Kele pigs.

【Objective】 The aim of this experiment was to compare the slaughter performance and meat nutritional composition of different genders of wild-blooded yaks in Gaize county of Ngari prefecture of Tibet.【Method】 Ten wild-blooded yaks in good condition and grazing naturally (half males and half females,aged 5-6 years,body weight 299.00 kg±30.50 kg and 247.00 kg±10.20 kg,respectively),were randomly selected from Gaize county of Ngari prefecture.Slaughter trials were conducted to study the slaughter performance,carcass traits and meat nutritional quality characteristics of wild-blooded yaks.【Result】 The pre-slaughter live weight,carcass weight,net meat weight,bone weight and tube circumference of male yaks with wild-blooded in Ngari prefecture were significantly or extremely significantly higher than those of female yaks (P＜0.05 or P＜0.01),while other slaughter and body condition indicators were not significantly different (P＞0.05).The carcass length,carcass width,carcass depth,carcass breast depth,hind leg circumference,thigh meat thickness and eye muscle area of male yaks were significantly or extremely significantly higher than those of female yaks (P＜0.05 or P＜0.01),while backfat thickness of female yaks was extremely significantly higher than that of male yaks (P＜0.01).The moisture content of male yak beef was significantly higher than that of female yaks (P＜0.05),the intramuscular fat content of female yaks was extremely significantly higher than that of male yaks (P＜0.01),there was no significant difference in protein,cholesterol,minerals,vitamin content and amino acid composition between the different genders (P＞0.05).And the amino acid ratio was rational for high quality protein source.In terms of fatty acid composition,C15∶0,C16∶0,C16∶1,C17∶0,C18∶0,C18∶1n9c,C20∶0,SFA,MUFA and TFA in female yak beef were significantly or extremely significantly higher than those in male yak beef (P＜0.05 or P＜0.01),and PUFA/SFA in male yak beef were significantly higher than that in female yak beef (P＜0.05).【Conclusion】 Gender had a certain influence on the slaughter performance and meat quality of wild-blooded yak in Ngari prefecture.The carcass traits of male yak was more prominent.The high intramuscular fat,high-quality amino acid composition and rich UFA content of the longissimus dorsi muscle of female yak made its meat quality and nutritional value slightly better than that of male yak,which provided basic data for the targeted breeding of new breeds of wild-blooded yak and the development of domestic yak purification and breeding innovation.

The gut and reproductive tracts of hens are colonized by vast and complex microbial communities.The dynamic processes of this microbiome development are closely associated with different developmental stages of the host,which play significant roles in the host’s immune function,nutrient absorption,endocrine regulation,reproduction and hatching processes,as well as egg quality.The balance of gut and reproductive tract microorganisms is crucial for the health and reproductive performance of hens,and chicks may be influenced by the gut and reproductive tract microorganisms transmitted by hens during hatching.However,the current research on the mechanism of the impact of gut and reproductive tract microorganisms on reproduction and hatching in hens is not yet in-depth and systematic.The author mainly introduces the dominant microorganisms in the gut and reproductive tracts of hens at different stages,as well as the colonization patterns of microorganisms in different segments of the gut and reproductive tracts.At the same time,the relationship between gut and reproductive tract microorganisms,and the impact of gut and reproductive tract microorganisms on egg quality are also discussed.And the impact of microorganisms on the reproductive and hatching process,the pathways of microbial transmission between parents and offspring,the microbial community structure at different developmental stages of chicken embryos,and the relevant factors affecting the hatching process are explained.The aim of this article is to identify the core microbial communities of hens that regulate the reproductive process and affect hatching rate as a unique identifier for selecting high hatching rate breeders,to gain a deeper understanding of the functional structure of gut and reproductive tract microorganisms in poultry and provide insights into improving poultry production and reproductive efficiency.

【Objective】 The purpose of this study was to identify the number of unannotated transcripts,splicing types,new protein coding and molecular structure of genes response to viral infection in porcine PK15 cells after polyI:C stimulation,which would lay a foundation on the function of these unannotated transcripts for further study.【Method】 Porcine PK15 cells were divided into control group and experimental group,with 3 replicates in each group.The experimental group was added with PolyI:C at a final concentration of 20 μg/mL,and the control group was added with an equal volume (2 μL) of PBS.The two groups were stimulated at 37 ℃ and 5% CO₂ for 6 h,respectively.After then,Nanopore sequencing was performed to identify the total transcripts and differentially expressed genes of the two groups.GO function analysis was performed on the differentially expressed genes to further screen the genes response to viral infection.The total transcripts were compared with the Ensemble annotated transcript sequences,and unannotated transcripts were identified.The unannotated transcripts genes response to viral infection were compared with their corresponding Ensemble annotated transcripts,and the splicing types and encoded proteins of unannotated transcripts were analyzed.【Result】 After PolyI:C stimulation,a total of 61 505 protein-coding transcripts were identified in the two groups,of which 39 497 were annotated in the Ensemble database,and 22 008 were unannotated transcripts,accounting for 35.78% of the total.At the same time,71 differential protein-coding genes were identified in the two groups.Compared with control group,57 genes were up-regulated and 14 genes were down-regulated in experimental group.GO functional enrichment analysis showed that these differentially expressed genes were enriched into 20 biological processes,of which the first three most enriched biological processes were virus defense response,type Ⅰ interferon signaling pathway and response related to virus.Among the 24 genes response to viral infection,16 genes had unannotated transcripts.Among them,the number of unannotated transcripts of CCL5,IFI6,BST2 and MX1 genes was more than the total number of transcripts annotated by Ensemble,and most of the unannotated transcripts produced new protein sequences.Unannotated transcripts of 9 genes including IFIT3,OAS2,RSAD2,CCL5,IFI44,CD40,IFI6,BST and MX1 were differentially expressed.【Conclusion】 This study systematically identified the molecular characteristics of unannotated transcripts of genes response to viral infection in porcine PK15 cells stimulated by PolyI:C.The differentially expressed unannotated transcripts of 9 genes,including IFIT3,OAS2,RSAD2,CCL5,IFI44, CD40,IFI6,BST and MX1 might play an important biological role,which provided a basis for further analysis of the complex transcriptional regulation mechanism of host genes in antiviral response.

【Objective】 The aim of this experiment was to analyze the transcriptional differences of immune key genes and signaling pathway molecules in host’s bursa of Fabricius after in-ovo immunization with Newcastle disease virus TS09-C strain in chicken embryos using transcriptomics,and explore the immune related molecular regulatory responses in the host’s bursa of Fabricius after immunization.【Method】 Sixty 18 embryo SPF chicken embryos were randomly divided into immunization group and control group,with 30 embryos per group.The immunization group inoculated NDV TS09-C strain into chicken embryos through the amniotic cavity,the dose was 10^3.0 EID₅₀/piece and the injection volume was 0.1 mL,while the control group was inoculated with equal volume PBS using the same method.The viral load of lung,thymus,spleen and bursa of Fabricius tissue was detected at different time points after immunization.The bursa of Fabricius tissue with the highest viral load was selected for transcriptome sequencing and analysis.Immune related differentially expressed genes were screened and the main functions and pathways they involved were analyzed.【Result】 The virus could be detected in bursa of Fabricius tissue 1 day after immunization,and the viral load reached the highest level 3 days after immunization.Transcriptome analysis was performed on the bursa of Fabricius 3 days after immunization.Compared with control group,there were 453 up-regulated genes and 98 down-regulated genes in immunization group,including 36 up-regulated genes and 10 down-regulated genes related to immunity.Immune related differentially expressed genes were mainly enriched in the immune related pathways of autophagy and cytokine-cytokine receptor interactions.Differentially expressed gene protein-protein interaction analysis showed that BCL2 enriched in the autophagy pathway interacted with other immune related genes,and was involved in functional annotation such as B cell activation,differentiation,and immune system development.【Conclusion】 The results of this study revealed the potential target genes and pathways regulating the activation and differentiation of B cells in bursa of Fabricius of chicken embryo immunized by NDV TS09-C strain,and provided new information for further study of the molecular mechanism of B lymphocyte regulation after in-ovo vaccination by NDV TS09-C strain.

【Objective】 The experiment was aimed to understand the prevalence of Swine influenza virus (SIV) in Guangdong and explore its molecular biological characteristics.【Method】 Nasal swab and lung tissue samples of pigs suspected to be infected with Swine influenza virus were collected from a pig farm in Guangdong for virus isolation and identification,genetic evolution and key amino acid site analysis.【Result】 The samples were tested positive for Swine influenza virus nucleic acid by Real-time quantitative RT-PCR.In the red blood cell agglutination test,the virus exhibited agglutination with chicken red blood cells,with a hemagglutination titer of 1∶128.The results of sequencing analysis of eight gene segments were analyzed by BLAST comparison and construction of evolutionary trees,and HA and NA genes belonged to the Eurasian avian-like Swine influenza virus (H1N1) branch,PA,PB1,PB2,NP and M genes belonged to the pdm/09 branch,and NS gene belonged to the North American triple reassortant branch,therefore,the isolate in this study belonged to the G4 genotype Eurasian avian-like Swine influenza virus,and was named A/swine/Guangdong/CJM2/2022 (H1N1).Key amino acid site analysis showed that the cleavage site sequence of HA protein of the isolate was PSIQSR/GL,which had the molecular characteristics of a typical low pathogenicity influenza virus.The amino acids at position 190,225 and 226 of the receptor binding site of HA gene were D,E and Q,respectively,which suggested that it had the potential ability to bind to the human salivary acid receptor and it also had the potential to bind avian sialic acid receptors.None of the amino acid residues of NA gene were mutated,suggesting that the isolate was sensitive to neuraminidase inhibitors such as oseltamivir and zanamivir.However,the mutations at three amino acid sites in M gene were V27A,A30T and D44A,suggesting an increased resistance to amantadine-based drugs.【Conclusion】 The strain isolated and identified in this study was a G4 Eurasian avian-like Swine influenza virus.The analysis of key amino acid sites suggested that this strain had the characteristics of adapting to replication and enhancing virulence in mammals.The results of this study provided reference data for the prevention and control of swine influenza in Guangdong.

【Objective】 The aim of this study was to verify the joint regulatory relationship between DNA methylation and mRNA expression of candidate genes in the brain tissue of piglets infected by Glaesserella parasuis (GPS),in order to provide a theoretical basis for revealing the apparent pathogenic mechanism of meningitis caused by GPS in piglets.【Method】 Six 28-day-old weaned piglets were randomly divided into control and GPS groups.The piglets in GPS group were intraperitoneally injected with 1 mL of 2×10⁹ CFU/mL GPS SH0165 solution,and the piglets in control group were intraperitoneally injected with the same amount of normal saline,and the brain of the piglets were collected 7 days later to extract DNA and RNA.The mRNA expression of 4 candidate genes (LYPD1,PITPNM1,SYP and ACVR1B) were detected by Real-time quantitative PCR,and bisulfite sequencing PCR (BSP) and methylation-specific PCR (MSP) were used to detect the DNA methylation changes of 4 genes in brain tissues of piglets before and after GPS infection.【Result】 This study successfully applied the MSP method to DNA methylation sequencing of genes,making it not limited to fixed-point detection of methylation sites.The results of Real-time quantitative PCR showed that compared with control group,the mRNA expression of LYPD1,PITPNM1,SYP and ACVR1B genes in GPS group were significantly or extremely significantly down-regulated (P＜0.05 or P＜0.01).The results of DNA methylation sequencing showed that the DNA methylation level of 4 genes were up-regulated except for ACVR1B gene.【Conclusion】 Except for ACVR1B gene,the DNA methylation of LYPD1,PITPNM1 and SYP genes was inversely associated with mRNA expression,and the DNA methylation changes in brain of piglet after GPS infection had different regulatory patterns for the expression of 4 candidate genes.

【Objective】 The extracellular domain (N-terminal fragment,CD2v-N) and intracellular domain (C-terminal fragment,CD2v-C) of African swine fever virus (ASFV) CD2v protein were expressed by Escherichia coli expression system,and the antigenicity of the two proteins was analyzed,so as to provide experimental basis and guidance for the development of ASFV detection methods.【Method】 Recombinant prokaryotic expression plasmids of CD2v-N and CD2v-C were constructed,the CD2v-N and CD2v-C proteins were expressed and purified in vitro,and the expressed proteins were identified by Western blotting.New Zealand White rabbits were inoculated with the expressed CD2v-N and CD2v-C proteins intramuscularly for three times,each time at an interval of 2 weeks 14 d after each immunization,the specific antibody levels of CD2v-N or CD2v-C were determined by ELISA.The purified CD2V-N and CD2v-C proteins were then used as coated antigens to detect CD2v-N or CD2v-C specific antibodies in 95 sera of ASFV-infected pigs by ELISA,and the levels of immune response induced by CD2v-N or CD2v-C in pigs were compared.【Result】 Recombinant proteins CD2v-N and CD2v-C were expressed in inclusion bodies and soluble forms,respectively,with relative molecular masses of 22.9 and 34.0 ku,and could bind specifically to porcine anti-ASFV serum.14 d after the primary immunization with the CD2v-N,no specific antibodies were detected in the serum of rabbits,whereas the specific antibodies with high titer were detected in the rabbits after the primary immunization with CD2v-C. 14 d after the third immunization,the titers of CD2v-N and CD2v-C were 1∶125 000 and 1∶10⁷,respectively.The level of CD2v-C-specific antibodies in the serum of ASFV-infected pigs was significantly higher than that of CD2v-N (P＜0.05).【Conclusion】 The intracellular and extracellular domains of CD2v were expressed,and the antigenicity of the former was higher than that of the latter.The intracellular domain of CD2v was more advantageous as a target for the research and development of ASFV immunological detection techniques.The results of this study had important guiding significance for the research and development of ASFV detection methods.

【Objective】 This study was aimed to elucidate the physiological and biochemical characteristics,virulence and drug sensitivity of the pathogen causing Haemophilus parasuis disease in piglets from a pig factory in Jingzhou,China,so as to provide scientific basis for the prevention and treatment of this disease in the future.【Method】 The pathogenic bacteria were isolated from infected piglets and identified by morphological observation,physiological and biochemical identification,PCR identification,16S rRNA gene sequencing,serotype identification and multi-sequence site typing (MLST).The pathogenicity and drug resistance of the isolate were analyzed by virulence gene detection,pathogenicity test of mouse,drug sensitivity test and traditional Chinese medicine treatment.【Result】 Through morphological observation,physiological and biochemical experiments,16S rRNA sequence alignment and phylogenetic tree analysis,the isolate was identified as Haemophilus parasuis.Serotype and MLST analysis confirmed that the isolate was serotype 10 and ST-299.Virulence gene test showed that the isolate had 15 virulence genes such as CapD,vta1 and vta2.The isolate could cause obvious lesions in many organs of the infected mice by intraperitoneal injection and had strong pathogenicity.The detection of drug resistance genes showed that the isolate had β-lactam resistance gene bla_OXA and fluoroquinolone resistance gene gyrA.The drug sensitivity test showed that the isolate was sensitive to 10 kinds of antibiotics,such as ceftriaxone,neomycin and minocycline,and to 3 kinds of traditional Chinese medicines including realgar,Tussilago farfara and Buthus martensii.The traditional Chinese medicine treatment showed that realgar and Buthus martensii had obvious therapeutic effect on mice infected by the isolate.【Conclusion】 In this study,a strain of Haemophilus parasuis serotype 10 was successfully isolated which could cause bleeding and necrosis of multiple organs in infected mice,and was sensitive to 10 kinds of antibiotics and 3 kinds of traditional Chinese medicines.It was found that realgar and Buthus martensii were effective in treating mice infected by the isolate.The results provided a reference for future prevention and treatment of Haemophilus parasuis.

Porcine reproductive and respiratory syndrome virus (PRRSV) infection is known to cause reproductive disorders in sows,respiratory disease in piglets,and reduce semen quality in boars,resulting in significant economic losses to the global pig industry.PRRSV is unable to replicate on its own and relies on the host metabolic system for all stages of its life cycle.Host cells,in turn,regulate their metabolic processes to hinder PRRSV replication and maintain their normal physiological functions.Both lipid metabolism and glucose metabolism play crucial roles in PRRSV infection.As an enveloped virus,PRRSV is particularly reliant on lipid metabolism systems.Lipids are involved in various stages of the PRRSV life cycle,including adsorption,entry,replication,assembly and release,it is also associated with cellular inflammation,immunity and apoptosis.Glucose metabolism can also interfere with PRRSV life activities,thereby promoting or inhibiting PRRSV replication.The roles of fatty acids,cholesterol,phospholipids,lipid droplets and lipid rafts in lipid metabolism are reviewed,along with the involvement of glycolysis and the tricarboxylic acid cycle in glucose metabolism in PRRSV-infected host cells,so to provide a theoretical basis for elucidating the pathogenic mechanism of PRRSV and contribute to the development of vaccines and anti-PRRSV drugs.

【Objective】 The objective of this study was to prepare chicken E3 ubiquitin ligase FBXL8 polyclonal antibody and to investigate the impact of chicken-derived FBXL8 on the replication of Avian leukosis virus subgroup A (ALV-A) in chicken embryonic fibroblasts (DF-1).【Method】 The FBXL8 gene was amplified by PCR using DF-1 cell cDNA as a template,followed by construction of the recombinant plasmid pET-28a-FBXL8.The recombinant plasmid pET-28a-FBXL8 was transformed into E.coli expression strain RIL for induction and purification.The recombinant protein His-FBXL8 was utilized to immunize 10-month-old New Zealand White rabbits in order to prepare polyclonal antibodies.Western blotting and indirect ELISA were used to identify the immunogenicity and titer of the polyclonal antibody.Overexpression primers and interference sequences targeting FBXL8 gene were designed according to the published FBXL8 gene sequence.Furthermore,the overexpression plasmid psi-flag-FBXL8 and interference plasmid pLKO.1-FBXL8-shRNA were constructed.Western blotting was used to detect the expression effect.Successful overexpression plasmid and interference plasmid were constructed to transfect DF-1 cells,followed by infection with ALV-A after 24 hours.The expression levels of structural proteins P27 and GP85 of ALV-A were detected by semi-quantitative PCR and Western blotting.【Result】 The FBXL8 gene fragment with the size of 1 182 bp was successfully amplified by PCR,and the prokaryotic expression recombinant plasmid pET-28a-FBXL8 was successfully constructed.The induced expression fusion protein His-FBXL8 was 43 ku.Western blotting and indirect ELISA results showed that the prepared rabbit polyclonal antibody of FBXL8 could specifically recognize foreign protein,and the titer of the antibody was 1∶ 64 000.FBXL8 overexpression plasmid and interference plasmid were successfully constructed,which could effectively overexpress and knock down FBXL8 in DF-1 cells.Semi-quantitative PCR and Western blotting results showed that overexpression of FBXL8 inhibited the replication of ALV-A,while knockdown of FBXL8 promoted its replication.【Conclusion】 The FBXL8 polyclonal antibody had good specificity and reactivity.In DF-1 cells,FBXL8 negatively regulated the replication of ALV-A.The results provided an important reference for further exploring the biological characteristics of FBXL8,and laid a foundation for revealing the molecular mechanism of FBXL8 against ALV-A infection.

【Objective】 The purpose of the present study was to obtain the pathogenic bacteria and their main biological characteristics of diseased Micropterus salmoides,and provide technical support for clinical disease prevention and control.【Method】 Pathological changes of the naturally diseased Micropterus salmoides were observed firstly.After that,bacteria were isolated and identified through Gram staining,biochemical testing and 16S rDNA sequencing analysis.Pathogenicity of the isolate was performed on mice to explore the pathological lesions.Furthermore,PCR and Kirby-Bauer (K-B) disk methods were used to detect the common virulence genes and analyze the drug resistance characteristics of the isolated bacteria,respectively.The draft genome was obtained by second-generation sequencing technology,and its molecular characteristics were analyzed using bioinformatics methods.【Result】 Swollen hepatocytes,vacuolar degeneration and hemorrhagic necrotic lesions were observed in the naturally infected Micropterus salmoides.A dominant bacterial strain was isolated from the diseased Micropterus salmoides,which exhibited milky white,semitransparent,and with a diameter of approximately 1 mm colony and β hemolysis on the blood plate.Gram staining showed negative short bacilli,with blunt and round ends.Through biochemical tests and 16S rDNA sequencing,it was confirmed that the isolated strain was Aeromonas caviae,which was named SCMS.The mortality rate of experimental mice was 100% within 24 h after inoculation with SCMS.And the pathological characteristics were similar to those of naturally infected Micropterus salmoides.Nine virulence genes were detected in the SCMS strain,including Aha, Ahp, Aer, Alt, gcaT, Act, Exu, Ela and eprCAI genes.Drug sensitivity test results showed that the isolated bacteria showed resistance or moderate sensitivity to most tetracycline and macrolide drugs.The whole genome sequencing showed that the isolated strain had a total length of 4 329 602 bp and a GC content of 61.5%.The genome information was registered as JARFUQ000000000 in GenBank.Similarity analysis showed that the isolated strain had high similarity with human and food sources of Aeromonas caviae.The comparison of Virulence Factor Database (VFDB) showed that the isolate strain carried multiple virulence genes related to adhesion,invasion,and secretion systems etc.Also,7 drug resistance genes were harbored in the isolated strain when annoted by the drug resistance gene database(CGE).Inconsistency phenomenon was found between partial genes and its drug resistance phenotypes.【Conclusion】 In this experiment,virulence and drug resistance genes of an Aeromonas caviae strain isolated from diseased Micropterus salmoides were analyzed through whole genome sequencing technology,and all these findings could provide technical support for clinical disease prevention and control of Micropterus salmoides.

【Objective】 This experiment was aimed to determine the cause of death of Tupaia belangeri during the breeding process,analyze the biological characteristics,pathogenicity and drug resistance of the pathogen,and develop prevention and control methods.【Method】 Pathological dissection was performed on the dead Tupaia belangeri and the pathological changes of each major organ were recorded,including lung,liver,spleen and intestine.Single colonies were isolated from the main diseased organs using the streak plate method,and the morphology of the colonies was observed.At the same time,Gram staining was used to observe under the microscope,followed by biochemical and drug resistance tests,and pathogenicity test.Further analysis was conducted on the isolated strains using 16S rRNA gene sequencing and bacteria genome sequencing.【Result】 A carbapenem-resistant Pseudomonas aeruginosa strain was isolated from the spleen and intestine of the deceased Tupaia belangeri chinensis.The isolate presented a gray-white colony producing green water-soluble pigments in LB agar medium,and was Gram-negative under microscope.Biochemical tests further confirmed that it was a non-fermenting bacterium.Sequence evolution analysis showed that it had a high similarity of 99% with the soil source Pseudomonas aeruginosa F4 in GenBank.The drug sensitivity test results showed that the isolated strain had certain resistance to carbapenem antibiotics such as imipenem and meropenem,as well as quinolone antibiotics such as levofloxacin.The pathogenicity test result showed that the median lethal dose (LD₅₀) of the isolated strain to adult Kunming mice was 3.382×10⁸ CFU.The organs and tissues of dead mice were isolated for pathological observation,and inflammatory damage and necrosis of the lungs,liver,spleen,intestine,etc.were observed.Bacterial isolation was conducted again on various organs,and based on 16S rRNA sequence analysis,Pseudomonas aeruginosa could be found in the spleen,intestine and blood of dead mice,with high pathogenicity.The whole genome sequencing results confirmed that the isolate strain had genes related to carbapenem resistance and shared similar virulence factor genes with the highly pathogenic reference strain of Pseudomonas aeruginosa.【Conclusion】 This study isolated one carbapenem-resistant Pseudomonas aeruginosa from the deceased Tupaia belangeri,providing a new theoretical basis for the study of microbial pathogenicity and domestication of Tupaia belangeri.

【Objective】 This study was aimed to improve the water solubility and dissolution rate of the insoluble veterinary drug fenbendazole (FBZ) and improve the therapeutic effect of the drug in clinical application.【Method】 Fbendazole-methyl-β-cyclodextrin inclusion complex (FBZ-Me-β-CD) was prepared by constant temperature magnetic stirring method and freeze-drying technique.Through the design of single factor orthogonal test,the optimum preparation technology of inclusion complex was studied with the inclusion rate and recovery rate as the index.According to the solubilization effect of 5 different cyclodextrins on drugs,the best inclusion material was determined by phase solubility test.High performance liquid chromatography (HPLC) was used to detect the solubility,inclusion rate and in vitro dissolution rate of the inclusion complex.Finally,the phase structure of the inclusion complex was characterized by differential scanning calorimetry (DSC) and scanning electron microscopy (SEM).【Result】 The optimum preparation process of inclusion complex was as follows:Stirring time was 3 h,rotational speed was 600 r/min,molar ratio of subject to guest was 1∶3,temperature was 50 ℃.The best inclusion material was methyl-β-cyclodextrin.The solubility of the inclusion complex in water was 12.02 mg/mL,which was 40 000 times that of fenbendazole (0.3 μg/mL).The dissolution rate of the inclusion complex in vitro was 7 times that of fenbendazole.The average recovery and inclusion rate of the inclusion compounds were 89.50% and 29.20%,respectively.By scanning electron microscopy and differential scanning calorimetry,fenbendazole and cyclodextrin were identified as amorphous inclusion states.【Conclusion】 In this study,the solubility and dissolution rate of the insoluble drug fenbendazole were improved by preparing the cyclodextrin inclusion complex of fenbendazole,and the preparation method was simple and easy,which was conducive to the further study of fenbendazole.

【Objective】 The aim of the experiment was to prepare a nanogel for topical delivery of kitasamycin for skin wound infections.【Method】 Chitosan/gelatin/poly(vinyl alcohol) nanogel were developed by electrostatic interaction between the gel materials under the action of Schiff base using genipin as cross-linking agent and loaded with the drug kitasamycin.The encapsulation rate,drug loading,swelling properties,nanoparticle size,Zeta potential,microstructure scanning,and other characterisation structures and biosafety were tested.The in vitro release effect and wound healing activity of nanogel were evaluated.【Result】 In the present study,the kitasamycin nanogel was prepared in the composition of 0.25 g of kitasamycin,0.4 g of chitosan,0.3 g of gelatin,0.2 g of polyvinyl alcohol,and 0.2 g genipin per 25 mL of the gel,and the remaining amount of deionized water,the appearance properties were light blue nanogel,without precipitation and drug precipitation,the swelling degree was high,the drug loading was 13.6%,and the encapsulation rate was 83.1%.Scanning electron microscopy showed that the nanogel formed a three-dimensional mesh structure with dense and uniformly distributed pores.The average particle size was 28.56 nm,Zeta potential was 16.33 mV,with good biological safety.The nanogel could release the drug in vitro for 48 h,which was significantly longer than that of the kitasamycin solution.In addition,the nanogel could reduce the wound size of mice by 96%,which was helpful for wound healing.【Conclusion】 In this study,kitasamycin nanogels were successfully prepared,and their potential ability to control drug release and promote skin wound healing was demonstrated by in vitro and in vivo evaluations.

【Objective】 The purpose of this experiment was to clarify the distribution of the main pathogenic bacteria,their resistance and virulence genes,and guide the rational use of drugs in the clinical treatment of mastitis in Hu sheep farms.【Method】 43 clinical mastitis milk samples were collected from 6 large-scale sheep farms.Bacterial morphology identification,biochemical identification and 16S rDNA sequence analysis were used to determine the main pathogenic bacteria causing mastitis in Hu sheep.Drug resistance was detected using paper diffusion method,and related virulence genes were detected using PCR method.【Result】 The Gram staining results showed that Gram positive cocci and Gram negative bacilli were obtained. On the Baird Parker agar plate,Gram positive cocci exhibited rounded edges and black colonies in the middle.Biochemical tests showed that the isolated strains were positive for mannitol,hydrogen peroxide and rabbit plasma coagulation tests.After 16S rDNA sequencing comparison results showed that a total of 31 strains were highly similar to Staphylococcus aureus.Gram negative bacilli presented black purple colonies with metallic luster on the Eosin Methyl Blue agar plate,while on the MacConkey agar plate,pink colonies appeared.Biochemical tests showed that the isolated strains were positive for indole and methyl red tests,and negative for VP and citrate utilization tests.After 16S rDNA sequencing comparison,a total of 12 strains were highly similar to Escherichia coli.The drug sensitivity test results showed that the isolates of Staphylococcus aureus were sensitive to drugs such as ciprofloxacin,levofloxacin and cefotaxime,but resistant to penicillin,erythromycin and streptomycin.The isolates of Escherichia coli were sensitive to drugs such as ciprofloxacin and levofloxacin,and were resistant to penicillin and erythromycin.Seven virulence genes were detected in Staphylococcus aureus,namely PVL, Hlb, seb, clfA, clfB, fnbB and icaD genes,but Hla, sea and fnbA genes were not detected.Six virulence genes were detected in Escherichia coli,namely hlyA, etrA, fasA, eaeA^a, exhA^a and stx1 genes,but no cnf1, aer, estA and stx2 genes were detected.【Conclusion】 The main pathogens causing mastitis in large-scale sheep farms were Staphylococcus aureus and Escherichia coli,which carried rich virulence genes.It was recommended to prioritize the use of ciprofloxacin and levofloxacin for treatment.This article provided scientific basis for clinical treatment of this disease.

【Objective】 This study was objective to explore the target and mechanism of chrysin and naringenin against Porcine epidemic diarrhea virus (PEDV),and provide theoretical basis for further developing new anti-PEDV therapeutic drugs with chrysin and naringenin.【Method】 Online databases of PharmMapper,TCMSP,SEA Search Server and STITCH were used to obtain the potential targets of chrysin and naringenin,at the same time GeneCards database was searched to obtain the targets of PEDV on the host,and Draw Venny Diagram online program was used to obtain the intersection targets of chrysin,naringenin and disease.The target protein-protein interaction (PPI) network was constructed by STRING database and Cytoscape 3.9.1 software,and the key targets were screened,and the GO function and KEGG pathway enrichment of the targets were analyzed.The molecular docking of core targets and small molecules was carried out by AutoDock Vina v 1.2.0 software,and the binding energy and binding mode of chrysin and naringenin with target proteins were analyzed,and the docking results were visualized by PyMOL v 2.5.The effects of chrysin and naringenin on the expression of core target proteins were detected by Real-time quantitative PCR.【Result】 The results showed that there were 12 potential anti-PEDV targets of chrysin,among which albumin (ALB),estrogen receptor 1 (ESR1) and transforming growth factor β-1 protein (TGF-β1) might be the core targets of chrysin against PEDV.Naringenin had 18 potential anti-PEDV targets,among which ALB,Caspase-3 (CASP3) and peroxisome proliferator-activated receptor γ (PPARG) might be the core targets of naringenin against PEDV.The anti-PEDV target of chrysin involved 21 biological processes,5 cellular components and 6 molecular functions and obtained 11 signal pathways,while naringenin anti-PEDV targets involved 31 biological processes,8 cellular components and 19 molecular functions, and obtained 13 signal pathways.There was a strong interaction between the core targets and the two natural compounds due to hydrogen bonding and hydrophobic interaction.Chrysin and naringenin could extremely significantly reduce the expression of Caspase-3 (P<0.01),which might antagonize the apoptosis induced by PEDV by affecting the expression and activation of Caspase-3,and they extremely significantly increased the expression of TGF-β1 protein (P<0.01),which might treat PEDV infection by affecting the expression of TGF-β1 and anti-PEDV-induced inflammation.【Conclusion】This study revealed the mechanism of anti-PEDV effects of chrysin and naringenin through potential core targets ALB,ESR1,TGF-β1 and ALB, Caspase-3,PPARG,respectively, and IL17,PI3K-Akt and AMPK signaling pathways, providing new ideas for the development of novel anti-PEDV drugs.

【Objective】 This study was aimed to determine the pathogen and drug sensitivity of a suspected Salmonella infection in a broiler farm in Guangdong province,and provide scientific basis for formulating effective prevention and control and purification measures and rational antibiotic using program.【Method】 In this study,Salmonella isolation and culture,Gram staining,biochemical test,invA-specific gene PCR identification,16S rRNA identification and serotyping were performed on liver and intestinal samples of several sick chickens from a large-scale chicken farm in Guangdong province.K-B drug sensitivity method was used to detect the sensitivity of the strains to 20 kinds of antibiotics.The drug resistance genes and virulence genes were detected by PCR.【Result】 In isolation and culture,it could be seen that colorless transparent,small dewdrop-like colonies or smooth,round,transparent and moist colonies with black center could be found.The suspected colonies were pink in Gram staining,scattered in distribution and slightly round at both ends,no capsule,no spore. The biochemical test showed gas production,acid production at the bottom,hydrogen sulfide production was black,and alkali production on the inclined surface turned red.PCR was performed to amplify the suspected colonies DNA,and the invA specific gene amplified a band of about 285 bp,and the 16S rRNA amplified a band of about 1 500 bp.In this study,a total of 8 strains of Salmonella Senftenberg were isolated and identified.The results of drug susceptibility test showed that 8 strains had no resistance to ceftazidime,gentamicin,amikacin,kanamycin,minocycline,doxycycline and polymyxins B.However,the resistance rates to penicillin,cephalexin,vancomycin,erythromycin,lincomycin,ampicillin,piperacillin,cefazolin,cefuroxime sodium,ceftriaxone,cefoperazone,streptomycin and tetracycline were high,ranging from 87.5% to 100%,and the isolates were all multi-drug resistant bacteria, qnrS and stcM resistance genes were detected in 7 strains,and no resistance genes were detected in 1 strain.The test results of virulence genes showed that detection rates of invA,hilA, sipA,sipC,ssrA and stnP1 genes were 100%,and sopB and ssaR genes were 50.0% and 62.5%,respectively.【Conclusion】 All isolates of chicken-derived Salmonella Senftenberg had multi-drug resistance and were sensitive to 7 species drugs,including ceftazidme and amicarcin,but the carrying rates of six virulence genes invA,hilA,sipA,sipC,ssrA and stnP1 were 100%.The results of this study supplemented the epidemiological data of Salmonella of chicken origin and provided theoretical reference for the prevention and control and purification of clinical Salmonella.

Equine,especially sport horses,tend to suffer from locomotor system injury.Once the injury occurs,it will lead to varying degrees of lameness,and it is difficult to completely repair the damaged tissue,which often leads to a significant decline in horse motor performance and poor prognosis,and is one of the main reasons for the early end of sports career of sports horses.Platelet-rich plasma (PRP) is a regenerative medicine formulation that promotes tissue repair and regeneration by activating platelets to release large amounts of growth factors.Many studies and clinical reports have shown that PRP has a certain therapeutic effect on locomotor injuries,and is most commonly used in the treatment of equine tendon,ligament injury and osteoarthritis,and is widely used in Europe and the United States and other countries.In addition,PRP also has certain antibacterial effect and is expected to be used in the treatment of septic arthritis.It also shows a certain therapeutic potential for laminitis and other diseases with limited response of traditional treatment.In the field of equine sports medicine,the mechanism and scope of application of PRP are not well understood in China,and there is a lack of research reports on the use of PRP in the treatment of equine locomotor system injury in clinical practice.The author reviewed the preparation and classification of PRP,the clinical application and research progress of PRP in the treatment of equine locomotor system injury,and summarized the application scope and development trend of PRP in the treatment of equine locomotor system injury,in order to provide references for domestic veterinarians and related practitioners to apply PRP.