【Objective】 The aim of this study was to knockout Krüppel-like factor 4 (KLF4) gene in porcine small intestinal epithelial cells using CRISPR/Cas9 technique,and investigate the effects of KLF4 gene knockout on cell viability and cell cycle.【Method】 Three pairs of sgRNAs (sgRNA1,sgRNA2 and sgRNA3) were designed in exon 1 of porcine KLF4 gene.The double stranded DNA after annealing was ligated with the linearized pGK1.1 vector,and the products was transferred into Escherichia coli Top10 competent cells for identification.The recombinant vectors were transfected into porcine intestinal epithelial cells (IPEC-J2).The sequences adjacent to the target sites were amplified by PCR,and the knockout efficiency of sgRNA was verified by sequencing.The positive cell clones were selected by Cruiser^TM Enzyme digestion,and the knockout sequences were checked by TA clone sequencing.The expression of KLF4 protein in gene knockout cells was detected by Western blotting.The changes of cell viability and cell cycle of KLF4 gene knockout cells were analyzed by CCK-8 and flow cytometry.【Result】Sequencing of the recombinant vectors showed that sgRNAs were successfully ligated with the pGK1.1 vector.Analysis of knockout efficiency demonstrated that all three sgRNAs could lead to gene knockout,of which sgRNA3 achieved higher knockout efficiency.Two positive cell clones were selected by Cruiser^TM Enzyme digestion.TA clone sequencing results indicated that 116 and 137 bp sequences were deleted in the two alleles of KLF4 gene,respectively.Western blotting results showed that KLF4 protein was not expressed in KLF4 gene knockout cells.Analysis of cell viability and cell cycle showed that KLF4 gene knockout extremely significantly inhibited the cell viability (P＜0.01),and induced cell cycle arrest at G0/S transition.【Conclusion】 KLF4 gene knockout IPEC-J2 cells were constructed in this study using CRISPR/Cas9 technique.KLF4 gene knockout could inhibit cell viability and result in cell cycle arrest at G0/S transition. KLF4 gene knockout cells could provide the materials for further exploring functions and molecular mechanisms of KLF4 gene.

【Objective】 The purpose of this experiment was to systematically analyze the fatty acid desaturases (FADS) gene family in Qinchuan cattle,clarify the spatiotemporal expression patterns of these members in various tissues and different differentiation stages of preadipocytes,and lay a foundation for further exploring the function and regulatory roles of FADS gene family in bovine fat deposition.【Method】 Different tissue samples of Qinchuan cattle were collected,the preadipocytes were isolated from perirenal fat,and the CDS region sequences of FADS gene family were amplified by PCR,and the sequence characteristics were predicted by bioinformatics software.Real-time quantitative PCR was used to detect the temporal and spatial expression profiles of FADS gene family members in different tissues of adult cattle and during the differentiation of prerenal adipocytes.【Result】 The CDS region of 3 members of FADS gene family (FADS1,FADS2 and FADS3) was amplified successfully with the size of 1 425,1 335 and 1 332 bp,respectively.The phylogenetic tree results showed that the amino acid sequences of FADS gene family members of Qinchuan cattle had the highest similarity with Bos indicus,and were far related to Mus musculus.The results of bioinformatics prediction showed that the FADS gene family were stable hydrophilic proteins,and the secondary structure was mainly composed of alpha helix,and the tertiary structure was consistent with the predicted secondary structure.FADS1 and FADS2 were mainly located in the endoplasmic reticulum (44.4% and 44.4%),and FADS3 was mainly located in the cell membrane (39.1%).Real-time quantitative PCR results showed that the expression of FADS1 and FADS3 genes were higher in muscle tissue,and the expression of FADS2 gene was the highest in liver, and significantly higher than that in other tissues (P<0.05).With the adipogenic differentiation of adipocytes,compared to other differentiation time, the expression of FADS1 and FADS2 genes were the highest on days 2 (P<0.05),while the expression of FADS3 gene was the lowest on days 2 (P<0.05).【Conclusion】 FADS1, FADS2 and FADS3 of FADS gene family in Qinchuan cattle were successfully cloned, which were highly conserved Bos indicus, Capra hircus and Ovis aries and other mammals. FADS gene family were relatively stable hydrophilic proteins, FADS1 and FADS3 genes were highly expressed in muscle, and FADS2 gene was highly expressed in liver. In the process of adipogenic differentiation of adipocytes, FADS1 and FADS2 gene expressions were the highest and FADS3 gene expression was the lowest on days 2 of differentiation. The results laid a foundation for further research on the mechanism of FADS gene family influencing fat deposition in beef cattle by regulating adipocyte differentiation.

【Objective】 This experiment was aimed to detect the differential expression of miRNA-185 and vascular endothelial growth factor A (VEGFA) in the placental tissue of dairy cows in the normal expulsion group and retained fetal membranes (RFM) group through in vivo and in vitro experiments,as well as the expression distribution of VEGFA in the placental tissue of dairy cows.It verified and clarified the close correlation and targeted regulatory relationship between miRNA-185 and VEGFA and retained fetal membranes in dairy cows and provided theoretical and experimental basis for the in-depth study of the role of miRNA-185 in the occurrence of retained fetal membranes in dairy cows.【Method】 Real-time fluorescence quantitative PCR method was used to detect the gene expression of miRNA-185 and VEGFA in the placental tissue of dairy cows with normal and retained fetal membranes.Fluorescence in situ hybridization (FISH) was used to detect the specific and mRNA differential expression of VEGFA in the placental tissue of dairy cows.Dual luciferase reporter genes was used to clarify the targeted regulatory relationship between miRNA-185 and VEGFA,Real-time fluorescence quantitative PCR and Western blotting methods were used to detect the mRNA and protein differential expression of VEGFA after in vitro transfection of miRNA185 mimics,miRNA-185 inhibitor and miRNA-185 NC.【Result】 Real-time fluorescence quantitative PCR results showed that compared with dairy cows with normal removal of placenta,the expression levels of miRNA-185 and VEGFA in dairy cows with retained fetal membranes were extreme downregulation (P＜0.01).FISH result showed that VEGFA mRNA was mainly expressed in dairy cow endometrial epithelial cells.VEGFA was a targeted regulatory protein of miRNA-185.Compared with the negative control group,the mRNA and protein expression levels of VEGFA were extreme downregulation after transfection with miRNA-185 mimics (P＜0.01),while the mRNA and protein expression levels of VEGFA were extremely upregulated after transfection with miRNA-185 inhibitor (P＜0.01).【Conclusion】 The expression of miRNA-185 and VEGFA in the placental tissue of dairy cows with retained fetal membranes significantly decreased.VRGFA was mainly expressed in endometrial epithelial cells,and miRNA-185 could participate in the occurrence and development of retained fetal membranes in dairy cows by targeting and regulating the expression of VEGFA.

【Objective】 This study was aimed to clone myogenin gene in Guizhou White goat and analyze the structure characteristics by bioinformatics,so as to provide an important reference basis for further exploring molecular regulation mechanism of muscle development of goat.【Method】 The blood genomic DNA of Guizhou White goat was used as template,four pairs of specific primers were designed,and the complete coding sequence of myogenin gene in Guizhou White goat was obtained by PCR amplification,Sanger sequencing and sequence assembly method.The molecular structure characteristics of myogenin gene,such as composition of nucleotide and amino acid sequence,phosphorylation sites,key domains and functional motifs were analyzed by BioEdit 7.0,NetPhos 3.1,Conserved Domains Search,SMART and Motif Scan softwares,respectively.The sequence similarity comparison of myogenin gene among 15 Artiodactyla species and phylogenetic tree construction were carried out by DNAStar Lasergene,DNAMAN 8.0 and Mega 5.0 softwares.【Result】 The length of myogenin gene sequence in Guizhou White goat was 2 424 bp(obtain GenBank accession No.:HZ53289),which was consisted of 3 exons,2 introns and partial of 5'-UTR and 3'-UTR,and was 471,82,122,765,589,135 and 260 bp in length,respectively.The length of coding region was 675 bp,which was encoded 224 amino acids.The deduced amino acid sequence analysis showed that 1-86 amino acids were the unique basic domain of myogenic determining factors family,81-139 amino acids were the helix-loop-helix DNA binding domain,and 160-170 amino acids were the low complexity regions.There were 21 phosphorylation sites and functional motif structure such as nuclear localization signal protein.Amino acid sequence alignment showed that six cysteine residues,one nuclear localization signal protein and one low complexity region were completely conserved among 15 Artiodactyla species.Sequence similarity and molecular phylogenetic analysis all showed that Guizhou White goat myogenin gene had the closest genetic relationship with Boer goat,Hu sheep and Marco Polo sheep.【Conclusion】 The myogenin gene of Guizhou White goat was successfully cloned,with a size of 2 424 bp in length and encoding 224 amino acids,which contained the Basic and H-L-H domains.These results could provide theoretical reference for further exploring the molecular regulation mechanism of muscle growth and development of goat.

【Objective】 This experiment was aimed to optimize the production process of xylanase using response surface methodology,so as to provide a reference basis for further large-scale production.【Method】 Employing single-factor experiments,the influence of six factors,including carbon source,nitrogen source,pH,temperature,inoculum size and agitation speed,on xylanase production by Bacillus cereus (B. cereus) W-3 was examined meticulously.The Box-Behnken response surface optimization design in Design Expert 10.0.3 software was applied.The fermentation conditions were systematically optimized by varying combinations of wheat bran,ammonium chloride,pH,temperature,inoculum size and agitation speed.A mathematical model for enzyme production in relation to these factors was established using a quadratic response surface regression method.The impact levels of each factor on enzyme production were determined through regression analysis.【Result】 The addition of wheat bran,ammonium chloride,pH and temperature were the main effect factors affecting fermentation.Through single-factor optimization,it had been determined that B. cereus W-3 ferments to produce xylanase under the following conditions:Wheat bran concentration of 35 g/L,ammonium chloride concentration of 5 g/L,pH 8.0,a temperature of 70 ℃,an inoculum size of 2%,and an agitation speed of 210 r/min. Having built upon the single-factor optimization,further response surface optimization led to the discovery of superior fermentation parameters.The optimized fermentation conditions included 33.0 g/L wheat bran,5.1 g/L ammonium chloride,pH 8.3,a temperature of 73 ℃,an inoculum size of 2%,and an agitation speed of 210 r/min. Under the optimized fermentation conditions,xylanase enzymatic activity produced by B. cereus W-3 reached 523.24 U/mL.【Conclusion】 In this experiment,the B. cereus W-3 fermentation conditions was optimized by response surface methodology.Under the conditions of 33.0 g/L wheat bran,5.1 g/L ammonium chloride,pH 8.3,73 ℃,2% inoculum size and 210 r/min,a significantly higher xylanase activity of 523.24 U/mL could be obtained,representing a 2.32-fold increase compared to pre-optimization level (225.53 U/mL).

Oxidative stress can lead to the imbalance between oxidation and antioxidation in the body,resulting in excessive reactive oxygen species (ROS),causing damage to cells and tissues,inflammation and other diseases,affecting the growth and development of the body.Superoxide dismutase (SOD) is an antioxidant metal enzyme that exists in living organisms.It can catalyze superoxide anion radical (O₂^―) through disproportionation reaction,thereby eliminating excessive ROS in animals.Studies have found that SOD not only has a specific and high efficiency in antioxidant function,slows down the aging of the body,but also improves the intestinal health production performance of animals and regulates the immune function of the body to a certain extent.In the case of large-scale use of intensive livestock and poultry breeding and production machines,the damage caused by radiation to animals needs to be paid attention to.Effectively utilizing the anti-radiation function of SOD to alleviate or improve the impact of radiation on the body is worth further exploration.At present,the main additives for relieving oxidative stress of the body are vitamins,trace elements,plant extracts,etc.,while the use of SOD to relieve oxidative stress and improve production performance of animals is relatively few studies,mainly due to the high extraction cost,large molecular weight,easy inactivation of natural SOD.However,with the emergence of synthetic SOD,the cost of preparing SOD is reduced,and it has the characteristics of high activity and easy absorption of small molecules,which makes the application of SOD in livestock and poultry production gradually increase,and more potential of SOD can be tapped in the future,and SOD can be more widely used in animal production.In this paper,the physiological functions of SOD such as anti-oxidation,anti-aging,anti-inflammation,anti-radiation and regulating immunity were summarized,in order to provide more theoretical basis for the application of SOD in animal production.

Stem cells are a kind of pluripotent cells with strong proliferation and regeneration potential,which can regulate various functions in the body with low immunogenicity,and have great application potential in the field of animal husbandry.Mesenchymal stem cells (MSCs) are a kind of pluripotent cells,possessing all the commonalities of stem cells,with a wide range of sources,easy separation,low moral and legal constraints,and gradually advancing from basic research to clinical application stage.Through migration and paracrine mechanisms,MSCs play a regulatory role in vivo,such as resisting pathogen invasion,stabilizing immune response,reducing cell stress,and promoting tissue regeneration.However,the biological characteristics of MSCs from different tissue sources are different,and their surface markers are an important basis for identification.At present,the related research of MSCs has gradually expanded from the field of human and pet to animal husbandry,and has also shown strong application potential in large animals such as cattle and horses.With its anti-inflammatory repair ability,it can effectively protect the animal body,promote the prognosis of diseases,and enhance the economic value of animals.But because the relevant research is not deep enough,many problems can not be solved,which makes the large-scale application is limited.In order to further enhance the application potential of MSCs and accelerate the transformation from laboratory to practical application,research in the fields of cell culture,gene editing and biomaterials is also constantly expanding.Therefore,the author systematically reviewed relevant literature,introduced the source,characteristics and mechanism of MSCs,and summarized the research progress and application potential of MSCs in animal husbandry,in order to provide new ideas for the application of MSCS in animal husbandry.

【Objective】 This study was conducted to examine the effect of walnut green husk (WGH) extracts on in vitro rumen fermentation and microbiota community in sheep,in order to elucidate its role in regulating rumen fermentation.【Method】 The WGH extracts obtained using ethyl acetate (Ea),deionized water (Dw),and 50% hydroalcohol (Ha) were added at concentrations of 0,0.125%,0.25%,0.50%,and 1.00% (weight of substrate) and fermented in vitro for 24 h using a high concentrate ration (the ratio of concentrate/roughage was 65∶35) as substrate.Gas production (GP) was recorded during fermentation,and at the end of the fermentation,the fermentation parameters,substrate degradation rate,lactate (LA) concentration and related enzyme activities were analyzed.Additionally,the changes in rumen bacterial community were assessed using high-throughput sequencing of the 16S rDNA V4 region.【Result】 Compared with control group,pH,dry matter degradation rate (DMD),acetate/propiontate (A/P ratio),and the activities of 6-phosphofructokinase (PFK),hexokinase (HK) and pyruvate kinase (PK) in Ea group were extremely significantly increased (P＜0.01),the 24-hour cumulative gas production (GP_{24 h}),protozoa number,and the concentrations of ammonia nitrogen (NH₃-N),acetate,propiontate,valerate,total volatile fatty acids (TVFA) and LA were extremely significantly or significantly desreased (P＜0.01 or P＜0.05).Compared with control group,pH,GP_{24 h},neutral detergent fiber degradation rate (NDFD),the levels of propiontate,isobutyrate valerate,and LA,and the activities of PFK,HK and PK in Dw group were extremely significant or significantly decreased (P＜0.01 or P＜0.05),while the A/P ratio exhibited an extremely significant increase (P＜0.01).As for Ha group,GP_{24 h},DMD,NDFD,the levels of isobutyric acid,butyrate and isovalerate,and A/P ratio were extremely significantly or significantly decreased reduced (P＜0.01 or P＜0.05),the protozoa count and the levels of NH₃-N and propiontate were extremely significantly or significantly increased (P＜0.01 or P＜0.05).16S rDNA sequencing showed that compared with control group,Alpha diversity index and the relative abundances of Firmicutes,Bacteroidotes and Rikenellaceae_RC9_gut_group in Ea group were extremely significantly or significantly increased (P＜0.01 or P＜0.05),whereas the relative abundances of Proteobacteria,Succinivibrio and Succiniclasticum were exhibited an extremely significant or significant decrease (P＜0.01 or P＜0.05).The Shannon index and the relative abundances of Bacteroidotes,Rikenellaceae_RC9_gut_group and uncultured rumen bacterium in Dw group were extremely significantly or significantly decreased (P＜0.01 or P＜0.05).Alpha diversity index and the abundance of Firmicutes in Ha group were extremely significantly deceased (P＜0.01),and the relative abundances of Succinivibrio,Prevotella and Succiniclasticum were significantly or extremely significantly increased (P＜0.05 or P＜0.01).【Conclusion】 The addition of ethyl acetate extract of walnut green husk in the diet could effectively improve rumen fermentation in sheep,with the best results observed when added at a dosage of 0.25% based on substrate weight.

【Objective】 This experiment was conducted to study the effects of organic acid combined with essential oil,tannic acid and compound enzyme preparation on growth performance,serum biochemical indexes and intestinal health of weaned piglets,so as to provide theoretical basis for their production and application in piglets.【Method】 240 Duroc×Landrace×Yorkshire weaned piglets (25-day-old) with body weight of 6.48 kg±0.68 kg were randomly divided into 5 groups with 6 replicates per group and 8 piglets per replicate.The dietary treatments were as follows,basal diets (CON),CON＋5 kg/t organic acid (OA),CON＋5 kg/t OA＋800 g/t essential oil (OAEO),CON＋5 kg/t OA＋500 g/t tannic acid (OATA),and CON＋5 kg/t OA＋500 g/t compound enzyme (OACE).The pre-feeding period was 3 days and the test period was 28 days.On the first day and the 29 th day,piglets were weighed with fasting and feed intake was recorded to calculate growth performance.Feed samples and feces samples were collected to determine the apparent digestibility of nutrients.Blood samples were collected to determine serum antioxidant and immune properties.The stomach and intestinal segments (duodenum,jejunum,ileum and cecum) were separated to measured the gastrointestinal pH.Jejunal mucosa was collected to determine digestive enzyme activity.The middle part of jejunum and ileum was taken for the determination of intestinal morphology.Cecal contents were collected for the determination of short-chain fatty acids.【Result】 ①Compared with CON and OA groups,the F/G of weaned piglets in OAEO group was significantly reduced (P＜0.05).②Compared with CON group,the serum SOD activity in OA and OAEO groups was significantly increased (P＜0.05),the serum GSH-Px activity in OATA group was significantly increased (P＜0.05),the serum MDA content was significantly decreased in four treatment groups (P＜0.05).③Compared with CON and OA groups,the serum IgG,IL-2 and IL-22 levels were significantly increased in OATA group (P＜0.05),the serum D-LA levels in OAEO,OATA and OACE groups was significantly decreased (P＜0.05).④Compared with CON group,the pH of stomach,duodenum and jejunum were significantly decreased in OAEO and OACE groups (P＜0.05),and the villus height of jejunum and ileum was significantly increased in four treatment groups (P＜0.05).Compared with CON and OA groups,the activities of α-amylase,sucrase and lactase in jejunum were significantly increased in OACE group (P＜0.05),the apparent digestibility of dry matter,crude ash,ether extract and phosphorus in OATA and OACE groups were significantly increased (P＜0.05).⑤Compared with CON and OA groups,the concentration of cecal acetic acid was significantly increased in OAEO group (P＜0.05),the concentration of cecal isobutyric acid in OAEO,OATA and OACE groups was significantly decreased (P＜0.05).【Conclusion】 The combination of organic acid and essential oil had a better effect on improving the growth performance of weaned piglets, the combination of organic acid and tannic acid had a better effect on improving the antioxidant and immune performance of piglets, and the combination of organic acid and enzyme preparation has a better effect on improving the digestive function of piglets and improving the apparent digestibility of nutrients.

【Objective】 This study was aimed to explore the effects of compound fermentation of traditional Chinese medicine and Cyperus esculentus meals on growth performance,serum antioxidant and immune indexes in Hu sheep,so as to provide a basis for the development of new feed additives.【Method】 Forty fattening Hu sheep with the same body weight and good health were randomly divided into 2 groups (control and experiment groups) with 20 sheep in each group.Hu sheep in control and experiment groups were fed with basal diet and added with 2% compound fermentation on the basal diet,respectively.The pre-test period was 7 days and the experiment period was 42 days.The body weight was weighed on the 0 and 42nd days of this experiment,and the average daily gain was calculated.Daily feed intake was recorded and feed to gain ratio was calculated.At the 1st,21st and 42nd days of this experiment,the blood samples were collected through the jugular vein,serum was separated,and serum antioxidant and immune indexes were measured.【Result】 The final body weight,average daily gain and average daily feed intake of Hu sheep in experiment group were significantly higher than those in control group (P＜0.05).On the 21st day,compared with control group,the activities of catalase (CAT) and total antioxidant capacity (T-AOC),and the contents of immunoglobulin G (IgG) and γ-interferon (INF-γ) in serum of Hu sheep in experiment group were increased by 2.75%,3.46%,7.28% and 4.55% (P＜0.05), respectively, the content of malondialdehyde (MDA) was decreased by 12.72% (P＜0.05).On the 42nd day,compared with control group,the activities of CAT and T-AOC,and the contents of IgG and INF-γ in serum of Hu sheep in experiment group were increased by 3.17%,3.67%,9.77% and 11.91% (P＜0.05),respectively,the content of MDA was decreased by 15.10% (P＜0.05).【Conclusion】 Under the conditions of this experiment,the growth performance,antioxidant and immune function in Hu sheep could be improved effectively by adding 2% compound fermentation of Cyperus esculentus and traditional Chinese medicine in the diet,which could be used as a new feed additive in livestock production.

【Objective】 To explore the utilization of energy,protein and amino acid of the corn DDGS from 5 regions for Cherry Valley male ducks.【Method】 Ninety-eight healthy Cherry Valley male ducks with a body weight of 3.25 kg±0.25 kg at 18 weeks of age were cultivated in metabolic cages,one duck one cage.They were randomly divided into 7 groups comprised of 14 individuals and 1 per replicate.The ducks in 5 experimental groups were fed the diets with corn DDGS and corn starch at a ratio of 7∶3,and the DDGS were sourced from Jilin,the United States (crude),the United States (fine),Shandong and Liaoning,respectively.The ducks in corn starch group were fed on the 100% corn starch diet,while in endogenous group was fed nothing.According to the process of force-feeding,both force-feeding test and metabolic test were carried out,with all the excrement collected.Moreover,gross energy (GE),crude protein (CP) and the amino acid content of force-feeding feed and excreta were determined.【Result】 The CP content of corn DDGS sourced from Liaoning was significantly lower compared to the other 4 experimental groups (P＜0.05),while the CP content of crude and fine corn DDGS sourced from the United States was significantly lower relative to the Jilin group (P＜0.05).The GE of DDGS from the United States (crude) was significantly lower compared to the other 4 experimental groups (P＜0.05).The apparently available protein and truly available protein in the United States (fine) and Liaoning groups were significantly higher than those from the United States (crude) and Shandong groups (P＜0.05).The apparently metabolizable energy and truly metabolizable energy in the United States (crude) and Liaoning groups were significantly higher than in Jilin and Shandong groups (P＜0.05).The true availability of threonine,glycine,histidine,arginine,branch chain amino acid,phenylalanine and tyrosine of corn DDGS from Liaoning was significantly higher compared to the other 4 experimental groups (P＜0.05),and the true availability rates of proline,aspartic acid,serine,glutamic acid and alanine were the highest.【Conclusion】 There were significant differences observed in nutritional composition,energy,protein and amino acid utilization of corn DDGS among 5 different regions.The utilization of energy,protein and amino acid by Cherry Valley male ducks was in the following order:Liaoning ＞ United States (fine)＞United States (crude)＞Jilin＞Shandong.The crude protein content of corn DDGS in five different regions was ranked as Shandong＞Jilin＞United States (crude)＞United States(fine)＞Liaoning.

Echinacea is one of the most popular natural plants,which has multiple biological activities such as anti-inflammation,antioxidation and bacteriostasis.As a kind of natural medicine with strong immune activity and safety,Echinacea and its extract have become the research hotspot for scientists in different fields.Several groups of pharmacologically active compounds have been isolated from Echinacea,such as polysaccharides,glycoproteins, alkylamides and caffeic acid derivatives,etc.A large number of studies have demonstrated that they may exert their effects through multiple signaling pathways,such as the mitogen-activated protein kinase (MAPK),c-Jun N-terminal kinase (JNK),etc.,to exert immunomodulatory effects.Additionally,they may also exert antioxidant effects through nuclear factor E2-related factor 2-Kelch-like ECH-associated protein 1 (Nrf2-Keap1),nuclear factor kappa B (NF-κB),and others.In recent years,under the background of the increasing demand for natural raw materials,Echinacea and its extract also have a good application prospect in animal breeding.Based on animal experiments,Echinacea and its extract to a certain extent have been shown to promote animal growth,alleviate oxidative stress,improve reproductive performance,regulate immune response,enhance disease resistance and minimize the transmission risk,thereby ensuring animal health and optimizing production benefit.This natural farming way meets the needs of modern consumers for safe and healthy food,and also provides a viable option for the sustainable development of livestock and poultry farming.The authors reviewed the main chemical components,their biological activity and the mechanism of action,as well as the application status of Echinacea and its extract in livestock,poultry,and aquaculture,in order to provide theoretical reference for standardizing the extraction of active components of Echinacea,exploring the action mechanism of Echinacea and its extract in animal production,and further broadening its application in livestock,poultry and aquaculture.

【Objective】 The objective of this study was to investigate the impact of various proportions of whole plant silage maize meal on the growth performance,serum biochemical,antioxidant,and immune indicators of fattening pigs and to determine the optimal proportion of silage corn meal.【Method】 A total of 40 pigs with an average body weight of (39.03±5.53) kg of "Duroc×Landrace×Yorkshire" fattening pigs were selected and randomly divided into 4 groups with 10 pigs in each group.The control group was fed with basic diet (without whole plant silage corn meal),and the test groups were fed with 5% (group Ⅰ),10% (group Ⅱ) and 15% (group Ⅲ) whole plant silage corn meal test diet.The formal test lasted for 90 days.The initial and final body weights of each pig were recorded,along with average daily feed intake.Towards the end of the experiment,3 pigs were randomly selected from each group based on similar body weights,and 10 mL of blood was collected through the anterior vena cava to measure serum biochemical,antioxidant,and immune indicators.【Result】 ① Compared with control group,the feed-to-gain ratio (F/G) of fattening pigs in group Ⅱ was significantly reduced (P＜0.05),while the final body weight (FBW) and the average daily weight gain (ADG) of group Ⅲ were extremely significantly or significantly reduced (P＜0.01 or P＜0.05).② Compared with control group,the levels of serum globulin (GLB) of fattening pigs in group Ⅱ and serum triglycerides (TG) in group Ⅲ were significantly increased (P＜0.05),while the levels of serum aspartate aminotransferase (AST) in groups Ⅰ and Ⅱ were significantly reduced (P＜0.05).③Compared with the control group,the activity of serum superoxide dismutase (SOD) of fattening pigs in groups Ⅱ and Ⅲ were extremely significantly increased (P＜0.01),while the activities of the serum total antioxidant capacity (T-AOC) and glutathione peroxidase (GSH-Px) in groups Ⅰ and Ⅱ were extremely significantly increased (P＜0.01).The contents of serum malondialdehyde (MDA) in groups Ⅰ,Ⅱ and Ⅲ were significantly reduced (P＜0.01).④Compared with the control group,the serum IgG and IgM levels of fattening pigs in group Ⅱ were significantly increased (P＜0.05).The serum IL-1β content in group Ⅱ was significantly or extremely significantly reduced compared to control group and groups Ⅰ and Ⅲ (P＜0.05 or P＜0.01).【Conclusion】 Adding 10% whole plant silage corn meal to the diet could reduce F/G of fattening pigs,improve their antioxidant capacity and immunity.

【Objective】 The objective of this experiment was to compare the effects of different types of benzoic acid on growth performance,serum biochemical indices,pH in gastrointestinal contents and intestinal flora of weaned piglets.【Method】 A total of 72 Duroc ×Landrace ×York weaned piglets at aged of 23 days were allotted to 3 groups according to body weight (mean body weight 6.44 kg±0.23 kg) with 6 replicates,and 4 pigs (2 barrows and 2 gilts) per replicate.The pigs in control group were fed a basal diet,while in benzoic acid group (BA group) were fed the basal diet supplemented with 0.5% benzoic acid,and in controlled-release-coated benzoic acid group (CBA group) were fed the basal diet supplemented with 0.3% controlled-release-coated benzoic acid.The experiment lasted for 42 days,which was divided into two stages:Days 1 to 14 and days 15 to 42.Body weight of each piglets were weighed on the 1st,15th and 42th day of the trial period,feed intake and the fecal morphology were recorded each day,the blood were collected on the 14th day,and the gastrointestinal chyme and urine were collected on the 42th day,the performance,diarrhea degree,the activities of diamine oxidase (DAO),glutathione peroxidase (GSH-Px),catalase (CAT) and superoxide dismutase (SOD),the contents of malondialdehyde (MDA) and D-lactic acid(D-LA),the pH of gastrointestinal chyme and urine,the count of Lactobacillus,Escherichia coli,total bacteria in ileum and caecum were detected.【Result】 Compared with the control group,① the ADG and ADFI of piglets in 1-42 and 15-42 days were significantly increased in BA and CBA groups (P＜0.05),and the F/G of piglets in CBA group was significantly decreased at all stages (P＜0.05).② The diarrhea index of piglets in 1-14 days was significantly decreased in BA group (P＜0.05),and the diarrhea rate and diarrhea index of piglets in 1-14 and in 15-42 days were significantly decreased in CBA group (P＜0.05).Compared with BA group,the diarrhea index in 1-14 and 15-42 days and the diarrhea rate in 15-42 days were significantly decreased in CBA group (P＜0.05).③ The pH of digesta in stomach, duodenal and urine of piglets in BA group were significantly decreased (P＜0.05),and the pH of digesta in duodenal,jejunum,ileum and cecum of piglets in CBA group was significantly decreased (P＜0.05).④ The levels of DAO and MDA in serum of piglets in both BA and CBA groups were significantly decreased (P＜0.05),and the activities of GSH-Px,SOD and CAT in serum of piglets were significantly increased (P＜0.05),and the effects in CBA group were significantly better than those of BA group (P＜0.05).⑤ The number of Escherichia coli in ileum and cecum and the total number of bacteria in cecum both in BA and CBA groups were decreased,and the differences in CBA group were significant (P＜0.05).The number of Lactobacillus in ileum and cecum in BA and CBA groups were significantly increased (P＜0.05).【Conclusion】 The results suggest that dietary supplementation of 0.5% benzoic acid and 0.3% controlled-release-coated benzoic acid could promote growth performance and improve intestinal barrier function of weaned piglets.The application effects of 0.3% controlled-release-coated benzoic acid were better than that of 0.5% benzoic acid,which had the advantage of reducing cost and increasing efficiency.

Heat stress is one of the important factors affecting animal health and production,and rumen function is closely related to nutrient absorption,body health and production performance of ruminants.Heat stress can easily cause changes in rumen microbial structure,rumen epithelial structure and barrier function,which in turn affects rumen function in ruminants.Current studies have shown that some specific rumen microorganisms,metabolites,and rumen epithelial protein expression in the rumen can be used as potential markers to evaluate the magnitude of heat stress in ruminants.By screening the potential heat tolerance markers of rumen and accurately judging the degree of heat stress in the rumen,it is helpful to better understand the mechanism of heat resistance in ruminants,and is also of great significance for the study of rumen microbial structure,rumen metabolism and rumen function.Therefore,the authors mainly review the effects of heat stress on rumen microbial structure,rumen function and potential heat tolerance markers of rumen,so as to provide theoretical basis and guidance for practical production and application under heat stress conditions.

【Objective】 The study aimed to explore the effects of cardamomamine (CDN) on organ coefficients,serum biochemical indexes,antioxidant capacity and inflammatory factors in chronic heat-stressed Danzhou chickens.【Method】 200 1-day-old Danzhou hens were selected for the experiment and randomly divided into 5 groups:Control group (CON),heat stress group (HS),low-dose (L-CDN),medium-dose (M-CDN),and high-dose cardamom group (H-CDN).Each group had 4 replicates,with 10 chickens per replicate.The hens in CON and HS groups were fed a basic diet,while in L-CDN,M-CDN,and H-CDN groups were added 50,100,and 200 mg/kg cardamom to the basic diet,respectively.The experiment lasted for 42 days,with a pre-trial period of 21 days and a formal trial period of 21 days.During the formal trial period,the HS,L-CDN,M-CDN,and H-CDN groups were kept at a temperature of (36±2) ℃ from 09:00 to 16:00 each day.The rest of the time,the temperature was maintained at (25±2) ℃,while the CON group was kept at a temperature of (25±2) ℃ all day.At the end of 42 days,two Danzhou chickens with similar weights were selected from each replicate for slaughter.Serum was collected to detect biochemical indicators,antioxidant indicators,and serum inflammatory factors.The liver,spleen,thymus,and bursa of Fabricius were collected to calculate organ coefficients.【Result】 ①The levels of alanine aminotransferase (ALT) in the serum of Danzhou chickens in M-CDN and H-CDN groups were significantly lower than those in HS group (P＜0.05), while the levels of aspartate aminotransferase (AST), lactate dehydrogenase (LDH), total cholesterol (TC), and triglycerides (TG) in the serum of chickens in L-CDN, M-CDN, and H-CDN groups were significantly lower than those in HS group (P＜0.05). Except for the significantly lower TG content in H-CDN group compared to CON group (P＜0.05), there was no significant difference between H-CDN group and CON group (P＞0.05). ② Compared with HS group,the Fabricius index of Danzhou chickens in M-CDN and H-CDN groups were significant increased (P＜0.05), while the spleen index in H-CDN group was significant increased (P＜0.05). The liver index of all three experimental groups were decreased significantly (P＜0.05), and there was no significant difference between them and CON group (P＞0.05). Among them, the Fabricius index of M-CDN and H-CDN groups was significantly higher than that of L-CDN group (P＜0.05), and the spleen index of H-CDN group was significantly higher than that of L-CDN and H-CDN groups (P＜0.05). ③ Compared with HS group, the serum malondialdehyde (MDA) content of Danzhou chickens in the three experimental groups was significantly reduced (P＜0.05). The serum superoxide dismutase (SOD) activity of Danzhou chickens in M-CDN group and the serum SOD, catalase (CAT), and glutathione peroxidase (GSH-Px) activities of Danzhou chickens in H-CDN group were significantly increased (P＜0.05), and there was no significant difference compared with CON group (P＞0.05). The total antioxidant capacity (T-AOC) of the serum of Danzhou chickens in three experimental groups was significantly increased (P＜0.05), but still significantly lower than that of CON group (P＜0.05). The SOD activity in M-CDN and H-CDN groups was significantly higher than that in L-CDN group (P＜0.05), while the CAT and GSH-Px activities in H-CDN group were significantly higher than those in L-CDN and M-CDN groups (P＜0.05). ④ Compared with HS group, the serum interleukin-1β(IL-1β) content of Danzhou chickens in all three experimental groups were significantly reduced (P＜0.05). The serum IL-6 and tumor necrosis factor-α(TNF-α) contents of Danzhou chickens in M-CDN and H-CDN groups were significantly reduced, while the IL-10 levels were significantly increased (P＜0.05), and there was no significant difference compared to CON group (P＞0.05). There was no significant difference between the three experimental groups (P＞0.05).【Conclusion】 Cardamom added to feed could regulate some serum biochemical indexes of Danzhou chickens under heat stress,promote the development of immune organs to enhance immune ability,alleviate heat stress-induced liver injury,improve antioxidant ability,and alleviate heat stress-induced immune stress and inflammation.The appropriate supplement level of cardamom for chronic heat stress in Danzhou chickens was 200 mg/kg.

【Objective】 This experiment was aimed to study the effects of dietary chlorogenic acid and leucine supplementation on carcass traits,meat quality,backfat enzyme activity and serum biochemical indexes of fattening pigs and to explore whether there was a synergistic effect of chlorogenic acid and leucine.【Method】 A total of 24 (half male and half female) healthy crossbred fattening pigs (Duroc×Landrace×Large White) with a similar body weight (68.3 kg±1.3 kg) at 115 days old were selected,which were randomly divided into three groups,with eight replicates per group and one pig per replicate.The control group (group A) was fed a basic diet,while the chlorogenic acid group (group B) and the leucine＋chlorogenic acid group (group C) fed the basic diet supplemented with 0.05% chlorogenic acid and 0.25% leucine＋0.025% chlorogenic acid,respectively.The experiment lasted for 25 days.After the experiment,the pigs were weighed to calculate growth performance.Blood was collected from anterior vena cava and serum biochemical indexes were detected.After slaughter,carcass traits were determined,and the meat quality of longissimus dorsi muscle,lipid metabolism-related enzyme activity of backfat were measured.【Result】 Compared with group A,the growth performance of groups B and C showed no significant differences (P＞0.05).The slaughter rate and lean meat rate of fattening pigs in group B were significantly increased (P＜0.05),lean meat rate of group C was significantly increased (P＜0.05),and backfat thickness was significantly decreased (P＜0.05).45 min after slaughter,the pH of longissimus dorsi muscle in groups B and C was significantly increased (P＜0.05),the redness value of longissimus dorsi muscle in group B was significantly decreased (P＜0.05),and the crude fat content of biceps femoris muscle in groups B and C was significantly increased (P＜0.05).The serum glucose and malondialdehyde (MDA) contents in group B were significantly decreased (P＜0.05),and the activities of acyl-coenzyme A-cholesterol acyltransferase (ACAT) and lipoprotein lipase (LPL) in back adipose tissue were significantly increased (P＜0.05).Serum glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities and total antioxidant capacity (T-AOC) in group C were significantly increased (P＜0.05),and MDA content was significantly decreased (P＜0.05).The activities of ACAT,LPL and hormone sensitive lipase (HSL) in back adipose tissue were significantly increased (P＜0.05).Compared with group B,the slaughter rate of group C was significantly decreased (P＜0.05),the activities of serum GSH-Px,SOD and T-AOC level were significantly increased (P＜0.05),and the MDA content was significantly decreased (P＜0.05).【Conclusion】 Dietary 0.05% chlorogenic acid could increase the dressing percentage and carcass lean meat rate of fattening pigs.Adding 0.25% leucine and 0.025% chlorogenic acid could significantly promote intramuscular fat deposition,reduce backfat deposition,and improve antioxidant capacity.

【Objective】 The aim of this experiment was to investigate the effects of dietary apparent metabolizable energy (AME) level on growth performance,slaughter performance and plasma biochemical indices of Sansui ducks aged 1 to 28 days,and to estimate AME requirement based on regression model.【Method】 A total of 336 healthy one-day-old Sansui male ducks were divided into 6 groups with 7 replicates per group and 8 ducks per replicate.Ducks were fed corn-soybean meal diets with AME of 10.92,11.38,11.80,12.26,12.68 and 13.14 MJ/kg,respectively.The experiment period was 28 days.At the end of the experiment,the production performance,slaughter performance and plasma biochemical indexes were measured.【Result】 Dietary AME level significantly affected daily gain,feed conversion ratio and abdominal fat percentage (P<0.05),and with the increase of AME level,daily gain showed a linear and quadratic trend of increasing (P<0.05),feed conversion ratio showed a linear trend of decreasing (P<0.05),and abdominal fat percentage showed a linear trend of increasing (P<0.05).Dietary AME level had no significant effects on total energy intake,breast muscle percentage,leg muscle percentage,liver index and gizzard index (P＞0.05).Dietary AME level significantly affected plasma aspartate aminotransferase (AST) activity (P<0.05),but had no significant effects on other plasma biochemical indices (P＞0.05). Based on broken-line linear regression,the minimum AME requirements of Sansui duck aged 1 to 28 days for optimal feed conversion ratio,daily gain,and abdominal fat percentage were 11.83,11.42 and 12.24 MJ/kg,respectively.【Conclusion】 Dietary 11.38 MJ/kg AME could effectively improve daily gain,feed conversion ratio,and adbominal fat percentage.The optimal AME requirement of Sansui ducks aged from 1 to 28 days was 11.42 to 12.24 MJ/kg.

Squalene is a natural all-trans triterpene compound,which is the precursor of sterols,hopanes and triterpenes,and plays a key role in metabolism.Squalene plays an important role in improving animal growth,protecting intestinal health and improving stress resistance due to its good antioxidant,anti-inflammatory and antibacterial properties.Squalene can regulate nuclear factor E2-related factor 2(Nrf2) to promote the transcription of corresponding antioxidant enzymes and detoxification enzyme genes, improve the antioxidant level of the body, down-regulate the inflammatory response pathway through mitogen-activated protein kinase (MAPK),nuclear factor kappa-B (NF-κB),matrix metalloproteinases (MMPs) and peroxisome proliferator-activated receptor γ (PPARγ), and increase the expression of anti-inflammatory factors to terminate the inflammatory response.In terms of animal disease resistance,squalene can improve the immune function of animals,increase the level of antibodies in the serum,and thus improve the ability of animals to resist pathogens and poisons.In terms of maintaining intestinal health,squalene can effectively improve intestinal antioxidant function,maintain intestinal mechanical barrier form and permeability homeostasis,provide a place for intestinal probiotics to attach,and then significantly improve intestinal tolerance and digestive function of livestock and poultry.In addition,squalene plays an important role in improving animal reproductive performance,such as inducing estrus,improving semen quality and improving embryo health.In conclusion,squalene has good application value and research potential as a natural antibiotic substitute in animal production.In this paper,the development and biological functions of squalene were reviewed in order to provide theoretical basis for the incremental expansion of squalene production and its application in animal production.

【Objective】 The effect of catalase (CAT) on the production performance,antioxidant capacity,cecal microbe and metabolites in broilers was investigated in this study,for providing theoretical foundation in the application of CAT in animal production.【Method】 600 1-day-old Yellow Feathered broiler chickens were selected and randomly divided into 5 groups:Blank control group (CON),antibiotic group (Anti),and low,medium and high-dose CAT groups (CAT1,CAT2 and CAT3),with 6 replicates in each group and 20 chickens in each replicate.Among them,CON group was fed with basic feed,Anti group added 50 mg/kg chloramphenicol to the basic diet,and CAT1,CAT2 and CAT3 groups were supplemented with 100,150 and 200 U/kg CAT in the basic diet,respectively.The feeding trial lasted for 42 d.After the experiment,the production performance was measured,and serum and cecal chyme were collected for measuring antioxidant indicators,microbial communities and metabolite content.【Result】 Compared with CON group,the ADG and ADFI of each dose group of CAT were significantly increased (P＜0.05),reaching the level of the Anti group.However,increasing the CAT dose did not further improve these indicators.Compared with CON group,the serum CAT and superoxide dismutase activities were significantly increased in all dose groups of CAT and Anti group (P<0.05).The levels of malondialdehyde,protein carbonyl and 8-hydroxydeoxyguanosine were significantly reduced (P＜0.05),and the 8-hydroxydeoxyguanosine levels in all dose groups of CAT were consistent with those in Anti group.Cecal microbiota analysis showed that compared with CON group,the number of Proteobacteria,Lactobacillus and Faecalibacterium in each dose group of CAT significantly increased (P<0.05),but the number of Clostridium decreased significantly (P＜0.05).Compared with CON group,the levels of lactic acid,acetic acid and isovaleric acid in the cecal contents of each dose group of CAT were significantly increased (P<0.05),while the content of valeric acid was significantly reduced (P＜0.05);The levels of methylamine,tryptamine,putrescine,cadaverine,and total amine in each dose group of CAT were significantly reduced (P＜0.05);The levels of spermidine were significantly reduced in CAT2 and CAT3 groups (P<0.05),and a comparative analysis of CAT dose effects showed that the levels of putrescine,cadaverine and total amine in CAT2 and CAT3 groups were significantly lower than those in CAT1 group (P＜0.05).【Conclusion】 Adding 150 or 200 U/kg CAT to feed could promote animal growth and health.

【Objective】 The aim of this study was to explore the difference of nutritional value of Schizothorax wangchiachii(S. wangchiachii) with different sizes.【Method】 Three S. wangchiachii of large size (425.27 g±14.99 g) and 15 S. wangchiachii of small size (72.63 g±11.31 g) were selected.The back muscles (Among them,S. wangchiachii of small size randomly mixed into one sample every 5) were taken to measure moisture content,crude ash content,crude protein,and crude fat content.The composition and content of amino acids were determined by automatic amino acid analyzer,and the composition and content of fatty acids were determined by gas chromatograph.Essential amino acid nutrition was evaluated by amino acid score (AAS),chemical score (CS) and essential amino acid index (EAAI).【Result】 There were no significant differences in moisture and crude ash contents in muscle in S. wangchiachii of different sizes (P＞0.05),and the contents of crude protein,crude fat,total amino acid content(ΣTAA),total essential amino acid content(ΣEAA),and total fresh amino acid content(ΣDAA) in the muscle of the fish in large size group were significantly lower than those of small size group (P＜0.05).The values of ΣEAA/ΣTAA in fish muscle protein of two different sizes were 0.41 and 0.42,respectively,both greater than 0.4.The values of ΣEAA/ total non essential amino acids (ΣNEAA) were 0.71 and 0.72,respectively,both greater than 0.6.Methionine + cystine was the first limiting amino acid in muscle of two different sizes of S. wangchiachii,followed by valine.The contents of saturated fatty acids and monounsaturated fatty acids in small size group were significantly lower than those in large size group (P＜0.05),but the contents of polyunsaturated fatty acids,EPA+DHA,ω-3,ω-6 fatty acids in small size group were significantly higher than those in large size group (P＜0.05).【Conclusion】 From the contents of crude protein and crude fat,nutritional evaluation of essential amino acids and nutritional value of fatty acids,small size S. wangchiachii had higher nutritional value than those of large size fish.

Flavour is both an important part of the sensory evaluation of raw milk and a comprehensive expression of the intrinsic quality of dairy products.Corresponding to the senses of smell,taste and touch in human,the flavour profile of raw milk can be analyzed in three dimensions:Odour,taste and mouthfeel,respectively.Volatile compounds are not only the main source of flavour, but also a very important flavour contributor, only some of the volatile compounds contribute to the flavour formation.The main volatile compounds in raw milk include terpenoids,acids (such as C4-C12 fatty acids),ketones (such as methyl ketones),aldehydes (such as nonenal and heptenal),esters (such as γ-lactone),phenols (such as p-cresol) and sulfides (such as dimethyl sulfide).Volatile compounds are the determining factor in the flavour of raw milk,but raw milk is highly sensitive and easily disturbed by chemical substances,which makes the flavour of raw milk is difficult to control objectively,therefore flavour is highly susceptible to the quality of dairy products.Therefore,on the basis of ensuring the fresh flavour,it is necessary to strictly control the interference of various external factors on the flavour.Raw milk flavour is closely related to a variety of factors,such as diet,drinking water and barn environment.At present,diet is considered to be the most important and sensitive factor affecting the flavour of milk and dairy products,and the nutrients such as carbohydrates,fats and proteins in diet can affect the flavour substances in milk through different ways,thereby changing the flavour of raw milk.In summary,the diet largely determines the composition of volatile compounds that affect the flavour of raw milk.In this paper,the flavour characteristics of raw milk were elaborated from three dimensions of odour,taste and mouthfeel,and the main volatile compounds in raw milk and the effects of diet,drinking water and barn environment on the flavour of raw milk were reviewed,in order to provide a theoretical basis for the flavour and quality control of raw milk.

【Objective】 This study was aimed to investigate the association between the polymorphism of lipoprotein lipase (LPL) and Nudix hydrolase 3 (NUDT3) genes with intramuscular fat (IMF) content and backfat thickness in Beijing Black pigs,in order to guide the breeding of Beijing Black pigs at the molecular level.【Method】 Using 347 samples of the longissimus dorsi muscle in Beijing Black pigs,the IMF content and back fat thickness data were measured,and the high and low IMF content was grouped.Primers were designed based on NUDT3 and LPL gene sequences of pigs,and PCR amplification and sequencing were performed.The sequencing results were analyzed using SeqMan in DNAStar softwares.Duncan’s multiple test statistical analysis was used to analyze the association between different genotypes of NUDT3 and LPL genes and IMF content and backfat thickness in Beijing Black pigs.The gene expression difference was analyzed using Real-time quantitative PCR.【Result】 There were 1 missense mutation,3 synonymous mutations and 3 3'-UTR mutations in LPL gene,and 6 3'-UTR mutations in NUDT3 gene.The c.431 G＞C of LPL gene CDS was significantly associated with 6-7 rib back fat thickness,c.647 G＞C was significantly associated with thoracolumbar junction back fat thickness,and c.637 A＞G was significantly correlated with 6-7 rib back fat thickness,at the thoracolumbar junction back fat thickness and lumbar sacral junction back fat thickness in Beijing Black pigs (P＜0.05).The c.1683 A＞G of LPL gene 3'-UTR region was significantly correlated with backfat thickness at the thoracolumbar junction;c.1689 C＞A was significantly correlated with IMF content in Beijing Black pigs (P＜0.05).The mutation sites of NUDT3 gene all occured in 3'-UTR region,c.7204 G＞T was significantly correlated with IMF content and back fat thickness at the lumbar sacral junction,c.6068 C＞G was significantly correlated with IMF content and 6-7 rib back fat thickness,c.5729 G＞A was significantly correlated with IMF content in Beijing Black pigs (P＜0.05).Real-time quantitative PCR analysis showed that there was no significant difference in the expression of NUDT3 gene c.7204 G＞T between the high and low IMF content groups,and there was also no significant difference in the expression of LPL gene c.1689 C＞A between CC and AA genotypes (P＞0.05).【Conclusion】 The mutation sites of LPL and NUDT3 genes were significantly associated with IMF content in Beijing Black pigs,providing new molecular markers for meat quality breeding in Beijing Black pigs.

【Objective】 This study was aimed to discuss the effects of different doses of compound Chinese medicine extract on semen quality,reproductive hormone,testis morphology and sperm acrosome integrity in male rabbits,and provide a theoretical basis for improving reproductive performance of male rabbits and developing traditional Chinese medicine feed additives with this kind of function.【Method】 Twenty-four New Zealand male rabbits were randomly divided into 4 groups,with 6 rabbits in each group.The male rabbits in control,Ⅰ,Ⅱ and Ⅲ groups were given 0,150,300 and 600 mg/kg compound Chinese medicine extract,respectively.The experimental period was 12 weeks,and the feeding period was 8 weeks.Blood samples were collected at the 4th,8th and 12th week,and the semen samples were collected at the 8th,10th and 12th week for the determination of serum sex hormone and semen quality.【Result】 Compared with control group,the sperm density,sperm viability and the number of grade A sperm of rabbits in experimental group at 8-12 weeks were significantly increased (P＜0.05),and the rate of acrosome integrity of rabbits in Ⅲ group was significantly increased (P＜0.05).The sperm deformity rate of rabbits in Ⅲ group was significantly lower than that in control group at 10-12 weeks (P＜0.05).The surface capsule of the testicular tissue of rabbits in experimental group was composed of thick irregular dense connective tissue and a large number of basement membranes of seminiferous tubules were clearly visible,the epithelium of seminiferous tubule was composed of spermatogenic cells and Sertoli cells.There were a lot of spermatogonia and differentiated spermatocytes in the epithelium of seminiferous tubule.There are abundant sperm in lumen and no obvious abnormalities in stroma.At 4-12 weeks,The contents of follicle stimulating hormone (FSH),luteinizing hormone (LH) and testosterone (T) of serum in Ⅱ and Ⅲ groups were significantly higher than that in control group (P＜0.05).【Conclusion】 Compound Chinese medicine extract could improve the quality of male rabbit semen,increase the rate of sperm acrosome integrity,reduce the sperm deformity rate,improve the morphology of testis,promote the development of reproductive organs,and increase the contents of FSH,LH and T in serum.The 600 mg/kg compound Chinese medicine extract had a significant effect on reproductive physiology of male rabbits,and had a good application prospect in practical production.

Non-coding RNA (ncRNA) is an RNA molecule transcribed from the genome that does not code for proteins,including microRNA (miRNA),long non-coding RNA (lncRNA),circular RNA (circRNA) and RNA of unknown function.The function of ncRNA is to perform various regulatory functions in cells,such as participating in gene expression regulation,DNA repair,cell division and other biological processes.ncRNA can target and regulate gene transcription which in turn modulates muscle growth and development.Myofibers are the basic structural unit of muscle,and different muscle fiber-type of organisms act differently.Moreover,meat comprising different muscle fiber types varies in color,texture,nutrient content and water retention.Hence,it is significant to conduct research on muscle fiber-type conversion to achieve improved meat quality.Whereas,as a critical regulator in the transformation of myofiber types,ncRNA is a research hotspot that can not be ignored.In this paper,based on three kinds of ncRNA,which are miRNA,lncRNA and circRNA,the authors reviewed the concepts of ncRNA and muscle fiber-type and the current research status of ncRNA regulating the muscle fiber-type conversion,in order to provide important references for the application of ncRNA in improving the meat quality of livestock and poultry.

【Objective】 The experiment was conducted to explore single nucleotide polymorphisms (SNP) of matrix metallopeptidase 2 (MMP2) gene and its correlation with reproductive traits,and to screen genetic markers related to reproductive traits of Kele pigs.【Method】 A total of 212 healthy partriparous Kele sows were selected and the SNP of MMP2 gene were screened by direct sequencing.Using RNAfold online software to predict the secondary mRNA structure of different haplotypes of MMP2 gene.SPSS 22.0 software was used to analyze the correlation between MMP2 gene SNP and reproductive traits of Kele pigs.【Result】 1 SNP (g.30062403 C＞T) was detected in exon 2 and 2 SNPs (g.30065309 C＞T and g.30065312 C＞T) were detected in exon 4 of MMP2 gene,all with 3 genotypes (CC,CT and CT).The results of genetic analysis showed that g.30062403 C＞T was a low polymorphism,and g.30065309 C＞T and g.30065312 C＞T were moderate polymorphisms.Chi-square fitness test results showed that g.30062403 C＞T significantly deviated from Hardy-Weinberg equilibrium (P<0.05),and the other 2 SNPs were in Hardy-Weinberg equilibrium (P＞0.05).Linkage disequilibrium analysis showed that there was strong linkage disequilibrium between g.30065309 C＞T and g.30065312 C＞T.The analysis of haplotype mRNA secondary structure showed that the mRNA secondary structure of haplotype H1 was the same as the original sequence,and the other 3 haplotypes all caused changes in the mRNA secondary structure and minimum free energy.The results of association analysis showed that the litter number of TT genotype at g.30062403 C＞T was significantly higher than that of CC genotype,and the weaning litter weight was significantly higher than that of CT genotype (P<0.05).The number of weaned piglets of CC genotype at g.30065309 C＞T was significantly higher than that of TT genotype (P<0.05). The number of live litter and weaned piglets of TT genotype at g.30065312 C＞T was significantly higher than that of CT genotype (P<0.05).The diplotype H2H2 was the dominant diplotype of litter number,litter born alive,weaned piglets and weaning litter weight,while H1H4 was the dominant diplotype of litter weight at birth.【Conclusion】 There was a significant correlation between of MMP2 gene 3 SNPs and litter number,litter born alive,litter weight at birth,weaned piglets and litter weight at weaning,which could be used as genetic markers for molecular breeding.

【Objective】 This study was conducted to explore the effects of compound traditional Chinese medicine supplementation on production performance and reproductive function of forced molting laying hens.【Method】 A total of 600 forced molting Lohmann Pink laying hens at 980 days were randomly divided into 4 groups with 5 replicates per group and 30 laying hens per replicate,which were blank group (group C) and low-dose (L),medium-dose (M) and high-dose (H) compound traditional Chinese medicine groups,respectively.The hens in group C were fed a basal diet,and the hens in groups L,M and H were fed the basal diet supplemented with 0.25%,0.5% and 1% compound traditional Chinese medicine,respectively.The experiment lasted for 42 days.Laying rate,egg weight,feed intake and feed to egg ratio were recorded every day during the experiment.Blood,ovary and oviduct tissues of laying hens were collected on day 21 and 42,serum reproductive hormone levels were detected,reproductive organ index was calculated,and tissue sections of ovary and oviduct tissues were made to observe the tissue morphology.【Result】 After different dosages of compound traditional Chinese medicine were added to the basic diet,compared with group C,the laying rate of forced molting laying hens in all dosages of compound traditional Chinese medicine groups was significantly increased (P<0.05),and the ratio of feed to egg was significantly decreased (P<0.05).Day 42 of the test,the phenomenon of heterophil cell infiltration in the ovary of L and H groups was reduced,and the ovary index of each dose compound traditional Chinese medicine group was significantly increased on day 21 (P<0.05),and the ovary and oviduct index of H group was significantly increased on day 42 of the test (P<0.05).The contents of progesterone and prolactin in M and H groups were significantly increased on day 21 (P<0.05),the contents of progesterone,prolactin and follicle-stimulating hormone in M group were significantly increased on day 42 (P<0.05),and the contents of follicle-stimulating hormone in H group were significantly increased (P<0.05).【Conclusion】 Dietary addition of compound traditional Chinese medicine could improve the production performance and reproductive function of forced molting laying hens.

【Objective】 The purpose of this experiment was to obtain the effective genotypes for the identification of foreign ternary pig breeds and to establish a systematic identification scheme for the identification of foreign ternary pig germplasm.【Method】 In this study,homozygous loci of 613 Duroc pigs Illumina 60K chip,503 Landrace pigs Illumina core No.1 50K chip and 10 Yarkshire pigs resequencing data were statistically analyzed,and specific single nucleotide polymorphism (SNP) of Duroc,Landrace and Yarkshire pigs were screened.Ear tissue DNA of 120 pigs with different strains and varieties was used as template for PCR amplification and sequencing to verify the specific SNP,and the effective specific gene loci were screened.The identification schemes of foreign ternary purebred,binary,ternary and multiple backcross pigs were established through loci combination analysis.【Result】 A total of 6 SNPs specific to Landrace pigs,8 SNPs specific to Duroc pigs and 1 SNP specific to Yarkshire pigs were obtained by comparison of chip data.The effective SNPs were SSC13:57293969 A＞G,SSC11:47494227 T＞G,SSC6:110653413 T>C and SSC6:110653219 T＞C,respectively.According to the mutation types and genetic rules of the loci,the germplasm identification scheme of three-loci joint analysis was developed,and several pig breeds such as Landrace pure breed pigs and multi-backcross Landrace pigs were identified.【Conclusion】 In this study, the identification schemes of foreign ternary pure breed,binary,ternary and multiplex backcross pig breeds was developed by pig-specific SNP.The results provided reference and technical support for germplasm identification of foreign ternary pigs.

In vitro maturation is the main way for mammalian embryo engineering to obtain mature oocytes.The quality of oocytes directly affects early embryonic development,establishment and maintenance of pregnancy,and fetal development.The large number of small follicles on the surface of mammalian ovaries can provide oocytes that apply to embryo engineering,but the oocytes obtain from the ovary are at different stages that shows different mature capacity,and the quality of in vitro maturation of oocytes from small follicles are poor and deficient in developmental ability.How to improve the in vitro maturation ability of small follicle oocytes to fully tap into the genetic resources of excellent female animals and improve the efficiency of embryo engineering has become a research hotspot.Based on this,the review summarizes the research progress of in vitro maturation of small follicle oocytes in mammalian,compares the differences in accumulation of oocyte material,the differences in the composition and content of follicular fluid,and the differences between follicular granulosa cells and cumulus cells in large and small follicles,analyzes the reasons for the poor quality of mammalian small follicle oocytes in vitro maturation,analyzes and summarizes the measures to improve the developmental ability of mammalian small follicle oocytes in vitro maturation,including inhibition of nuclear maturation,pre-maturation culture of oocyte and co-culture of oocytes with granulosa and cumulus cells,which aimes at providing theoretical guidance for the research of the development regulation mechanism of small follicle oocytes and the improvement of developmental capacity.

【Objective】 In April 2023,an acute outbreak of infectious disease in goslings with mortality up to 25% occurred in a commercial goose farm in Xinxiang city of Henan province.To determine the potential causative agent of this disease,anatomical examination and laboratory pathogen diagnosis of dead goslings were performed.【Method】 The tissue samples of livers,spleen,kidneys,small intestine and cardiac blood and fibrous exudate from the outer layer of the liver were collected.Bacterial isolation and cultivation,Gram staining microscopy observation,16S rRNA gene identification and drug sensitivity testing were performed on samples of cardiac blood and fibrous exudate from the outer layer of the liver to determine the bacterial infection and drug sensitivity of infected geese. PCR/RT-PCR method was used to test the nucleic acid of common gosling viral infectious disease pathogens,and sequencing analysis was performed on the main structural protein genes of positive pathogens to determine the viral pathogens and their molecular epidemiology in infected geese.【Result】 In the bacterial test,the results showed that the isolated bacteria could grow as the morphology of translucent,neat edges,smooth colonies on blood agar medium.Gram-negative single and paired bacteria could be observed after Gram staining,which were consistent with the characteristics of Riemerella anatipestifer (RA).PCR amplification of 16S rRNA gene and BLAST analysis were further confirmed the isolate was RA.The drug sensitivity test revealed that the RA strain was highly sensitive to ceftriaxone and cefotaxime,and resistance to amoxicillin,tetracycline and polymyxin B. PCR and RT-PCR detection results for the common pathogens of goslings showed that all the samples were positive for Goose parvovirus (GPV) and genotype Ⅰ Goose astrovirus (GAstV-1), and no corresponding nucleotide fragments were observed for GAstV-2,Avian influenza virus (AIV) and Goose reovirus (GRV). The two identified virus strains were designated as GPV/HN-2023 and GAstV-1/HN-2023,respectively.Furthermore,the analysis of the main structral protein gene of GPV/HN-2023 and GAstV-1/HN-2023 revealed that GPV/HN-2023 was closely related to DY-16 strain and belonged to DY-16-like strain,with two unique amino acid mutations D248E and V314L in VP3 protein.GAstV-1/HN-2023 was closely related to GAstV-1 strains and grouped into GAstV-1 branch,with three unique amino acid mutations G47R,S207G and A628T in ORF2 protein.【Conclusion】 This study identified the mixed infection of GPV,GAstV-1,and RA as the cause of gosling disease in the goose farm through comprehensive diagnostic methods.The drug resistance of pathogenic bacteria RA and the genetic evolution and variation characteristics of viral pathogens GPV and GAstV-1 were analyzed,providing theoretical reference for the scientific prevention and control of gosling disease in Henan region.

【Objective】 Vitamin D₃ (VD₃) is an important steroid hormone involved in the regulation of various physiological activities in animals.Its bioactive metabolite 1,25 (OH)₂D₃ performs the biological function by binding to vitamin D receptor (VDR).Cloning of goose VDR gene CDS and preparation of its polyclonal antibody would provide the basic support for further research on the molecular mechanism of VDR-mediated VD₃ regulation of various physiological activities in geese.【Method】 The CDS sequence of VDR gene in geese was obtained by the gene cloning method,and the structure and function of the encoded protein were predicted by bioinformatic methods.The recombinant plasmid of VDR gene in geese was constructed by gene synthesis and sub-cloning methods,and then was transformed into Escherichia coli BL21(DE3) competent cells.The recombinant VDR protein in geese was obtained by induction with IPTG and purification with a nickel column.The rabbit anti-goose VDR polyclonal antibody was prepared with the purified recombinant VDR protein as an immunogen.The antibody titer was detected by indirect ELISA,and the antibody specificity was detected by Western blotting.【Result】 The CDS sequence of VDR gene in geese was successfully cloned with a length of 1 356 bp and was predicted to encode 451 amino acid residues.The coding protein shared a high identity with chicken VDR protein (91.4%),and no signal peptide and transmembrane domain were observed.The coding protein contained an N-terminal nuclear localization sequence and two conserved domains of DNA binding domain and ligand binding domain,suggesting that it belonged to the steroid/thyroid hormone receptor superfamily.The recombinant plasmid pET-30a(+)-VDR was successfully constructed and the recombinant VDR protein in geese was expressed.The purity of the purified recombinant protein was more than 90% and its size was 52 ku as expected.The rabbit anti-goose VDR polyclonal antibody was successfully prepared,and the titer was greater than 1∶1 093 500.Except for the liver tissue,the polyclonal antibody was able to recognize two isoforms of native VDR protein in kidney,duodenum,jejunum and ileum in laying geese.【Conclusion】 The CDS region of VDR gene in geese was successfully cloned,and the rabbit anti-goose VDR polyclonal antibody was prepared.The polyclonal antibody had a high specificity and could be used for further study of VDR-mediated biological function in geese.

In recent years,with the continuous development of mucosal vaccine and immunization technology,the reports of atomized vaccine and corresponding vaccination technology have gradually increased.Atomized vaccine is a kind of vaccine that makes liquid and solid immunogen into droplets (spray immunization) or aerosol particles (aerosol immunization) through atomizing device,and enters human and animal respiratory tract to induce the protective response.Atomized vaccine can induce mucosal immune response better,and at the same time,avoid the risk of disease transmission and pain caused by injection.Since the 1950s,the research on the spray immunization of Newcastle disease vaccine in chickens has opened the prelude of animal respiratory immunity.After that,the spray vaccination of infectious bronchitis vaccine was carried out.At present,the spray immunization of many poultry vaccines has been widely used.The research on aerosol immunization of swine vaccine started late and was more difficult.Since the end of 1970s,the corresponding aerosol immunization research has been carried out on classical swine fever vaccine,Actinobacillus pleuropneumoniae vaccine and Mycoplasma hyopneumoniae vaccine.Compared with animal vaccines,there are more reports on aerosol immunization of human vaccines,mainly focusing on measles vaccine,tuberculosis vaccine and influenza vaccine.Since 2019,the aerosol immunization of COVID-19 vaccine has developed rapidly,and it has been approved for emergency use in China.In this paper,the research status of atomized vaccines for animal and human is systematically stated,and the physical and chemical parameters,safety,tendency in vivo,local immune response in lung and protection effect of various atomized vaccines were summarized,and the future research on vaccine dosage forms,components,atomization generation devices,quality control,immunization program and effect evaluation were put forward.This review could provide a reference for the further research on respiratory mucosal vaccines.

【Objective】 This study was aimed to investigate the effect of deletion of galactosyltransferase gttA gene on the anchoring of surface proteins and pathogenicity of Listeria monocytogenes,so as to provide a reference to the pathogenic mechanism research of Listeria monocytogenes.【Method】 Homologous recombination methods were used to construct the gttA gene deletion strain, the growth and motility of the parental strain ScottA,the deletion strain ΔgttA and the complement strain CΔgttA were analyzed.The effects of gttA gene deletion on the anchoring of two surface virulence proteins InlB and ActA in Listeria monocytogenes were investigated by Western blotting.The adhesion and invasion test of colorectal adenocarcinoma cell line (Caco-2) and mouse infection test were used to evaluate the effect of gttA gene deletion on the adhesion and invasion ability and pathogenicity of Listeria monocytogenes.【Result】 The growth and motility of Listeria monocytogenes were not affected by the deletion of gttA gene.Western blotting results showed that after the deletion of gttA gene,the anchor quantities of virulence proteins InlB and ActA on the bacterial cell wall surface were extremely significantly decreased (P＜0.01).The results of cell adhesion and invasion assay showed that ΔgttA had an extremely significantly lower adhesion and invasion ability to Caco-2 cells than ScottA and CΔgttA (P＜0.01).The results of mouse infection test showed that the colonization ability of Listeria monocytogenes in liver and spleen of mice was extremely significantly or significantly weakened due to the deletion of gttA gene (P＜0.01 or P＜0.05).After the gttA gene was replenished,the level of adhesion and invasion in Caco-2 cells,and the colonization ability of the bacteria in liver and spleen were restored to the level of the parental strain ScottA.【Conclusion】 The deletion of gttA gene did not affect the growth and motility of Listeria monocytogenes,but affected the anchoring of the major virulence proteins on the surface of Listeria monocytogenes,which in turn weakened the ability of the bacterium to invade Caco-2 cell line and the pathogenicity of the bacterium in mice.

【Objective】 Two strains of monoclonal antibodies of Porcine rotavirus (PoRV) were used to establish PoRV colloidal gold test strip for rapid detection of PoRV.【Method】 The 40 nm colloidal gold particles were used to label the monoclonal antibody 4G5.The monoclonal antibody 5G4 and sheep anti-mouse IgG were coated on the surface of nitrocellulose membrane as the detection line and the control line,respectively.The colloidal gold test strip was established by optimizing the optimal pH of colloidal gold solution,the optimal usage amount of the labeled antibody and the detection antibody.The sensitivity,specificity,repeatability and stability of the strip were assessed.The coincidence rate of the strip and RT-PCR was calculated by detecting 50 pig fecal samples,and then the strip was put to clinical use by detecting 1 249 clinical samples.【Result】 The optimal parameters of the test strip were as follows:The optimal pH of colloidal gold solution was 8.0,the optimal usage amounts of labeled antibody 4G5 and detection antibody 5G4 were 14.4 μg/mL and 1 mg/mL,respectively.The strip could detect a 1∶2 000 dilution of PoRV OSU strain (genotype G5),the sensitivity was 10^4.5 TCID₅₀/mL,and the detection results of PoRV G9,G3 and G4 strains were all positive.And no cross reaction occured with Transmissible gastroenteritis virus (TGEV),Porcine epidemic diarrhea virus (PEDV),Porcine deltacoronavirus (PDCoV),Porcine pseudorabies virus (PRV) and Porcine circovirus type 2 (PCV2).The assay results showed that the strip had good repeatability in and between batches and stability.50 pig fecal samples were detected by the strip and RT-PCR,the results showed that the positive coincidence rate was 97.5% (39/40),the negative coincidence rate was 100% (10/10),and the overall coincidence rate was 98% (49/50).1 249 clinical samples which were collected from pig farms in Henan and Shanxi provinces in 2022 were detected by the test strip,and the positive rate of PoRV was 5.2%.【Conclusion】 The colloidal gold strip developed in this study had good sensitivity,specificity and repeatability,and high coincidence rate with RT-PCR method,and could be used for PoRV rapid detection in industrialized pig farm.

African horse sickness (AHS) is an acute and subacute insect borne infectious and highly contagious and rapidly fatal infectious disease of equids caused by African horse sickness virus (AHSV).It is characterized by symptoms such as fever,subcutaneous swelling,and internal organ hemorrhaging.AHS is classified as a notifiable disease by the World Animal Health Organization and is also categorized as Class Ⅰ animal diseases in China.Recent data from the World Animal Health Information System indicates that AHS has been spreading in Africa and neighboring Southeast Asian regions,which are in close proximity to China.The situation is considered extremely severe.China’s official strategy for the prevention and control of AHS involves strict monitoring of equids with a focus on border areas and ports,along with the timely removal of infected animals.This article provides a comprehensive overview of the advancements and recent research developments in the field of AHS diagnostics,both in terms of pathogenetics and serology.Pathogenetic diagnostic techniques encompass virus isolation,RT-PCR,isothermal amplification,and gene chip technology.Virus isolation remains the classical gold standard for definitive diagnosis.RT-PCR and Real-time quantitative RT-PCR techniques are commonly employed in laboratory settings.Isothermal amplification methods such as loop-mediated isothermal amplification (LAMP),recombinase polymerase amplification (RPA),and recombinase-aided amplification (RAA) are suited for rapid on-site diagnosis.Gene chip technology allows for the simultaneous detection of multiple pathogens.In the realm of serological diagnostics,methods include ELISA,virus neutralization tests,and complement fixation tests.Among these,RT-PCR,Real-time quantitative RT-PCR,and ELISA are recommended as the primary diagnostic methods by both the World Animal Health Organization and the "African Horse Sickness Diagnostic Techniques" (GB/T 21675—2022) guidelines.In recent years,the emergence of high-throughput metagenomic techniques based on next-generation sequencing has enabled simultaneous quantitative detection of various pathogens,greatly enhancing diagnostic accuracy and efficiency of dieases.The development and application of new technologies,such as cloud-based platforms and the Internet of Things,in disease intelligence monitoring and warning systems,promise to offer more scientifically rigorous and precise technical support for the monitoring and prevention of AHS.

【Objective】 This study was aimed to screen the traditional Chinese medicine (TCM) against avian Infectious bronchitis virus (IBV) and verify therapeutic effect,so as to provide a reference for clinical medication.【Method】 The chicken embryo culture method was used to study the anti-IBV screening test which selected 24 single TCM and 6 commercially available compound TCM.The experimental procedure was to inject TCM in SPF chicken embryos first and then inoculate the virus 2 h later or inoculate the virus first and then inject TCM 2 h later.The variance (ANOVA) analysis of chicken embryo weight was performed with SPSS 20.0 software,and Real-time quantitative PCR was used to assist in determining the therapeutic effect.【Result】 ANOVA on the net weight of chicken embryos showed that there was significant difference between Platycodon grandiflorus (Jacq.) A.DC and positive control group (P＜0.05),and there was no significant difference with negative control group(P＞0.05),indicating that Platycodon grandiflorus (Jacq.) A.DC had preventive and curative effects.The viral load in allantoic fluid by Real-time quantitative PCR showed that Platycodon grandiflorus (Jacq.) A.DC,Houttuynia cordata Thunb,Xuanfeibaidu prescription,Gynostemma pentaphyllum (Thunb.) Makino,Taraxacum mongolicum Hand.-Mazz and Glycyrrhiza uralensis Fisch could inhibit the proliferation of IBV, and the inhibitory effect of Platycodon grandiflorus (Jacq.) A.DC was the best.【Conclusion】 Platycodon grandiflorus (Jacq.) A.DC had the remarkably effective for treating IBV,which laid a reference for the in-depth study of the mechanism of Platycodon grandiflorus (Jacq.) A.DC.

【Objective】 The purpose of this study was to investigate the role of Rab family proteins in the infection of Porcine pseudorabies virus (PRV) in host cells.【Method】 Porcine renal epithelial cells (PK15) were used as the test model to construct Rab gene shRNA library,and purinomycin was used to screen stable cell lines that inhibited porcine Rab gene expression.Real-time quantitative PCR was used to detect cell knockdown efficiency,and flow cytometry was used to detect the effect of knockdown Rab gene on PRV proliferation.Western blotting was used to detect the expression levels of PRV-gB protein,and Real-time quantitative PCR was used to detect the expression levels of PRV-gB,PRV-TK genes,and related inflammatory factors after Rab gene knockout.【Result】 A shRNA library with 13 Rab gene knockdown cell lines was successfully constructed.Flow cytometry analysis showed that knockdown of Rab gene significantly inhibited PRV-GFP proliferation (P<0.05).Viral titer and Western blotting test results showed that Rab gene knockdown extremely significantly inhibited the generation of progeny virus and the expression of PRV-gB protein (P<0.01).The results of Real-time quantitative PCR showed that the expression of PRV-gB and PRV-TK genes could be significantly inhibited after knocking down Rab gene (P<0.05 or P<0.01).The expressions of IFN-β,ISG15,IL-1β and IL-18 genes were significantly or extremely significantly increased after knocking down Rab14 and Rab27 genes (P<0.05 or P<0.01).【Conclusion】 Knockdown of Rab gene could inhibit PRV proliferation.The results laid a foundation for further research on the role of Rab gene in PRV life cycle.

Livestock and poultry are easy to suffer from virus infection during the breeding process,which causes serious economic losses to the breeding industries.In addition,some viruses belong to zoonotic pathogens (including Japanese encephalitis virus and Foot-and-mouth disease virus),which pose potential threats to the public health of livestock workers and consumers.Therefore,the prevention and control of livestock and poultry viruses not only can reduce economic losses in the breeding industry,but also is very important for safeguarding public health.Histone deacetylases (HDACs) belong to the epigenetic modification enzymes,which affect gene expression by regulating the histone acetylation process, so as to participate in various life activity processes.Numerous studies have shown that HDACs participate in various animal virus infection processes by interacting with viral proteins or influencing cellular signaling pathways,and HDAC inhibitor treatment can inhibit the replication of some viruses (such as Pseudorabies virus and Marek’s disease virus),suggesting that HDACs can serve as broad-spectrum antiviral drug targets against animal virus infection.Deeper investigation of the involvement of HDACs in livestock and poultry virus infection is of great significance for the prevention and control of livestock and poultry virus infection.The author briefly introduces HDACs and their inhibitors,mainly summarizes the roles and mechanisms of HDACs in animal virus infection.This review will provide reference for in-depth research on the regulation of HDACs in livestock and poultry virus infection,and provide guidance for developing novel antiviral agents against virus infection.

【Objective】 To understand the immune protection effects of different commercial Marek’s disease vaccines,and provide valuable data for Marek’s disease prevention and control,this study analyzed the immune protection of 7 batches/varieties (A-D,F-H) of CV1988 or CVI988＋HVT commercial vaccine.【Method】 A total of 140 1-day-old SPF chickens were randomly divided into 9 groups,groups G01 to G07.G01 to G07 (15 chickens/group) were immunized with Marek’s disease vaccine A-D and F-H according to the manufacturer’s recommended dose,respectively,while groups G08 (challenge control group,15 chickens) and G09 (blank control group,20 chickens) were not immunized.At 7 days of age,the chickens of groups G01 to G08 were intraperitoneally injected with Md5 strain (1 000 PFU/bird).All chickens were raised to 120 days of age.The protective effect of each vaccine was evaluated through the examination of virus plaque numbers,vaccine replication,weight differences post-challenge,clinical manifestations,and pathological changes.【Result】 The virus plaque number of all 7 vaccine batches confirmed to national standard (2 000 PFU/feather),and the virus plaque number of vaccine F (6 467 PFU/feather) was the highest.The replication rate of vaccine F was faster than other vaccines.After challenged with Md5 strain,the weight of the challenge control group chickens were decreased,and the unvaccinated chickens challenged with Md5 had 93.3% (14/15) mortality,and 100% (15/15) MD lesions.Compared with blank control group,there was no significant difference in body weight in groups G01 to G07 (P>0.05). The mortality of vaccinated chickens in groups G01-G07 were 0/14,3/15,5/15,0/14,0/13,0/15 and 1/15,respectively.The MD lesion proportion of vaccinated chickens in groups G01-G07 were 5/14,8/15,7/15,5/14,3/13,3/15 and 5/15,respectively.The results of immune challenge protection test showed that 7 batches of Marek’s disease vaccine had a certain protective effect on chickens,and the protection index of these vaccines were 45.7%-73.3%.【Conclusion】 Different manufacturer’s Marek’s disease vaccine demonstrated comparable protective effect against Md5 strain of MDV.Furthermore,the bivalent vaccine exhibited higher protection (73.3%) compared to the monovalent vaccine.

Streptococcus agalactiae (S. agalactiae) is a widely distributed zoonotic pathogen.It can survive indefinitely in mammary glands of cows,by forming a biofilm that allows them to adhere and persist in the mammary gland,concomitantly enhancing resistance to host immune defense.S. agalactiae causes sub-clinical mastitis,which is one of the most common pathogens leading to mastitis in dairy herds worldwide,which causes huge economic losses to dairy industry.In addition,S. agalactiae can also infect various animals such as Camelus and Oreochromis niloticus,causing serious deterioration in animal welfare,and there is a possibility of indirect transmission between humans and animals. S. agalactiae is a Gram positive opportunistic pathogen that can asymptotically colonize the gastrointestinal and urogenital tracts of healthy adults.Infection of newborns can lead to life-threatening sepsis and meningitis,often accompanied by severe neurological sequelae.In recent years,the incidence rate of adult S. agalactiae infection is on the rise,especially for pregnant women,the elderly and people with low immunity.It can cause skin and soft tissue infections and more serious septicemia,meningitis,endocarditis and other diseases.Although the symptoms can be partially alleviated by treatment with antibiotics,the emergence and spread of drug-resistant strains have gradually reduced the effectiveness of treatment,and the control of S. agalactiae infection becomes more and more difficult.In this paper,the author summarized the pathogenicity of S. agalactiae to humans and different animals,in order to provide reference for the prevention and control of S. agalactiae.

【Objective】 This study was aimed to investigate and analyze the drug resistance of diarrheal piglets in a pig farm and the molecular characteristics and the co-transmission mechanism of bla_CTX-M-65 and mcr-1 drug resistance genes positive Escherichia coli (E. coli) in Guangdong province,so as to provide scientific basis for drug resistance monitoring and risk prevention and control.【Method】 E. coli strains were isolated and identified from 90 intestinal samples of diarrhea pigs in a pig farm in Zhaoqing,Guangdong province.ESBLs and mcr-1 genes were detected by PCR,and subtyping of CTX-M ESBLs was determined by sequencing.The minimum inhibitory concentration (MIC) of strains to 12 antibiotics was performed using agar double dilution method and broth microdilution method.Genetic relatedness of bla_CTX-M-65 and mcr-1 genes co-carrying strains was analyzed by pulsed field gel electrophoresis (PFGE) and multiple locus sequence typing (MLST).The ability of plasmid horizontal transfer was determined by conjugation transfer,and plasmid replicon types were detected by PCR-based replicon typing.The localization of bla_CTX-M-65 and mcr-1 genes and the size of plasmids were determined by S1-PFGE and Southern blotting.The recombination event of plasmid was determined by whole-genome sequencing and bioinformatics analyses.【Result】 A total of 86 strains of E. coli were isolated and identified.44 strains carried CTX-M ESBLs genes,bla_CTX-M-55 gene was the predominant subtype (n=16,36.4%),followed by bla_CTX-M-65 (n=10,22.7%),bla_CTX-M-27 (n=8,18.2%),bla_CTX-M-15 (n=5,11.4%),bla_CTX-M-79 (n=2,4.5%),bla_CTX-M-14 (n=2,4.5%) and bla_CTX-M-24 (n=1,2.3%).Of 10 bla_CTX-M-65 positive strains,8 were confirmed co-carrying mcr-1 gene,presenting multidrug-resistance phenotypes,including resistance to gentamicin,florfenicol,aztreonam and other antibiotics.The 8 strains were divided into 6 ST types,contained 7 PFGE profiles.A total of 6 transconjugates co-carrying bla_CTX-M-65 and mcr-1 genes were obtained.The bla_CTX-M-65 and mcr-1 genes in all 6 conjugates were located on the IncHI2 (253 kb) plasmids.The resulting 323 kb plasmid was a fusion of a 253 kb IncHI2 plasmid and a 69 kb IncFⅡ plasmid,which was formed during conjugation transfer.Sequence analysis of plasmids revealed that IS26 mediated the recombination of the IncHI2 plasmid and the IncFⅡ plasmid.【Conclusion】 IncHI2 plasmid mediated the widely spread of bla_CTX-M-65 and mcr-1 genes.IS26 led to the fusion of IncHI2 plasmid and IncFⅡ plasmid through integration with the target site GTTTCACT.Such recombination events of plasmids played an important role in expanding the antibiotic resistance spectrum,accelerating the spread of resistance genes,making multidrug resistance more serious and posing a threat to public health security.This study provided a basis for elucidating the transmission mechanism of multi-drug resistant E. coli.

【Objective】 The study was aimed to identify the pathogens of the death of broiler chickens suspected to be infected by Clostridium perfringens in a farm in Nantong,Jiangsu.【Method】 The livers,spleens,lungs,kidneys and contents of intestines of the dead chickens were collected aseptically.Bacterial isolation and purification were carried out through anaerobic cultivation,and the isolated strains were preliminarily identified by Gram staining microscopy.Genus and evolutionary relationship were determined by 16S rRNA gene sequence analysis,toxin type was determined by PCR amplification of toxin genes,pathogenicity and drug resistance phenotypes were determined by animal pathogenicity test and drug susceptibility test,and drug resistance genes were detected.【Result】 9 suspected strains were isolated from different organs of 4 dead chickens,and the isolates were black on the identification medium of Clostridium perfringens.16S rRNA analysis showed that the similarity between the 9 isolates and Clostridium perfringens was up to 99.60%.The 16S rRNA gene phylogenetic tree showed that 4 isolates were closely related to chicken-derived Clostridium perfringens of Pakistan (GenBank accession No.:MN365136.1),and 5 isolates were closely related to chicken-derived Clostridium perfringens of South Africa (GenBank accession No.:OR494055).The results of toxin typing showed that 5 strains were type A,2 strains were type F and others were type E.The results of pathogenicity test showed that the bacteria were lethal.The drug sensitivity tests showed that 9 strains were sensitive to cephalosporin antibiotics.Resistance genes analysis showed that macrolide erm(B) (77.8%),tetracycline tetA(P) (55.6%),tetB(P) (66.7%) genes were the main resistance genes.【Conclusion】 In this study,different toxin-type Clostridium perfringens were isolated from the same dead chicken,and the drug resistance of the isolated bacteria was serious.This result provided a new idea for the prevention and treatment of chicken Clostridium perfringens in veterinary clinic.

【Objective】 The aim of this study was to investigate the mechanism of action of Sihuang antidiarrheal granules in the treatment of piglets diarrhea basing on network pharmacology and molecular docking techniques.【Method】 The active ingredients and corresponding targets of Scutellaria baicalensis,phellodendron,rhubarb,coptis,licorice and isatis root were collected in the TCMSP database.The targets related to piglets diarrhea were collected from the GeneCards database.The collected targets were standardized by the UniProt database.Shared targets were obtained through Venn online mapping site.Cytoscape 3.9.1 was used to create a drug target network diagram to obtain the main active ingredients.Protein-protein interactions (PPI),GO function and KEGG pathways analysis were performed on the shared targets to produce the PPI network diagram and obtain the core targets.3D structure files of the main active ingredients and targets were obtained from PubChem and PDB databases.Molecular docking was performed by Discovery Studio 2019 Client software.【Result】 The main active ingredients of Sihuang antidiarrheal granules were wogonin,β-sitosterol,acaciain,dehydrotanshinone ⅡA and (R)-canadine.The core targets are caspase 3(CASP3),tumor necrosis factor (TNF),interleukin-6 (IL6),vascular endothelial growth factor A (VEGFA) and signal transducer and activator of transcription 3 (STAT3).The results of GO function analysis indicated that the biological functions of the main targets of action of Sihuang antidiarrheal granules were enriched to the entries of RNA polymerase Ⅱ promoter transcriptional regulation,apoptosis,angiogenesis,IL8 production,cytokine activity,and growth factor activity.The results of KEGG pathway analysis showed that the main targets of the action of Sihuang antidiarrheal granules were enriched in signal transduction,immune system,viral infection and other related molecular signaling pathways.The molecular docking results showed that wogonin with TNF,acaciain,dehydrotanshinone ⅡA and (R)-canadine with STAT3 showed good docking activity.【Conclusion】 Sihuang antidiarrheal granules might act on the targets of IL6,VEGFA,STAT3,TNF and CASP3 through the main active ingredients such as wogonin,β-sitosterol,acaciain,dehydrotanshinone ⅡA and (R)-canadine.In this way,it could have anti-inflammatory and antioxidant effects on piglet diarrhea through the pathways in cancer,lipid and atherosclerosis,and human cytomegalovirus infection,reflecting the multi-component,multi-target and multi-pathway characteristics of traditional Chinese medicine.

【Objective】 The aim of this experiment was to isolate and identify the pathogenic bacteria causing the acute death of South China tiger,determine its pathogenicity and drug sensitivity,and provide scientific basis for the prevention and control of the disease in the future.【Method】 Morphological and molecular biology were used to determine the types of isolated strains,and the pathogenicity test and drug sensitivity test of the isolated strains were carried out.【Result】 A strain of bacteria was isolated from the intestine of dead South China tiger cubs.The isolated bacteria showed gray-white colonies on the fresh blood agar plate and white colonies on the MacConkey plate.Microscopically,it was Gram-negative bacilli,named FJ/Tiger201802.Combined with the results of 16S rRNA sequencing,it was identified as Providencia alcalifaciens.Phylogenetic analysis showed that FJ/Tiger201802 was closely related to HXM38 (MZ734428.1) in the same evolutionary branch,and the nucleotide similarity of 16S rRNA gene was 99.5%.The mice inoculated with the isolated strain showed symptoms such as listlessness,loose hair,clumping,and secretions in the eyes.The median lethal dose (LD₅₀) of the isolated strain to mice was 1×10^8.2 CFU.The results of pathological anatomy and histopathology showed that the pathogenicity of FJ/Tiger201802 was strong,mainly affecting the liver,lung,intestine and other tissues and organs of mice.The results of drug sensitivity test showed that ampicillin,aztreonam,cefuroxime,cefoperazone,ceftriaxone,cefepime,ceftazidime,cefoxitin,ofloxacin,streptomycin,tobramycin and gentamicin had a strong inhibitory effect on the growth of the isolated strain in vitro,and the isolate were not sensitive to cefazolin and cefalotin.【Conclusion】 In this study,a strain of Providencia alcalifaciens from South China tiger was isolated,which could cause infection in mice.The results provided a reference for the clinical diagnosis and treatment of Providencia alcaligenes.