【Objective】 This study integrated transcriptomic and proteomic profiling to identify candidate genes associated with cashmere fiber shedding in cashmere goats,elucidating genetic regulatory mechanisms and establishing a theoretical framework for molecular marker-assisted breeding. 【Method】 Six 2-year-old female half-sib Hanshan White cashmere goats were randomly divided into control and melatonin (MT)-implanted groups,with three biological replicates per group.Skin tissues were collected during natural shedding in May (D5),MT-induced premature shedding in April (M4),and secondary shedding in August (M8).RNA and proteins were extracted for transcriptomic sequencing (RNA-Seq) and Label-free DIA-based proteomic quantification to identify differentially expressed genes (DEGs) and proteins (DEPs),followed by functional enrichment analysis and protein-protein interaction (PPI) network construction. 【Result】 In M4 vs D5 comparison group,332 DEGs and 47 DEPs were identified,including overlapping genes P4HA2 and DLX3,with DEGs and DEPs significantly enriched in pathways related to skin development,hair follicle cycling,PPAR signaling pathway,and PI3K-Akt signaling pathway,leading to the identification of nine key genes such as DLX3,FOXN1,and COL1A1.In M8 vs D5 comparison group,345 DEGs and 120 DEPs were identified,with overlapping genes CNDP1,KRTAP11-1,and OAT,enriched in skin development,hair follicle development,hair cycle,PPAR,and calcium signaling pathway,highlighting six key genes including KRTAP11-1,OAT,FOXN1,etc.The M8 vs M4 comparison group showed 193 DEGs and 51 DEPs primarily involved in cell projection regulation,PI3K-Akt signaling,focal adhesion,etc.with three key genes (COL1A1,COL1A2,and S100A4) identified.Among them,cysteine and methionine metabolic pathways in shedding groups were continuously activated,while the COL1A1 and FOXN1 genes repeatedly appeared in multiple comparison groups,suggesting that they might be involved as core regulatory factors in abnormal hair follicle cycles. 【Conclusion】 This study revealed distinct genetic regulatory patterns during MT-induced cashmere shedding in April and August.In the April shedding group,two overlapping genes (P4HA2 and DLX3) and two unique genes (KRT32 and DKK1) were identified,while the August secondary shedding group exhibited three overlapping genes (KRTAP11-1,OAT,and CNDP1) and one unique gene (S100A4).Persistent activation of cysteine and methionine metabolism pathways was observed across shedding groups.The results provided critical insights into the molecular mechanisms governing cashmere shedding characteristics in cashmere goats,establishing a theoretical foundation for precisely regulating hair follicle cycle and optimizing MT-induced shedding techniques alongside cashmere elongation strategies.

【Objective】 The transcriptome sequencing was performed on the skin tissue of the scapular region in Tianzhu White yaks at different age stages,the differentially expressed genes (DEGs) at different age stages were screened,and the function and interaction relationship of DEGs were analyzed,so as to provide a theoretical basis for analyzing the molecular mechanism of skin tissue development process in yak adapting to the alpine environment. 【Method】 The skin tissue of the scapular region in Tianzhu White yaks in good health and growth condition at the ages of 0.5,2.5,and 4.5 years were collected for RNA-Seq.The DEGs were screened with the thresholds of Q＜0.05 and |log₂FoldChange|＞1.Then,GO function and KEGG pathway enrichment analysis were conducted for DEGs.Seven DEGs were randomly selected for Real-time quantitative PCR to verify RNA-Seq results. 【Result】 Among the 605 obtained DEGs,364,153,and 88 DEGs were screened in 2.5 vs 0.5,4.5 vs 0.5,and 4.5 vs 2.5 groups.GO functional enrichment analysis showed that DEGs in 2.5 vs 0.5 group were related to the immune response included positive regulation of immune response,keratinization,neutrophil chemotaxis and chemokine activity,DEGs in 4.5 vs 0.5 group were related to the immune response included positive regulation of chemokine secretion,chemokine activity and response to bacteria,and DEGs in 4.5 vs 2.5 group were related to the immune response included immune response and peptidase inhibitor activity.KEGG pathway annotation results indicated that DEGs in 2.5 vs 0.5 and 4.5 vs 0.5 groups were significantly enriched in immune-related pathways such as the interaction between viral proteins and cytokines and cytokine receptors,IL-17 signaling pathway,and cytokine-cytokine receptor interaction,and DEGs in 4.5 vs 2.5 group were significantly enriched in ABC transporters, Staphylococcus aureus infection,complement and coagulation cascade reaction,etc.Through the enrichment results,30 genes related to skin immune regulation were screened out,and the protein-protein interaction network analysis map was made by STRING database and Cytoscape software,and IL-6,IL-1A,and CCL20 with higher scores were screened as the core proteins.The results of Real-time quantitative PCR were consistent with the expression trend of RNA-Seq,indicating that RNA-Seq results were reliable. 【Conclusion】 The number of DEGs in skin tissues of Tianzhu White yaks at different age stages were revealed in this study,as well as the function and pathway of DEGs.Through analysis,it was believed that pathways such as the IL-17 signaling pathway were associated with the development and regulation of the skin immune function in Tianzhu White yaks,and key regulatory genes such as IL-6,IL-1A and CCL20 were screened.The results provided a reference for comprehensively understanding the regulation mechanism of the skin immune function and related genes in Tianzhu White yaks.

【Objective】 This study was aimed to investigate the transcriptional activity of the core promoter of vitronectin (VTN) gene in sheep rumem epithelial cells and the effects of CCCTC-binding factor (CTCF) gene overexpression on the expression of VTN and related genes. 【Method】 The core region of VTN gene promoter in sheep was predicted using online bioinformatics analysis software,and six pairs of promoter deletion fragment primers were designed.DNA was extracted from blood samples in Hu sheep to clone six promoter deletion fragments upstream of VTN gene CDS region.Luciferase recombinant plasmids L1-basic,L2-basic,L3-basic,L4-basic,L5-basic,and L6-basic were constructed and transfected into sheep rumen epithelial cells.Luciferase reporter gene assays were conducted to verify the location of promoter activity regions.The binding sites between transcription factor CTCF and VTN gene promoter were predicted using online bioinformatics analysis software.RNA was extracted from sheep rumen epithelial cells for reverse transcription,and the CDS region of CTCF gene in sheep was cloned to construct CTCF gene overexpression vector.The vector was co-transfected with VTN gene promoter recombinant plasmids L1-basic,L2-basic,L3-basic,and L4-basic into sheep rumen epithelial cells,with single plasmid transfection as control groups.Luciferase reporter gene assays were performed to determine the core region where transcription factor CTCF binds to VTN gene promoter.Real-time quantitative PCR was used to analyze the relative expression of CTCF,VTN,C-MYC,GLUT1,and SP1 genes in CTCF gene overexpression transfection groups. 【Result】 Six recombinant luciferase reporter vectors in the promoter region of VTN gene in sheep were successfully constructed.The results of bioinformatics prediction and luciferase reporter gene assays indicated that the core promoter of VTN gene might be located at ―933/―440 bp,and transcription factor CTCF potentially interacted with the core region of VTN gene promoter.After CTCF gene overexpression,compared with control group,the relative expression of VTN,C-MYC,and GLUT1 genes were significantly decreased (P＜0.05),while the expression of SP1 gene showed no significant difference (P＞0.05). 【Conclusion】 In sheep rumen epithelial cells,transcription factor CTCF might predominantly bind to the promoter region (―439/+26 bp) and core region (―933/―440 bp) to influence VTN gene expression.CTCF gene overexpression affected the transcription of VTN,C-MYC,and GLUT1 genes,suggesting that CTCF gene potentially influenced the growth,proliferation,and metabolism of sheep rumen epithelial cells.

Induced pluripotent stem cells (iPSCs) are a type of stem cells generated by introducing exogenous transcription factors into somatic cells,thereby activating the endogenous pluripotency network.iPSCs exhibit high similarity to embryonic stem cells (ESCs) in terms of morphology,functionality,and molecular expression,demonstrating extensive self-renewal and differentiation capabilities.Compared with ESCs,iPSCs offer advantages such as relative ease of acquisition,lower risk of immune rejection,and fewer ethical concerns,providing new approaches for disease modeling,drug screening,regenerative medicine,and other fields.Therefore,iPSCs hold significant research value and broad market potential.In alignment with the "One Health" initiative,the emergence of canine induced pluripotent stem cells (ciPSCs) not only opens up novel stem cell-based therapies for canine clinical diseases but also promotes advancements in human regenerative medicine,given that dogs serve as one of the best translational models for human diseases.However,it is noteworthy that ciPSCs still lack standardized and highly efficient production protocols,and issues such as incomplete transgene silencing or reactivation are prevalent.Factors such as safety,cost,and feasibility continue to limit the widespread clinical application of ciPSCs.This review summarizes the reprogramming strategies for ciPSCs,discusses the challenges and possibilities in their generation,and explores the promising applications of ciPSCs in veterinary medicine,aiming to provide insights for further refining the establishment of ciPSCs.

【Objective】 This study was conducted to investigate the effects of different dose of condensed tannins (CT) on growth performance,liver and intestinal morphology and the intestinal flora of Lateolabrax maculatus. 【Method】 Four diets were prepared by adding 0 (CT0),1 (CT1),3 (CT3) and 5 (CT5) g/kg of CT in the basal diet,respectively.Fish (initial weight of 4.38 g±0.01 g) were randomly divided into four groups,with three replicates in each group and a total of 35 fish in each replicate.The feeding trial was lasted for 45 days.At the terminal of the feeding trial,the total quantity,total weight and total food intake of each tank were counted to calculate the growth performance.Three fish were randomly selected from each tank,and the liver and intestine were dissected to prepare paraffin sections and analyze the tissue morphology.Another three fish were randomly selected from each tank to isolate the intestinal contents and determine the composition and diversity of intestinal bacteria. 【Result】 Compared with CT0 group,fish in CT1 group had similar growth performance (P＞0.05),the final body weight,weight gain,specific growth rate,and feed intake of fish in CT3 and CT5 groups were lower (P＜0.05),whereas the feed coefficient was higher (P＜0.05).In CT1,CT3 and CT5 groups,the liver showed varying degrees of vacuolization,fibrosis,and inflammatory infiltration,with the most severe tissue damage observed in CT5 group.Dietary inclusion of CT caused intestinal damage,with fish in CT5 group showing severe atrophy of the intestinal villi and obvious structural damage.At the phylum level,the intestinal microbiota of fish was mainly composed of Proteobacteria and Firmicutes.The relative abundance of Proteobacteria presented a trend of first decreasing and then increasing but was not significant (P＞0.05),while the relative abundance of Bacteroidetes,Chlorobi and Chloroflexi presented a trend of first increasing and then decreasing,and they were higher in CT3 group (P＜0.05).At the genus level,the intestinal microbiota of fish was mainly composed of Methylobacterium and Ralstonia.Compared with CT0 group,the relative abundance of Methylobacterium in CT1,CT3,and CT5 groups were significantly decreased (P＜0.05).The relative abundances of Bacteroides,Faecalibacterium,Roseburia and Sutterella in CT3 group were significantly increased (P＜0.05).Compared with CT0 group,the Shannon index in CT3 group was significantly increased (P＜0.05). 【Conclusion】 Dietary inclusion of 1 g/kg of CT did not alter the growth performance,whereas 3 and 5 g/kg of CT significantly reduced the feed intake and growth performance,damaged the morphology of liver and intestine,and interfered with the intestinal microflora composition of Lateolabrax maculatus.

【Objective】 The aim of the experiment was to investigate the effects of dietary addition of yeast cultures on the growth traits,blood physiological and biochemical indices,slaughter performance,rumen fermentation parameters and conventional nutrient composition of lamb in the F1 generation of Aohu (Australian White sheep♂×Hu sheep♀) lambs,so as to provide references for the scientific rearing of Aohu crossbred lambs. 【Method】 Fourteen 3-month-old F1 weaned male lambs of Aohu with good health and similar body weight (22.50 kg±1.40 kg) were randomly divided into two groups,with 7 lambs in each group.The lambs in control group were fed a basal diet,and in experimental group were fed the basal diet supplemented with 1.0% yeast culture (air-dried basis).The pre-feeding period was 10 d,and the experimental period was 60 d.On the day of 0,30 and 60,all sheep were weighed and body size indexes were measured.On the ending of the experiment,blood was collected to determine blood physiological and biochemical indexes,and slaughter performance,rumen fermentation parameters and longissimus dorsi routine nutritional components were measured after slaughter. 【Result】 ①Fattening 30 days,the body slant length of lambs in experimental group was increased by 7.58% compared with that of control group (P＜0.01).②Compared with control group,the pre-slaughter live weight of lambs in experimental group was increased by 14.30% (P＜0.05),and the carcass fat content (GR) value was increased by 7.78% (P＜0.01).③Compared with control group,the content of albumin (ALB) in blood of lambs in experimental group was increased by 9.03% (P＜0.01),the content of triglyceride (TG) was decreased by 56.25% (P＜0.01),the activity of aspartate aminotransferase (AST) was increased by 28.13% (P＜0.05),the content of total protein (TP) was increased by 8.58% (P＜0.05),and the content of red blood cell (RBC) and hemoglobin concentration (HGB) were decreased by 7.48% and 8.39% (P＜0.05),respectively.④Compared with control group,the pH of rumen fluid in experimental group was increased by 14.08% (P＜0.05),and the moisture content of longissimus dorsi muscle was decreased by 1.37% (P＜0.05). 【Conclusion】 Adding 1.0% yeast culture to the ration of fattening lambs could promote the homeostasis of the organismal internal environment,improve the health level and rumen internal environment,prevent the occurrence of rumen acidosis,and further improve the meat quality.

【Objective】 This study was conducted to explore the effects of different levels of fermented tea on laying performance of laying hens after forced moulting. 【Method】 480 Hy-Line Grey molted hens aged 695 days(moulting was initiated at 581 days) were randomly assigned to four groups,each with 8 replicates and 15 hens per replicate.The hens in control group were fed a basal diet,while in experimental groups Ⅰ,Ⅱ,and Ⅲ were fed the basal diet supplemented with 2%,4%,and 6% fermented tea,respectively.The testing phase was 42 d.During the trial,daily records were kept of egg rate,egg weight,and feed-egg ratio was calculated.On days 21 and 42 of the trial,egg,blood,and ovarian tissue samples were collected by replicate group to assess indicators related to egg quality,serum reproductive hormone levels,and ovarian antioxidant capacity. 【Result】 Compared with control group,①During days 0-21 of the trial, the laying rate of laying hens in experimental groups Ⅱ was significantly increased (P＜0.05). During days 22-42 of the trial, the laying rate of laying hens in experimental group Ⅰ was significantly increased (P＜0.05),and the feed-egg ratio of experimental groupⅠand groupⅡ was significantly decreased (P＜0.05). ②On day 21 of the trial,the yolk ratio of eggs in experimental groups Ⅱ and Ⅲ was significantly decreased (P＜0.05),and the eggshell thickness of eggs in experimental groups Ⅰ,Ⅱ,and Ⅲ were significantly decreased (P＜0.05).On day 42 of the trial,the Haugh units of eggs in experimental groups Ⅰ,Ⅱ,and Ⅲ were significantly increased (P＜0.05),the yolk ratio of eggs in group Ⅱ was significantly increased (P＜0.05),and the eggshell thickness of eggs in experimental groups Ⅰ,Ⅱ,and Ⅲ were significantly decreased (P＜0.05).③On day 21 of the trial,in the ovarian tissue,the activity of catalase (CAT) in experimental group Ⅲ was significantly decreased (P＜0.05),and the total antioxidant capacity (T-AOC) in experimental groups Ⅱ and Ⅲ were significantly increased (P＜0.05).On day 42 of the trial,in the ovarian tissue,the activity of glutathione peroxidase (GSH-Px) in experimental group Ⅱ was significantly increased (P＜0.05).Meanwhile,the activity of CAT and T-AOC in experimental groups Ⅰ,Ⅱ,and Ⅲ were significantly increased (P＜0.05).④On day 21 of the trial,serum progesterone (PROG) concentration in experimental group Ⅱ significantly was increased (P＜0.05).On day 42 of the trial,serum concentrations of follicle-stimulating hormone (FSH),luteinizing hormone (LH),and PROG in group Ⅱ,and prolactin (PRL) in group Ⅲ were significantly increased (P＜0.05). 【Conclusion】 Fermented tea supplements might improve laying performance,egg quality,ovarian antioxidant capacity,and serum reproductive hormone levels in laying hens after forced molting.The most effective results were observed at 4% fermented tea leaves supplementation.

【Objective】 To investigate the effects of millet (Zhangzagu) replacing part corn on slaughtering performance,meat quality,immune and antioxidant indexes of Ira rabbits,and to select the appropriate proportion of millet. 【Methods】 180 30-day-old Ira rabbits,half male and half female,were randomly divided into 6 groups with 5 replicates per group and 6 rabbits per replicate.The rabbits were fed experimental diets containing 0 (control group),5% (group Ⅰ),10% (group Ⅱ),15% (group Ⅲ),20% (group Ⅳ) and 25% (group Ⅴ) millet, respectively,with 5 d of pre-test period and 55 d formal test period.After the experiment,2 rabbits were selected from each repeat,and 5 mL of blood was collected from ear vein to determine the serum immune and antioxidant index.After slaughtering,slaughtering performance and organ weight were determined,and longissimus dorsi muscle were collected to detect meat quality traits. 【Results】 ①The full carcass weight and half carcass weight of Ira rabbits in experimental groups Ⅲ and Ⅳ were higher than those of control group (P＜0.05),there was no significant difference in slaughter rate between the groups (P＞0.05).The liver index and sacculus rotundus index of groups Ⅲ and Ⅳ were both higher than those of control group (P＜0.05).②Compared with control group,the cooking loss of the longissimus dorsi muscle of rabbits was significantly reduced,and the fat content was significantly increased in experimental groups Ⅲ and Ⅳ (P＜0.05).The essential amino acids and flavor amino acids content of longissimus dorsi muscle in all experimental groups had an increasing trend，but the difference was not significant (P＞0.05).③In terms of serum immune indexes,the serum immunoglobulin G content of experimental groups Ⅲ and Ⅳ were higher than that of control group (P＜0.05).Compared with control group,groups Ⅰ and Ⅴ,the immunoglobulin A content of groups Ⅱ, Ⅲ and Ⅳ were significantly increased (P＜0.05).The serum interferon-α content of group Ⅲ was significantly increased compared to control group (P＜0.05),complement protein 4 content was significantly higher than that of control group,groups Ⅰ and Ⅱ (P＜0.05).In terms of serum antioxidant indexes,the contents of serum glutathione peroxidase and superoxide dismutase in experimental group Ⅲ was significantly increased compared with those in control group and experimental group Ⅰ(P＜0.05),and the content of serum glutathione peroxidase in experimental group Ⅳ was also higher than that in control group and experimental group Ⅰ(P＜0.05). 【Conclusion】 Considering the above results,replacing corn with 15% Zhangzagu could improve the slaughter performance of Ira rabbits,enhance meat quality,and strengthen immunity and antioxidant capacity.

【Objective】 The aim of this study was to explore the requirements for metabolizable energy (ME) and crude protein (CP) of Qingyuan partridge chickens from 1 to 30 days. 【Method】 A total of 2 160 healthy 1-day-old female Qingyuan partridge chickens with similar body weights were randomly assigned to 9 groups,with 6 replicates per group and 40 birds per replicate.A two-factor design with;
3. levels of ME and 3 levels of CP was employed.The ME levels were set at 12.77,12.35,and 11.93 MJ/kg,while the CP levels were set at 21.5%,20.5%,and 19.5%.The experimental period lasted for 30 days.The average daily weight gain (ADG),average daily feed intake (ADFI),and feed-to-weight ratio (F/G) of the chickens from 1 to 30 days of age were calculated.At the end of the experiment,five birds were randomly selected from each replicate.Two of these birds were used for blood sampling and breast muscle collection to determine serum biochemical indicators,as well as the CP and crude fat content in the breast muscle.The remaining three birds were used to measure body composition and the deposition rates of energy and CP. 【Result】 ①As the dietary ME level decreased from 12.77 MJ/kg to 11.93 MJ/kg,the ADG of Qingyuan partridge chickens was significantly decreased (P＜0.05),while the F/G was decreased with increasing ME levels (P＜0.05).Chickens in the high ME group had significantly lower ADFI (P＜0.05),whereas those in the low ME group had significantly lower final body weight (P＜0.05).② Serum analysis revealed that chickens in the high ME group had significantly higher total cholesterol levels than those in the low ME group (P＜0.05).When the ME level was reduced to 11.93 MJ/kg,serum uric acid content was significantly decreased (P＜0.05).Additionally,when the dietary CP level was reduced to 19.5%,serum uric acid content was also significantly decreased (P＜0.05).An interaction between ME and CP levels was observed for serum total cholesterol and uric acid content in Qingyuan partridge chickens (P＜0.05).③ Moreover,when the ME level decreased from 12.77 MJ/kg to 11.93 MJ/kg,the crude fat content in the breast muscle of Qingyuan partridge chickens was significantly decreased (P＜0.05),whereas the CP content in the breast muscle was significantly increased (P＜0.05).④ In terms of body composition,when the ME level was reduced from 12.77 MJ/kg to 11.93 MJ/kg,the energy and crude fat content in the body of Qingyuan partridge chickens were significantly decreased (P＜0.05).The body crude fat content was also significantly decreased when the CP level was at its lowest (P＜0.05).⑤The deposition rates of energy and CP in Qingyuan partridge chickens were significantly decreased with decreasing ME levels (P＜0.05).Chickens in the high and intermediate CP groups showed significantly higher energy and CP deposition rates than low CP group (P＜0.05).An interaction between ME and CP levels was observed for energy and CP deposition rates in Qingyuan partridge chickens (P＜0.05), with higher CP deposition rates when ME levels was highest and CP levels were lower.⑥ Using BW^0.75/ADG as the independent variable to establish the metabolic energy and crude protein requirement models for Qingyuan partridge chickens. Based on the models, the recommended ME and CP requirements for 1-30-day-old Qingyuan partridge chickens were 12.28 MJ/kg and 20.07%, respectively. 【Conclusion】 In summary, the growth performance of 1-30-day-old Qingyuan partridge chickens was predominantly influenced by dietary ME levels, while serum biochemical indicators and body composition were jointly regulated by the interactive effects of dietary ME and CP levels.By establishing requirement models, the ME and CP needs for this stage were determined as 12.28 MJ/kg and 20.07%, respectively.

【Objective】 This experiment was conducted to explore the effects of compound enzyme preparation on nutrient apparent digestibility,milk composition,serum biochemical indicators,rumen fermentation parameters and rumen microbial flora in periparturient dairy cows. 【Method】 A total of 18 periparturient Holstein dairy cows of the first-lactation and in good healthy were selected and randomly divided into 2 groups,with 9 cows in each group.The cows in control group were fed the basal diet.The test group added 0.2% compound enzyme preparations (amylase,cellulase,pectinase and protease) to the basal diet according to the feed intake.The adaptation period was 10 days and the experimental period was 42 days.During the experiment,feces of periparturient dairy cows were collected for the detection of nutrient apparent digestibility,milk samples were collected for the detection of milk composition,and blood was collected for the determination of serum biochemical indicators.In addition,rumen fluid was collected for the detection of rumen fermentation parameters and microbial flora. 【Result】 Compared with control group:①The apparent digestibility of dry matter (DM),crude protein (CP) and neutral detergent fiber (NDF) in the feces of dairy cows in test group were significantly increased (P＜0.05).② The milk protein rate and lactose rate of dairy cows in test group increased by 14.69% and 13.09% respectively (P＜0.05),the milk fat rate and the content of non-fat solids increased,and the milk yield decreased (P＞0.05).③ Ten days before prenatal,the contents of albumin (ALB) and triglyceride (TG) in serum of dairy cows in test group increased significantly (P＜0.05).④ The contents of acetate,propionate,butyrate and total short-chain fatty acids in rumen fluid of dairy cows in test group were significantly increased (P＜0.05), pH was significantly decreased (P＜0.05),and the ammonia nitrogen (NH₃-N) content showed a decreasing trend (P＞0.05).⑤ At the phylum level,the relative abundances of Firmicutes and Actinobacteriota in rumen of dairy cows in test group were significantly increased (P＜0.05),while the relative abundance of Bacteroidota was significantly decreased (P＜0.05).At the genus level,the relative abundance of Pseudobutyrivibrio,norank_f_Ruminococcaceae,Olsenella,Lachnospira,Lachnospiraceae_NK4A136_group,Blautia and Atopobium were significantly increased (P＜0.05),while the relative abundances of Treponema was significantly decreased (P＜0.05). 【Conclusion】 The addition of 0.2% compound enzyme preparation (amylase,cellulase,pectinase and protease) to periparturient dairy cow rations could significantly increase the dietary digestion and utilization rate,significantly improve the dairy quality,and at the same time significantly increase the content of short-chain fatty acids and regulate the structure of beneficial microbial flora in rumen.

【Objective】 The experiment aimed to analyze the differences in the composition and abundance of intestinal microbiota in piglets with different birth weights during the lactation period,identify the key microbiota markers that affect subsequent development,and provide a scientific basis for the formulation of piglet health care strategies. 【Method】 Ten second-time pregnant sows with similar body conditions were selected and fed uniformly.After the piglets were born,one low birth weight piglet and one normal birth weight piglet were selected in each litter according to their body weight to form a normal birth weight piglet group (NBWP,1.58 kg±0.04 kg) and a low birth weight piglet group (LBWP,0.98 kg±0.02 kg),with 10 piglets in each group and half males and half females. Fecal samples were collected from piglets in each group at 3,7,14 and 21 days old.DNA was extracted,quality-filtered,and clustered into OTUs.Species composition was analyzed through database comparison,and Alpha and Beta diversity were calculated using QIIME2.PL-DA analysis was performed using the R language mixOmics package. 【Result】 At the ages of 3,7,14 and 21 days,the body weight of piglets in NBWP group was extremely significantly higher than that in LBWP group (P＜0.01).NBWP group obtained 2 478 OTUs,and LBWP group obtained 1 721 OTUs.The changing trends of Alpha diversity and Beta diversity in the two groups at the same age were similar,but the microbiota diversity and abundance in LBWP group were significantly lower than those in NBWP group.The dominant bacterial phyla in the intestines of the two groups of piglets were all Firmicutes,Bacteroidetes and Proteobacteria.At 3 days of age,the relative abundance of Firmicutes in the intestinal tract of piglets in LBWP group was significantly higher than that in NBWP group,and the relative abundance of Proteobacteria was significantly lower than that in NBWP group.Bacteroides, Lactobacillus and Prevotella were the dominant bacterial genera in the intestines of piglets in the two groups.The relative abundance of Bacteroides in the intestines of piglets in LBWP group was higher than that in NBWP group and the relative abundance of Lactobacillus was lower than that of NBWP group.Further studies havd found that the weight differences between piglets in LBWP and NBWP groups were closely related to the abundance changes of Bacteroidetes,Firmicutes and other bacteria. 【Conclusion】 The difference in the birth weight of piglets had an impact on the early colonization process of intestinal microorganisms,and thereby affected the growth and development process of piglets.This study explored the differences in intestinal microbiota among piglets with different birth weights,which could provide a reference for subsequent regulation of intestinal microbiota to improve the growth and development status of piglets.

【Objective】 This study was aimed to compare and analyze the differences in meat quality and blood physiological-biochemical indices between healthy and fattening culled cattle,providing a scientific basis for improving health management and meat quality optimization in fattening cattle. 【Method】 Forty healthy cattle and 40 fattening culled cattle were selected.Blood samples were collected before slaughter to measure physiological and biochemical indices.After slaughter,the samples of longissimus dorsi muscle between the 11th and 13th ribs of the left carcass were collected to determine meat quality indices,including color (L^*,a^* and b^* values),pH,cooking loss rate,shear force,pressed water loss rate,and texture. 【Result】 Compared with fattening culled cattle,healthy cattle exhibited significantly lower pH and cooking loss rate (P＜0.05),but significantly higher L^* and b^* values (P＜0.05).For blood physiological indices,healthy cattle showed significantly higher levels of red blood cell count (RBC),hemoglobin (HGB),hematocrit (HCT),eosinophils (EOS),platelet count (PLT),and plateletcrit (PCT) (P＜0.05).Regarding blood biochemical indices,healthy cattle had significantly higher levels of albumin (ALB),total protein (TP),glucose (GLU),calcium (Ca),total cholesterol (TC),and urea (UREA) (P＜0.05).Correlation analysis revealed that pH of beef in healthy cattle was extremely significantly positively correlated with TP level in blood (P＜0.01),and extremely significantly or significantly negatively correlated with TC and PCT levels (P＜0.01 or P＜0.05).Shear force was negatively significantly correlated with TC level (P＜0.05).In contrast,b^* value of beef in fattening culled cattle was positively correlated with GLU level in blood,pressed water loss rate showed a positive correlation with ALB level in blood,cooking loss was negatively correlated with monocytes,and pressed water loss rate was negatively correlated with PCT in blood. 【Conclusion】 Fattening culled cattle generally exhibited lower blood indices (such as hemoglobin,calcium,and glucose) and meat quality parameters (such as pH and cooking loss rate) compared with healthy cattle.Significant correlations were identified between blood indices and meat quality,providing a reference for optimizing meat quality in fattening cattle.

【Objective】 The purpose of this experiment was to compare and evaluate the egg quality and nutritional value of eggs from four local chicken breeds in Southern China. 【Method】 Chongren partridge chicken,Wumeng crested chicken,Yao chicken,Danzhou chicken and Hy-line Brown laying (control) were taken as the research objects,and 50 chickens of each breed were selected.Under the same feeding conditions,90 eggs were randomly selected for each breed.The egg quality and the contents of nutrients such as crude fat,crude protein,trace elements,amino acids and fatty acids in the eggs were compared and analyzed. 【Result】 The egg weight of Hy-line Brown laying was significantly higher than that of local breeds (P＜0.05),while the egg weight of Chongren partridge chicken and Danzhou chicken were significantly lower than those of other breeds (P＜0.05).The egg yolks of Chongren partridge chicken and Wumeng crested chicken were darker in color,which was significantly different from those of Danzhou chicken and Hy-line Brown laying (P＜0.05).The eggshell strength of Danzhou chicken was significantly lower than that of Chongren partridge chicken,Wumeng crested chicken and Hy-line Brown laying (P＜0.05).In terms of nutritional components,the cholesterol content in Chongren partridge chicken eggs was significantly lower than that in other breeds (P＜0.05).The calcium content in the eggs of Danzhou chicken was significantly higher than that of Yao chicken and Hy-line Brown laying (P＜0.05),and the iron content was significantly higher than that of other breeds (P＜0.05).The phosphorus content in the eggs of Hy-line Brown laying was significantly higher than that of other breeds (P＜0.05).In addition,the vitamin E content in the eggs of Wumeng crested chicken and Danzhou chicken was significantly higher than that of other breeds (P＜0.05).The content of glutamic acid in the eggs of Wumeng crested chicken was significantly higher than that of other breeds (P＜0.05),and the content of polyunsaturated fatty acids was significantly higher than that of Chongren partridge chicken,Danzhou chicken and Hy-line Brown laying (P＜0.05). 【Conclusion】 The local chicken breeds in Southern China generally performed well in terms of nutritional value and flavor,especially in terms of cholesterol and branched-chain amino acid composition.These results had important reference value for the further development and utilization of local chicken breeds.

【Objective】 This experiment aimed to study the effect of sodium butyrate and Ampelopsis grossedentata flavonoids in the basal diet of Holstein weaned calves on their production performance,diarrhea and serum indicators. 【Method】 28 Australian-Canadian F1 Holstein female calves of similar age and body weight were randomly divided into 4 groups with 7 heads each.The calves in control group (group Ⅰ) were fed the basal diet,in experimental groups were supplemented with 3 g/kg DM sodium butyrate (group Ⅱ),8 g/kg DM Ampelopsis grossedentata flavonoids (group Ⅲ) and 3 g/kg DM sodium butyrate + 8 g/kg DM Ampelopsis grossedentata flavonoids (group Ⅳ),respectively.The pretest period was 10 d and the pilot period was 60 d.After the experiment,the body weight and body size of weaned calves were measured，and body size index，the average daily gain (ADG),daily dry material food intake (DMI) and feed to gain ratio (F/G) of calves in each group were calculated.The fecal stool of each calf was collected and the fecal moisture rate was measured.The fecal situation of each calf in each group was observed and recorded every day,and the diarrhea rate of calves was measured and fecal score was performed.Blood samples from tail root vein were collected to determine the serum biochemical indicators. 【Result】 ①The final body weight and ADG of calves in group Ⅲ were significantly higher than those in groupⅠ (P＜0.05),and DMI was significantly higher than groups Ⅰ,Ⅱ and Ⅳ (P＜0.05).②The withers height of calves in group Ⅲ was significantly higher than that of groups Ⅰ and Ⅳ (P＜0.05),the pipe circumference of calves in groups Ⅲ and Ⅳ were significantly higher than that of group Ⅰ,and pipe circumference index of calves in group Ⅳ was significantly higher than that of group Ⅰ (P＜0.05);③At 60 days,the fecal moisture rate of calves in groups Ⅱ and Ⅲ were significantly lower than that in group I (P＜0.05)，and the average fecal scores and diarrhea rate of calves in group Ⅲ were significantly lower than that of group Ⅰ (P＜0.05).④The content of cholesterol (CHOL) in serum of calves in groups Ⅱ and Ⅲ were significantly higher than that in groups Ⅰ and Ⅳ (P＜0.05),the content of insulin-like growth factor-1 (IGF-1) in serum of calves in groups Ⅱ and Ⅲ were significantly higher than that in group Ⅰ (P＜0.05). 【Conclusion】 The addition of 8 g/kg DM Ampelopsis grossedentata flavonoids in the basal diet could reduce the diarrhea and improve the growth performance of Australian-Canadian F1 Holstein weanded calves.

【Objective】 This study was aimed to investigate the effects of yeast peptide on immune function,digestive and metabolic performance and intestinal microorganisms of Sichuan White geese. 【Method】 A total of 120 healthy Sichuan White geese at 1 day of age with equal amount of male and female,were randomly divided into 2 groups,with 6 replicates in each group and 10 geese in each replicate.Geese in control group were fed a basal diet,geese in experimental group were supplemented with 200 mg/kg of yeast peptide on top of the basal diet.Geese had access to feed and water ad libitum in the 70 d of the experiment period.At the end of the test,jugular blood was collected to determine IgA,IgM,IgG contents and lysozyme activity,and spleen,thymus,bursa of Fabricius,liver,pancreas,muscularis propria,glandularis propria,duodenum,jejunum and ileum were collected to determine the immune function,digestive performance,intestinal morphology and microorganisms of the geese. 【Result】 Compared with control group,① IgM content and lysozyme activity in serum,splenic index and bursa of Fabricius index of geese in yeast peptide group were significantly increased (P＜0.05);② The liver index, ileum index,and duodenum length of geese in yeast peptide group were significantly increased (P＜0.05),but there was no significant effect on digestive enzymes activity and the length of jejunum and ileum of geese (P＞0.05);③The villus height,V/C and mucosal layer thickness of duodenum and ileum of geese in yeast peptide group were significantly increased (P＜0.05),while the mucosal layer thickness of jejunum and crypt depth of ileum were significantly lower (P＜0.05);④ The relative abundance of Proteobacteria, Parasutterella, Clostridium_ⅩⅣb, Sutterella,Sporobacter,Pseudoflavonifractor and Roseburia in intestines of geese in yeast peptide group were significantly increased (P＜0.05).The LEfSe analysis results showed that compared with control group,the abundance of Sutterellaceae,Burkholderiales,Betaproteobacteria,Helicobacteraceae,Campylobacterales and Epsilonproteobacteria were significantly increased in yeast peptide group (P<0.05). 【Conclusion】 The addition of 200 mg/kg of yeast peptide to the feed under the conditions of the present study improved the immune function and digestive performance of geese to a certain extent,as well as improved the intestinal microbial composition and promoted intestinal health.

Omega-3 fatty acid is a type of polyunsaturated fatty acids that are widely present in nature and an important component of cell membranes,which mainly come from some marine fish and plant seeds such as flaxseeds.Omega-3 fatty acid can change the composition of cell membrane lipids,regulate the biosynthesis of eicosanoids,control gene expression and cell signal cascades,and have a positive impact on the health of animals.Adding an appropriate amount of Omega-3 fatty acid to the diet in dairy cows has significant benefits.It can effectively enhance the immunity of dairy cows,reduce the incidence of mastitis and reproductive diseases,and improve their reproductive performance.In addition,Omega-3 fatty acid can optimize the rumen microbial flora,promote the digestion and absorption of nutrients,and thereby increase milk production and milk quality.This article reviews the sources and in vivo transformation process of Omega-3 fatty acid,and focuses on summarizing their regulatory effects on the immune system,mastitis,reproductive performance and milk production performance of dairy cows,with the aim of providing a reference for the research and application of Omega-3 fatty acids as feed additives in dairy cow farming.

【Objective】 Jersey is a worldwide famous dairy cattle breed with characteristics of small body size,high milk components and good resistance.This study analyzed the factors affecting the lactation performance of Jersey cattle,aiming to reveal the performance level and population characteristics of production performance of Jersey cattle in Yunnan. 【Method】 Based on the dairy herd improvement (DHI) records of Jersey cattle in a large-scale dairy farm in Yunnan,the influencing factors of lactation performance traits were analyzed by fitting a linear mixed model,and the lactation curve of Jersey cattle was fitted using WOOD model and the lactation curve parameters were calculated. 【Result】 The mean of daily milk yield,fat percent,protein percent,lactose percent and somatic cell score of Jersey cattle in Yunnan was 26.37 kg,5.19%,3.72%,5.05% and 2.84,respectively.The test season,test year and lactation stage had significant effects on daily milk yield,fat percent,protein percent,lactose percent,somatic cell score and milk urea nitrogen content (P＜0.05),and parity had a significant effect on the above lactation performance traits except for fat percent and milk urea nitrogen content(P＜0.05).The average degree of fitness (R²) of the lactation curve in Jersey cattle was 0.78.Based on fitting results by the WOOD model,the average 305 d milk yield of Jersey cattle was 7 997.04 kg,the average peak milk yield and peak day were 34.30 kg and 71.91 d,respectively. 【Conclusion】 This study revealed the population characteristics of lactation curve and six lactation performance traits such as daily milk yield,milk fat percent and milk protein percent,and found that Jersey cattle in Yunnan perform well and some of the non-genetic factors had significant effects on the lactation performance.The results could provide a reference for the farm management and genetic improvement of Jersey cattle in Yunnan.

【Objective】 The aim of this study was to investigate the expression of mitogen-activated protein kinase kinase 6 (MAP2K6) gene in longissimus dorsi muscle of Hu sheep at different developmental stages,and analyze the correlation between the polymorphisms of MAP2K6 gene and the growth traits of Hu sheep,so as to provide novel marker resources for the molecular breeding of growth traits in Hu sheep. 【Method】 Real-time quantitative PCR was utilized to analyze the expression of MAP2K6 gene in longissimus dorsi muscle of Hu sheep (n=15) at various developmental stages.The Illumina OvineSNP 50K BeadChip was used to detect the single nucleotide polymorphisms of MAP2K6 gene in Hu sheep (n=3 024).A general linear model was used to analyze the relationship between MAP2K6 gene SNP and growth traits in Hu sheep (n=1 974).The correlation coefficient between body weight and body size of Hu sheep was calculated by R language corrplot package. 【Result】 The results of Real-time quantitative PCR showed that the expression of MAP2K6 gene in longissimus dorsi muscle of Hu sheep gradually increased from birth to 4-month-old.The expression of MAP2K6 gene at 3 and 4-month-old was extremely significantly higher than that at birth,45-day-old,and 6-month-old(P＜0.01).Two SNPs (rs414959578 G＞A and rs426057803 A＞G) of MAP2K6 gene in Hu sheep were detected.The association analysis results indicated that rs414959578 G＞A of MAP2K6 gene had significant or extremely significant effects on the body weight,body height,body length,chest circumference,chest depth,chest width,cross height,and waist angle width at 5-month-old,as well as chest circumference and backfat thickness at 6-month-old in Hu sheep (P＜0.05 or P＜0.01).rs426057803 A＞G of MAP2K6 gene had significant or extremely significant effects on the cannon circumference at 3-month-old,and chest circumference,shin circumference,and cross height at 5-month-old,as well as the backfat thickness at 6-month-old in Hu sheep (P＜0.05 or P＜0.01).Correlation analysis results revealed that there was significant positive correlations between body weight and body size indicators in Hu sheep (P＜0.05).However,there was no significant correlation between body length and chest width/waist angle width at 6-month-old,or shin circumference at 5-month-old and waist angle width at 6-month-old (P＞0.05). 【Conclusion】 MAP2K6 gene was associated with the development of longissimus dorsi muscle in Hu sheep,and rs414959578 G＞A and rs426057803 A＞G had significant influence on the growth traits in Hu sheep.The results could provide a theoretical basis for identifying and utilizing molecular markers for growth traits in Hu sheep.

【Objective】 This study was aimed to explored the influence of different glycerol removal methods on the artificial insemination efficacy of thawed chicken semen,aiming to simplify the glycerol removal procedure for chicken semen. 【Method】 Semen was collected from Wenchang roosters using the abdominal-dorsal combined massage method,frozen,thawed,and then diluted with extender at ratios of 1∶2,1∶4,1∶6,and 1∶8 (V/V) to remove glycerol.The optimal dilution ratio was determined by assessing sperm motility after deglycerolization.The optimal proportions were used for one-step,two-step,and six-step dilution methods for deglycerolization.The effectiveness of each method was evaluated by assessing sperm motility,sperm recovery rate,and glycerol content in both seminal plasma and sperm.Subsequently,the deglycerolized semen from the three methods was used for artificial insemination trials to measure fertilization and hatching rates. 【Result】 Among various dilution ratios,the 1∶4 six-step dilution method for deglycerolization demonstrated superior sperm motility both post-deglycerolization and at 30 minutes post-deglycerolization compared with 1∶2,1∶6,and 1∶8 six-step dilution methods,although the differences were not statistically significant (P＞0.05).Among different deglycerolization methods with a total dilution ratio of 1∶4,the six-step dilution method achieved the highest post-deglycerolization sperm motility at 68.34%,but the differences among the three methods were not significant (P＞0.05).The glycerol removal rates of both seminal plasma and sperm in the two-step dilution method were extremely significantly higher than those of the other two methods (P＜0.01).The sperm recovery rate was higher with the six-step dilution method,but no significant differences were found among the three methods(P＞0.05).In terms of artificial insemination outcomes,the fertilization rate,live embryo rate,and hatching rate of hatching eggs in non-glycerol-removed group were all 2.25%,which were extremely significantly lower than those of the fresh semen group and the three glycerol-removed method groups (P＜0.01).Among the three glycerol removal methods,the six-step dilution method showed relatively higher fertilization rate,live embryo rate,and hatching rate of hatching eggs,but no significant differences were observed compared with the two-step dilution method (P＞0.05).The hatching rate of fertilized eggs in the two-step dilution method was similar to that of fresh semen,and the live embryo emergence rate was the highest,reaching 95.71%,but the overall differences were not significant (P＞0.05). 【Conclusion】 The 1∶4 two-step dilution method efficiently removed glycerol from frozen chicken semen and achieved favorable sperm recovery rate,sperm motility,and artificial insemination outcomes.Meanwhile,compared with the traditional six-step dilution method,the 1∶4 two-step dilution method had a simpler operational process,making it more suitable for application in chicken breeding production.

【Objective】 Granulosa cells (GCs) proliferation is regulated by many factors,such as miRNAs,miR-26a-5p was significantly enriched in the growing follicles of goats,which might have a regulated role during the folliclar development.This study was aimed to reveal the functions of miR-26a-5p in goat GCs,in order to enrich the molecular regulatory network in follicular development. 【Method】 Real-time quantitative PCR was used to analyze the expression of miR-26a-5p in goat follicles with different sizes.The ovarian GCs were cultured and identified in vitro.The proliferation of GCs was detected using EdU and MTT assays after with overexpressed and inhibited miR-26a-5p expression.The interaction between miR-26a-5p and the target gene PTEN was verified using a dual luciferase reporter system and Western blotting,respectively.Western blotting was used to analyze the expression of proteins related to PI3K/Akt signaling pathway in GCs after overexpression or interference with miR-26a-5p expression. 【Result】 The expression of miR-26a-5p was detected in goat follicles of varying sizes,including in oocytes.miR-26a-5p was significantly enriched in GCs of small-to-medium follicles (2-3 mm in diameter) compared with GCs from follicles of other diameters (＜2,4-5,and ＞5 mm) (P＜0.05).In in vitro cultured GCs,the proportion of proliferating cells was significantly higher in miR-26a-5p overexpression group than that in control group (transfected with NC mimics) (P＜0.05).Conversely,transfection with miR-26a-5p inhibitor significantly reduced the proliferation rate compared with NC inhibitor group (P＜0.05).Dual-luciferase reporter assays revealed that compared with mutant group,miR-26a-5p overexpression significantly reduced luciferase activity by targeting the seed sequence in the 3'-UTR of PTEN gene (P＜0.05).Western blotting results showed that compared with NC mimics group, miR-26a-5p overexpression significantly downregulated the expression of PTEN protein in GCs (P＜0.05).Furthermore,elevated phosphorylated the expression of p-AKT protein and an increased p-AKT/AKT ratio were still detectable 96 h after miR-26a-5p overexpression (P＜0.05). 【Conclusion】 miR-26a-5p directly targeted and suppressed the expression of PTEN gene in ovarian GCs,thereby activated the AKT protein and PI3K/Akt signaling pathway,which promoted GCs proliferation.

【Objective】 The aim of this study was to investigate the effects of melatonin (MT) on sperm motility and enzymatic levels of Blue foxes (Vulpes lagopus) semen during cryopreservation,so as to provide a theoretical basis for the application of melatonin in semen cryopreservation. 【Method】 Semen samples were collected from nine healthy male Blue foxes.After quality assessment,the samples were pooled and divided into six groups:A fresh semen control group (non-cryopreserved) and five melatonin (MT) concentration gradient cryopreservation treatment groups (0,0.05,0.1,0.15,and 0.2 mmol/L).The fresh semen control group was diluted at a 1∶9 ratio and directly analyzed.Each MT freezing treatment group was supplemented with the corresponding MT concentration in an equivalent dilution medium prior to cryopreservation.Post-thaw,parameters including sperm viability,wobble (WOB),beat cross frequency (BCF),and the activities of mitochondrial,antioxidant enzyme activity,and acrosomal enzyme were evaluated. 【Result】 The partial indices in MT freezing treatment group were significantly lower than those in fresh sperm group (P＜0.05).In terms of motility,the sperm viability,WOB,BCF,average path velocity (VAP),curvilinear velocity (VCL),and straight line velocity (VSL) of Blue foxes in 0.05 mmol/L MT group were significantly higher than those in other MT freezing treatment groups (P＜0.05).In terms of energy metabolism,the ATP content and Complex Ⅰ activity of of Blue foxes sperm in 0.05 and 0.1 mmol/L MT groups were significantly higher than those in 0 and 0.2 mmol/L MT groups (P＜0.05).In terms of oxidative stress,the ROS activity of Blue foxes sperm in 0.05 and 0.1 mmol/L MT groups was significantly lower than that in 0 and 0.15 mmol/L groups (P＜0.05).The activities of superoxide dismutase (SOD),catalase (CAT),and heme oxygenase (HO) of Blue foxes sperm in 0.05 mmol/L MT group were significantly higher than those in 0,0.15 and 0.2 mmol/L MT groups (P＜0.05).In terms of fertilization ability,the activities of esterase,acrosin and HAase of Blue foxes sperm in 0.05 and 0.1 mmol/L MT groups were significantly higher than those in other MT freezing treatment groups (P＜0.05). 【Conclusion】 Under the conditions of this experiment,adding an appropriate amount of MT to the frozen diluent of semen in Blue foxes could effectively improve the sperm motility,antioxidant capacity and fertilization ability of frozen sperm,with 0.05 mmol/L as the optimal concentration.

【Objective】 The purpose of this paper was to investigate the regulatory mechanism of miR-1343 in porcine ovarian granulosa cells.The target genes of miR-1343 were predicted and analyzed by the bioinformatics method，and the effects of reproductive hormone on miR-1343 expression were explored in porcine ovarian granulosa cells. 【Method】 Mature sequences of miR-1343 were downloaded from the miRbase online database for sequence conservative analysis.Porcine ovarian granulosa cells were isolated and cultured,which were seeded into six-well plates.At 70%-80% cell confluence,the granulosa cells were treated with 0 IU/mL (control group) and 20 IU/mL (experimental group) follicle-stimulating hormone (FSH),with 3 replicates per group,24 h after addition,the cells were harvested,and the expression of miR-1343 was detected by Real-time quantitative PCR.The PROMO online software was utilized to predict the transcription factor binding sites for miR-1343 in pigs.The target genes of miR-1343 were predicted using TargetScan,miRDB,and Starbase database.GO function and KEGG pathway enrichment analysis were performed for the target genes using DAVID online database.The protein-protein interaction (PPI) of target genes was analyzed using STRING database,and regulatory network was constructed using Cytoscape software. 【Result】 The mature sequences of miR-1343 were highly conserved among multiple species.The relative expression of miR-1343 in experimental group was extremely significantly down-regulated compared with the control group (P＜0.01).There were many transcription factor binding sites such as Sp1,SMAD3,SMAD4,and FOXO4 in the promoter region of miR-1343.A total of 278 common target genes were predicted using different databases.GO function annotation analysis results revealed that target genes of miR-1343 were mainly enriched in phosphorylation,positive regulation of transcription by RNA polymerase Ⅱ,protein phosphorylation,and positive regulation of DNA-templated transcription for biological process,nucleus,cytoplasm,cytosol,and nucleoplasm for cellular component,and ATP binding,protein homodimerization activity,protein serine/threonine kinase activity and chromatin binding for molecular function.KEGG analysis results revealed that target genes of miR-1343 were significantly enriched in the Hippo signaling pathway,MAPK signaling pathway,and thyroid hormone signaling pathway,which were related to ovarian function.PPI analysis of miR-1343 target genes indicated that SYNJ1,PLCB1,and GSK3B had more targeted relationships with other proteins. 【Conclusion】 The expression of miR-1343 was influenced by FSH stimulation in granulosa cells.miR-1343 might participate in the development of porcine ovarian granulosa cells by regulating the expression of target genes such as PLCB1 and GSK3B.The results provided a reference for further exploring the regulatory mechanism of miR-1343 in porcine ovarian granulosa cells.

【Objective】 This study aimed to establish cryopreserving method of Fenneropenaeus chinensis and investigate the damage of sperm ultrastructure after cryopreservation. 【Method】 The sperm survival rate was detected by using eosin B staining.The suitable way was selected by comparing the effect of nine diluents (sterilized natural seawater,artificial seawater,decalcified artificial seawater,magnesium removal artificial seawater,de-potassic artificial seawater,5% sodium chloride,normal saline,D-Hanks and Hank’s balanced salt solution),three permeable cryoprotectants (DMSO,glycerol and propylene glycol),five non-permeable cryoprotectants(trehalose,glucose,maltose,vitamin C and bovine serum protein),four cooling procedures and four different resuscitation temperature (25,30,35 and 40 ℃),and the sperm ultrastructure was observed by transmission electron microscopy. 【Result】 ①The sperm survival rate after thawing of the sterilized natural seawater and artificial seawater groups were significantly higher than that of the other groups (P＜0.05).②The sperm survival rate of the experimental groups using glycerol as the permeable cryoprotectant were significantly higher than that of the other experimental groups (P＜0.05),with the best concentration being 15%.③The sperm survival rate after thawing of the experimental groups with the addition of non-permeable cryoprotectants such as trehalose and vitamin C were significantly higher than that of the other groups (P＜0.05).④The sperm survival rate after thawing of the P-1 and P-2 experimental groups using slow cooling were significantly higher than that of the other groups (P＜0.05),among which the P-1 experimental group with slow cooling from 0 ℃ to -20 ℃,medium-speed cooling from -20 ℃ to -80 ℃,and rapid cooling from -80 ℃ to -180 ℃ had the highest sperm survival rate.⑤The sperm survival rate after thawing of the 35 ℃ water bath thawing group was significantly higher than that of the other groups (P＜0.05).⑥The normal rates of the cryopreservated sperms were 79%,structural damage of injured sperm was mainly manifested as spike lost,inter membrane space augmented,membrane swelled,acrosomal cap lost,vacuolization of the cell nucleus observed. 【Conclusion】 The optimal ultra-low temperature cryopreservation method for Fenneropenaeus chinensis sperm involves using sterilized natural seawater as the diluent,15% glycerol as the cryoprotectant solution,supplemented with 0.25 mol/L trehalose.By employing a programmable cooling device with a three-step slow cooling protocol and thawing the frozen sperm in a 35 ℃ water bath,this approach effectively minimizes structural damage to Fenneropenaeus chinensis sperm during ultra-low temperature cryopreservation.

【Objective】 The intelligent detection of ewes licking lambs and the standing postures of lambs,the characteristics of the lamb standing process as well as the influence of ewes licking lambs on the standing of lambs were intended to be studied in this experiment. 【Method】 Videos of the licking behavior of 30 ewes and the standing process of lambs were collected to train the YOLOv5s model.20 multiparous single-fetus Saanen dairy goats and their newborn lambs were selected.Those with the licking time of ewes less than 120 s were divided into control group (Con group),and those with the licking time of ewes more than 120 s were divided into licking group (Lick group),with 10 in each group.The licking behavior of ewes and the standing postures of lambs were recognized based on the trained YOLOv5s algorithm.The characteristics of the standing process of lambs were analyzed by relying on the Farneback optical flow algorithm.The time and frequency of the ewes’ licking behavior and the lambs’ standing between consecutive frames were extracted by combining the two algorithms.Pearson correlation analysis was adopted,and One-Way ANOVA was used to explore the effect of lamb licking on lamb standing. 【Result】 ①The average precision of the YOLOv5s algorithm for identifying the licking posture was 97.90%.The average precision for identifying the lying posture of lambs was 90.70%.The average precision for identifying the posture of lambs attempting to stand was 82.60%.And the average precision for identifying the standing posture of lambs was 86.30%.②Through the analysis of the characteristics of the lamb standing process by the Farneback optical flow algorithm,the motion trajectory and direction changes of lambs during the process from lying down to standing could be effectively demonstrated.③The longest licking time was 1 462.00 s,the shortest was 0 s,with an average of 410.10 s.The time when lambs first attempted to stand ranged from 284.00 to 1 195.00 s,with an average of 529.25 s.The time when lambs first successfully stood ranged from 695.00 to 2 921.00 s,with an average of 1 464.35 s.The time for attempting to stand ranged from 323.00 to 1 785.00 s,with an average of 930.10 s.The minimum number of times that lambs attempted to stand was 11.00,the maximum was 46.00,with an average of 23.60 times.④The number of attempts of lambs in Con group to stand was significantly higher than that of lambs in Lick group.The duration of each attempt to stand by lambs in both Con and the Lick groups showed an increasing tendency as the number of attempts increased.⑤The total duration of ewes licking lambs was significantly negatively correlated with the duration of lambs’ attempts to stand,the duration of lambs’ first successful standing and the frequency of lambs’ attempts to stand (P＜0.05).⑥There was no significant difference in the time from birth to the first attempt to stand between lambs in Lick group and those in Con group (P＞0.05).The time for lambs in Lick group to attempt to stand was significantly shorter than that of lambs in Con group (P＜0.05).The time for lambs in Lick group to first successfully stand was significantly shorter than that of lambs in Con group (P＜0.05).The number of attempts of lambs in Lick group to stand was significantly less than that of lambs in Con group (P＜0.05). 【Conclusion】 The YOLOv5s model could accurately identify the postures of ewes licking lambs and the standing postures of lambs,and the Farneback optical flow algorithm could well analyze the characteristics of the lamb standing process.Moreover,the longer the licking time of ewes was,the higher the vitality of lambs would be,which could help lambs successfully stand up as soon as possible.

【Objective】 The purpose of this study was to explore the molecular genetic diversity and population genetic structure of Ashidan yak. 【Method】 Fifty-two Ashidan yaks (32 males and 20 females) were selected for the experiment，the sequences of their mitochondrial DNA D-loop (mtDNA D-loop) were amplified by PCR and sequenced.Based on nucleotide sequence variations,the maternal genetic diversity and population genetic structure of Ashidan yak were explored.PCR amplification and sequencing typing were performed on 32 Ashidan yak males using 5 Y-chromosome single nucleotide polymorphism (Y-SNP) markers (SRY4,USP9Y,UTY19,AMELY3 and OFD1Y10) and 1 Y-chromosome short tandem repeat (Y-STR) marker (INRA189)，to analyze the paternal genetic diversity and population structure composition. 【Result】 ①A total of 52 mtDNA D-loop sequences of Ashidan yak were obtained by PCR amplification and sequencing,with a length of 617-618 bp.49 SNPs were detected through comparative analysis,including 6 single polymorphic sites and 43 parsimony information sites.29 haplotypes were determined based on the nucleotide variations between sequences in the D-loop region.Maternal genetic diversity analysis showed that the haplotype diversity and nucleotide diversity of Ashidan yak were 0.954±0.017 and 0.018±0.009,respectively.Phylogenetic analysis revealed that Ashidan yak was composed of two maternal genetic lineages,MT-Ⅰ and MT-Ⅱ.MT-Ⅰ was the dominant branch,consisting of haplogroups A (H5,H6,H11-H14,H16-H18,H24-H27 and H29),B (H1,H3,H15 and H23),and E (H2 and H28),accounting for 68.97%.MT-Ⅱ was composed of haplogroups C (H4,H7,H9 and H21) and D (H8,H10,H19,H20 and H22),accounting for 31.03%.②Based on the combined typing of Y-SNP and Y-STR markers,6 Y chromosome haplotypes (H1Y1,H2Y1,H6Y1,H8Y1,H9Y1 and H11Y2) were identified in Ashidan yak.Paternal genetic diversity analysis showed that the haplotype diversity of the Y chromosome in Ashidan yak was 0.742±0.049.Phylogenetic analysis revealed that the 6 Y chromosome haplotypes of Ashidan yak were clustered into 2 paternal branches,Y1 and Y2,accounting for 65.62% and 34.38% respectively. 【Conclusion】 Ashidan yak had rich maternal and paternal genetic diversity,consisting of 2 maternal branches (MT-Ⅰ and MT-Ⅱ) and 2 paternal branches (Y1 and Y2),which was similar to the genetic structure composition of most yak breeds (populations) in China.

Songliao Black pig,as a premium crossbred variety of pigs in China,has unique genetic characteristics and economic value.Its breeding history can be traced back to the early 20th century.Songliao Black pig exhibits excellent adaptability and reproductive ability due to its prominent appearance features such as black fur,large ears and short legs.In terms of reproductive performance,Songliao Black pigs exhibit high conception rate and litter size.However,in terms of growth performance,although their growth rate and feed conversion rate are not as good as foreign breeds,their meat quality has obvious advantages,with delicious meat and moderate intramuscular fat content,which is highly favored by consumers.In recent years,the application of molecular biology technology has provided a new perspective for the study and breeding improvement of genetic characteristics in Songliao Black pigs.The author summarizes the breeding history,appearance characteristics,as well as the research progress in production performance,such as gene association analysis,the effects of dietary additives on growth and meat quality,and the progress in crossbreeding utilization of Songliao Black pigs.This review will provide theoretical supports that strengthen the protection and utilization of germplasm resource for promoting the sustainable development of special black pig industry.

【Objective】 The experiment aimed to construct tripartite motif 8 (TRIM8) gene knockout porcine small intestinal epithelial cells (IPEC-J2) using CRISPR/Cas9 technology and investigate the regulatory effect of TRIM8 knockout on Porcine epidemic diarrhea virus (PEDV) replication,provide genetic resources and theoretical basis for an in-depth understanding of the mechanism of TRIM8 gene in antiviral natural immunity and the development of new strategies for PEDV prevention and control. 【Method】 Three sgRNAs (sgRNA1,sgRNA2 and sgRNA3) were designed in the first exon region of the porcine TRIM8 gene transcript,annealed to form double-stranded DNA ligated with linearized pGK1.2 vector,and the products were transformed into Escherichia coli DH5α competent cells for identification.The recombinant vector was transfected into IPEC-J2 cells.Sequences near the knockout site were amplified by PCR,and the efficiency of sgRNA knockout was determined by sequencing.Positive cell clones were identified by T7 endonuclease Ⅰ (M0302) zymography,and the knockout sequences were identified by sequencing of the TA clones.The expression of TRIM8 protein in gene knockout cells was detected by Western blotting.The changes in PEDV replication after TRIM8 gene knockout were detected by 50% tissue culture infective dose (TCID₅₀) and Real-time quantitative PCR. 【Result】 Sequencing of the recombinant vector showed that sgRNAs were successfully ligated with pGK1.2.Analysis of knockout efficiency showed that only sgRNA2 among the three sgRNAs had knockout efficiency.Eight positive monoclonal cells were screened by T7 endonuclease Ⅰ (M0302) digestion of PCR products.Sequencing analysis of TA clone revealed that the sequences of the 2 alleles of the TRIM8 gene missed 5 and 9 bp,respectively.Western blotting results showed that no TRIM8 protein was expressed in TRIM8 gene knockout cells.TCID₅₀ and Real-time quantitative PCR results showed that compared to wild-type IPEC-J2 cells,knockout of TRIM8 gene significantly up-regulated the viral titer of PEDV (P＜0.05),and M gene and N protein of PEDV were also significantly up-regulated (P＜0.05). 【Conclusion】 In this study,TRIM8 knockout IPEC-J2 cells were constructed by CRISPR/Cas9 technology and it improved the ability of PEDV replication in host cells.TRIM8 knockout cells might provide materials for further investigation of TRIM8 gene function and molecular mechanisms regulating PEDV replication.

【Objective】 P33 protein is an important surface protein of Theileria orientalis,possessing good antigenicity and immunogenicity.The present study aimed to develop specific monoclonal antibodies against P33 protein using hybridoma technology,systematically evaluate their immunological characteristics,and screen monoclonal cell lines with stable antibody-secreting capacity,thereby laying a material foundation for establishing a P33 antigen-based serological diagnostic method for Theileria orientalis. 【Method】 The position of the signal peptide and B cell epitopes of P33 protein was predicted and analyzed by online software.P33 gene without signal peptide but with dominant antigenic epitopes was cloned into prokaryotic expression vectors pET-30a and pGEX-4T-1 to construct recombinant expression vectors pET-30a-P33 and pGEX-4T-1-P33,respectively.The recombinant plasmids were transformation into Escherichia coli BL21 (DE3) and BL21 competent cells for induction of expression.The expressed products were analyzed by SDS-PAGE and Western blotting.BALB/c mice were immunized with the purified pET-30a-P33 recombinant protein,and the specific monoclonal antibody was screened with the pGEX-4T-1-P33 recombinant protein.Antibody subclasses and titer were identified by ELISA.The antigenicity and the specificity of the monoclonal antibody were detected by Western blotting and indirect immunofluorescence assay (IFA). 【Result】 PCR and restriction enzyme digestion results showed that the two expression vectors pET-30a-P33 and pGEX-4T-1-P33 were successfully constructed.The results of SDS-PAGE and Western blotting showed that the recombinant proteins pET-30a-P33 and pGEX-4T-1-P33 with sizes of approximately 43 and 57 ku were successfully prepared and had good antigenicity.A hybridoma cell line (2H10A6) that stably secretes monoclonal antibodies against P33 protein was established through screening with the pGEX-4T-1-P33 recombinant protein.Monoclonal antibody subclass identification results showed that the antibody secreted by 2H10A6 was IgG1 subtype,and the antibody titer was 1∶6 553 600.Western blotting and IFA results showed that the 2H10A6 hybridoma cell line could specifically bind to the P33 protein of Theileria orientalis. 【Conclusion】 The recombinant P33 protein of Theileria orientalis was successfully obtained through a prokaryotic expression system in this study,and monoclonal antibody specifically recognizing the P33 protein was successfully generated.

【Objective】 This study aimed to comparatively analyze the differences in the composition and diversity of fecal flora between diarrheal and healthy forest musk deer under captive conditions,and identify the biomarker flora with significant differences,so as to provide a reference for the prevention and treatment of diarrhea in forest musk deer. 【Method】 Illumina NovaSeq 6000 platform was used to sequence the V3-V4 region of 16S rRNA gene in the total DNA of fecal flora of diarrhea (group D,n=10) and healthy (group H,n=10) forest musk deer,and the differences between groups D and H in the composition,diversity,biomarker flora and functions of the flora were analyzed. 【Result】 Alpha diversity analysis results showed that ACE and Chao1 indices of fecal microfiota of forest musk deer in group D were significantly higher than those of group H (P＜0.05),while the Simpson and Shannon indices were lower than those of group H,with a significant difference in the Simpson index between the two groups (P＜0.05).The results of Beta diversity showed that there was a significant difference in the community structure of flora between groups D and H (R²＝0.55，P＝0.001).The results showed that at phylum level,Firmicutes was the dominant phylum in both groups.The differential phyla included Bacteroidota,Proteobacteria and Actinobacteriota.At genus level,Ruminococcaceae_UCG_005 emerged as the common dominant genus in both groups,while the differential genera included Acinetobacter,Bacteroides and Rikenellaceae_RC9_gut_group.Group D contained 23 marker microbiota such as Olsenella,and group H had 15 marker microbiota such as Rikenellaceae_RC9_gut_group.Based on PICRUSt2 function prediction,it was found that compared with group H,group D was significantly increased in membrane transport,amino acid transport and metabolism,endocrine and metabolic diseases (P＜0.05),and significantly decreased in the biosynthesis of other secondary metabolites,cell growth and death,defense mechanisms (P＜0.05). 【Conclusion】 Diarrhea could significantly change the composition,diversity and functional abundance of the intestinal flora of forest musk deer.The abundance of Acinetobacter,Olsenella and Rikenellaceae_RC9_gut_group in the gut were characteristic markers of the intestinal health of forest musk deer.

【Objective】 This study aimed to investigate the sequence characteristics of Salmonella Typhimurium FliC protein,its prokaryotic expression,and effects on the proliferation and cytokine secretion of macrophage RAW264.7 cells. 【Method】 Specific primers were designed based on Salmonella Typhimurium ATCC 14028 gene sequence from GenBank,and FliC gene coding sequence was amplified by PCR using the Salmonella Typhimurium ATCC 14028 genome DNA as a template.Online bioinformatics tools were used to analyze the structural features of FliC protein.The abtained FliC gene was cloned into the prokaryotic expression vector pET-28a(+),and the recombinant plasmid pET-28a-FliC was transformed into Escherichia coli BL21(DE3) competent cells to construct the recombinant strain BL21 (pET-28a-FliC).The expression of the recombinant protein was induced with IPTG,and the expression conditions were optimized.The recombinant protein was purified by Ni-NTA and verified by SDS-PAGE and Western blotting.The purified recombinant protein was co-incubated with macrophage RAW264.7,then the proliferation of macrophages was detected by cytotoxicity test,and the cytokines secretion of macrophage was detected by ELISA. 【Result】 FliC gene with 1 488 bp was successfully amplified,and FliC protein (molecular formula:C₂₁₉₄H₃₅₉₇N₆₄₀O₆₄₁S₃) consisted of 495 amino acids with a molecular weight of approximately 52 ku.The protein was acidic,with high frequencies of Ala (12.3%) and Thr (11.5%) residues.It was hydrophilic,lacked a signal peptide and transmembrane domains,and contained 67 phosphorylation sites.The secondary structure of the protein consisted of alpha helix,beta sheet,beta turn and random coil,with the proportions of 39.80%,20.2%,4.24% and 35.76%,respectively.SDS-PAGE and Western blotting showed that the recombinant protein was partially soluble and had a good reactivity with flagellum serum from mice.The recombinant purified protein of 50 and 400 ng/mL could effectively stimulate the proliferation of macrophages,and the proliferation rate reached the highest at 24 h.At the same time,it could effectively stimulate macrophages to secrete cytokines interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α),with peak cytokine levels at 12 h. 【Conclusion】 FliC gene of Salmonella was successfully amplified.The high purity FliC protein was acidic,hydrophilic,which could promote the proliferation of macrophages and secretion of cytokines.This study laid a foundation for in-depth analysis of function of the Salmonella FliC protein.

【Objective】 Interleukin-8 (IL-8) was a key chemokine that plays a role in the immune response,and the increase of its level was closely linked to multiple infectious diseases and autoimmune conditions.It had been recognized as a detection target for active pulmonary tuberculosis,tuberculous meningitis,and bovine tuberculosis.This study aimed to develop monoclonal antibodies against bovine IL-8 and create a sandwich ELISA kit for the quantitative detection of IL-8 in bovine serum or plasma,offering tools for diagnosing and researching bovine tuberculosis (bTB) and other inflammation-associated diseases. 【Method】 The bovine IL-8 gene was cloned into pGEX-6P-1 and pcDNA3.1 vectors and expressed in Escherichia coli and 293F cells.Recombinant proteins were purified using GST affinity and nickel columns.Eukaryotic recombinant protein was used to immunize BALB/c mice,and monoclonal antibodies against bovine IL-8 were generated and screened via lymphocyte hybridoma technology.The optimal antibody pairs were identified through checkerboard analysis,and a sandwich ELISA detection method was established.IL-8 level was quantitatively assessed in plasma samples from shedding and non-shedding tuberculosis-infected cattle as well as healthy cattle to evaluate the method’s effectiveness in detecting tuberculosis in cattle. 【Result】 SDS-PAGE analysis showed that IL-8 produced in both prokaryotic and eukaryotic expression systems was expressed in a soluble form,with purified proteins exhibiting a purity exceeding 90%.Five monoclonal cell lines stably secreting IL-8 antibodies including 2B8,2F5,and so on, were obtained after three rounds of subcloning,all confirmed as IgG1 subtype.Checkerboard analysis revealed that 4G10 and 3D5-HRP were the optimal antibody pairs.The ELISA method established with 4G10 and 3D5-HRP was applied for quantitative detection of IL-8 level in plasma from cattle with varying infection statuses,demonstrating that IL-8 level in plasma of tuberculosis-infected cattle was extremely significantly higher than that in healthy cattle (P＜0.01). 【Conclusion】 The monoclonal antibodies against bovine IL-8 were successfully prepared and a sandwich ELISA detection method was established，which could realize the quantitative measurement of bovine IL-8,with potential applications in screening for tuberculosis in cattle and analyzing immune and infection status.

【Objective】 This study aimed to optimize the fermentation medium components of Haemophilus parasuis (Hps) serotype 12 H31 strain by response surface methodology,improve the number of viable bacteria,reduce the cost of vaccine production by fermentation of Haemophilus parasuis serotype 12 H31 strain,and provide reference for industrial production of Hemophilus parasuis vaccine. 【Method】 Employing single-factor experiments,the effects of bovine serum,yeast extract,nicotinamide adenine dinucleotide (NAD) and MgSO₄ on the viable bacteria count of Haemophilus parasuis serotype 12 H31 strain were investigated meticulously.After determining the optimal level ranges,suitable levels were selected for Box-Behnken design (BBD) response surface optimization design.The fermentation conditions were systematically optimized by different combinations of bovine serum,yeast extract,NAD and MgSO₄.The mathematical model between viable count and each component was established by quadratic response surface regression method,and the influence degree of each component on viable count was determined by regression analysis. 【Result】 Single factor optimization revealed that the optimal ranges of the various components added to the fermentation medium were as follows:When the viable count was high,bovine serum was 8%-10%,yeast extract was 0.4%-0.6%,NAD was 0.05%-0.1% and MgSO₄ was 0.3-0.5 g/L.Further application of surface optimization resulted in the determination of the optimal culture medium components for Haemophilus parasuis serotype 12 H31 strain after optimization, bovine serum concentration was 9.91%,MgSO₄ concentration was 0.21 g/L,yeast extract concentration was 0.59%,NAD concentration was 0.09%.Compared with the initial medium components,the viable count of the strain was increased from 1.2×10⁸ to 3.0×10⁹ CFU/mL,which was 24 times higher after optimization. 【Conclusion】 In this experiment,the response surface method was used to optimize the fermentation conditions of Haemophilus parasuis serotype 12 H31 strain.The optimized fermentation medium could improve the number of viable bacteria of Haemophilus parasuis well,and provide a reference for the industrial production of Haemophilus parasuis vaccine.

【Objective】 The experiment aimed to explore the bioinformatics characteristics of the spherical body protein 2 (SBP2) of Babesia bovis,and conduct prokaryotic expression and prepare polyclonal antibodies,providing a theoretical basis for the screening of Babesia bovis vaccines and diagnostic antigens. 【Method】 SBP2 gene of Babesia bovis was amplified and cloned. The phylogenetic tree of SBP2 protein of Babesia bovis was constructed using Mega 7.0.The phosphorylation sites and B-cell antigenic epitopes of the SBP2 protein in Babesia bovis were predicted and analyzed using bioinformatics methods such as IEDB Analysis Resource.The amino acid sequences of SBP2 protein and dense granules antigen (GRA) of Toxoplasma gondii were compared and analyzed. The prokaryotic expression vector pET-28a-SBP2 was constructed. The recombinant protein of SBP2 was induced to be expressed and purified.The reactivity of the recombinant protein of SBP2 was verified by Western blotting.Polyclonal antibodies were prepared by immunizing BALB/c mice with the recombinant protein SBP2 as the immunogen,and their titers were detected by indirect ELISA method. 【Result】 The phylogenetic tree showed that SBP2 protein of Babesia bovis in this study was most closely related to the amino acid sequence of the Thai strain (OM46855).Bioinformatics analysis revealed that SBP2 protein contained 17 B-cell antigenic epitopes and 31 phosphorylation sites,including 12 serine sites,14 threonine sites and 5 tyrosine sites.There was a strong correlation between the amino acid sequence of SBP2 protein of Babesia bovis and GRA protein of Toxoplasma gondii.The recombinant plasmid pET-28a-SBP2 was successfully constructed.The results of Western blotting showed that the molecular weight of the SBP2 recombinant protein was approximately 35 ku and it had good reactivity.The titer of the prepared polyclonal antibody was 1∶204 800. 【Conclusion】 This study deepened the understanding of SBP2 protein by predicting its biological characteristics.The SBP2 recombinant protein of Babesia bovis was successfully obtained using the prokaryotic expression system,and its polyclonal antibodies derived from mice were also acquired,laying a foundation for further research on the function of SBP2 and its role in the pathogenic mechanism of Babesia bovis.

【Objective】 The codon of the novel lectin PcLT gene sequence of Procambarus clarkii was optimized，and the physicochemical properties and structure of the protein encoded by this sequence were analyzed using bioinformatics software.The recombinant PcLT protein was expressed through the prokaryotic expression system,and the application effect in white spot syndrome virus disease of shrimp was evaluated. 【Method】 The PcLT gene sequence was obtained from GenBank (accession No.：JQ670880.1),optimized according to the codon preference of Escherichia coli.The physicochemical properties,secondary structure and tertiary structure of the protein were predicted by bioinformatics software.The PcLT gene fragment was ligated with the pET-32a(＋) vector by T4 ligase to construct the recombinant plasmid pET32a-PcLT.The recombinant protein PcLT was expressed in prokaryotic expression system of Escherichia coli BL21 (DE3).The protein was purified by Ni-column affinity chromatography.The recombinant protein PcLT was mixed with feed and fed to diseased shrimp infected with White spot syndrome virus (WSSV)，and to detect its application effect. 【Result】 PcLT gene encoded 169 amino acids，and PcLT protein was a stable and hydrophobic protein with a theoretical isoelectric point of 4.17 and a solubility of 0.645.It was a non-transmembrane protein,containing 27 phosphorylation sites and a secretory signal peptide.The secondary structure of PcLT protein mainly included alpha helix (26.63%),extended chain (21.30%) and random coil (52.07%),and the tertiary structure was mainly random coil.The recombinant plasmid pET32a-PcLT was successfully constructed,and the recombinant protein PcLT was expressed and purified.The molecular mass of PCLT protein was 33 ku.After feeding the shrimp with 100 mL PcLT recombinant protein for 5 days,the copy number of WSSV in the shrimp decreased,the virus detection result turned from positive to negative,and the shrimp ate the feed normally. 【Conclusion】 The recombinant protein PcLT was successfully expressed,and it had a good effect in the application against WSSV.This results provided a theoretical basis for the prevention and treatment of WSSV in shrimp aquaculture and related research.

【Objective】 Porcine circovirus type 4 (PCV4) is a newly discovered type of Porcine circovirus related to respiratory diseases,diarrhea and dermatitis-nephrotic syndrome of pigs in recent years.This study aimed to investigate the prevalence and genetic variation of PCV4 in pig farms in Henan province. 【Method】 From January 2020 to August 2023,504 porcine serum and tissue samples suspected of PCV4 infection collected from Henan province were detected by PCR method.Whole-genome amplification and sequencing were performed on PCV4 positive samples.The similarity comparison of the whole genome sequence of PCV4 was conducted using MegAlign program in DNAStar software.The amino acid sequences of Rep and Cap proteins were compared and analyzed by MegAlign and BioEdit software. 【Result】 Among 504 samples,the detection rate of PCV4 was 15.28% (77/504),and except for Xinyang,PCV4 was detected in all other cities.15 PCV4 whole genome sequences were obtained,the similarity of nucleotide sequences among them ranged from 97.7% to 99.9%,and the similarity with the 51 genomic sequences of PCV4 included in GenBank ranged from 97.7% to 100%.Amino acid sequence analysis showed that the main amino acid mutation in Rep protein was V239L,while the main amino acid mutations in Cap protein were S27N,R28G and L212M.Genetic evolution analysis showed PCV4 belonged to Circovirus of Circoviridae and had the highest affinity with Mink circovirus.Among 15 PCV4 strains,10 strains belonged to PCV4a,3 strains belonged to PCV4b,and 2 strains belonged to PCV4c. 【Conclusion】 PCV4 had been widely present in pig farms of Henan province,and PCV4a had become the main prevalent strain.This study had enriched the epidemiological data of PCV4 in Henan region,providing references for the prevention and control of PCV4 and further research.

【Objective】 The objective of this study was to establish an indirect ELISA (iELISA) method with good specificity and sensitivity for detecting antibodies against Schmallenberg virus (SBV). 【Method】 The full-length sequence of the SBV N protein-coding gene was cloned into pET-28a(+) plasmid to construct recombinant plasmid pET-28a-SBV.The target protein was expressed using the Escherichia coli expression system and purified using a His-tagged nickel column.After identification by SDS-PAGE and Western blotting,the purified SBV N protein was used as the detection antigen for coating to establish an iELISA method.The coating conditions,sealing fluid,sealing time,and incubation time of serum were optimized.The negative and positive critical value,specificity,sensitivity,and repeatability of the method were evaluated. 【Result】 SDS-PAGE results showed that the molecular weight of SBV N protein was 30 ku.Western blotting results demonstrated that the purified SBV N protein specifically reacted with SBV positive serum.The optimal conditions for the established SBV iELISA method were as follows:SBV N antigen coating concentration of 4 μg/mL,coating at 4 ℃ for 12 h,sealing with 1% gelatin at 37 ℃ for 1 h,serum dilution of 1∶200,incubation at 37 ℃ for 1 h,and enzyme-labeled secondary antibody dilution of 1∶6 000 with incubation at 37 ℃ for 1 h.The negative and positive critical value was 0.371.The iELISA method specifically reacted with SBV positive serum and showed no cross-reactivity with positive sera against BPIV3,BRV,BCoV,IBRV,BVDV and AKAV.The method exhibited high sensitivity,with a clear reaction observed even at a serum dilution of 1∶1 280.The coefficient of variation of inter-batch and intra-batch repeated tests was less than 10%.Compared with the commercial kit,the total concordance rate was 94.57%. 【Conclusion】 This study established an SBV iELISA method using SBV N protein as the detection antigen.The method demonstrated good specificity and sensitivity and could be applied for serological detection of SBV,providing an additional option for rapid diagnosis of SBV.

【Objective】 The purpose of the experiment was to screen plant extracts that had inhibitory effects on canine Escherichia coli (E.coli),provide a reference for the application of plant extracts in the prevention and control of bacterial diseases in dogs. 【Method】 Fifteen plant extracts including shikimic acid,rubusoside,chlorogenic acid,etc.,were selected.The antibacterial activity of these extracts against E.coli was determined using the hole-punching method to identify those with significant antibacterial effects.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of plant extracts to canine E.coli were measured using the double dilution method.The MIC dose of each plant extract was co-cultured with E.coli in a constant temperature shaker.Samples were taken periodically,centrifuged,and supernatants were collected.The absorbance at D_{600 nm},D_{260 nm} and D_{280 nm} values of the supernatant was used to assess the impact of different plant extracts on the growth curve and extracellular nucleoprotein of E.coli.Meanwhile,the activity of alkaline phosphatase (AKP) in the supernatant and lactate dehydrogenase (LDH) in the bacteria was measured using kits. 【Result】 Shikimic acid,rubusoside,chlorogenic acid,phloretin and salidroside showed good antibacterial effects against E.coli.Based on the size of the inhibition zone diameter,their antibacterial capacities ranked as follows:Shikimic acid＞chlorogenic acid＞rubusoside＞phloretin＞salidroside.The MICs of phloretin,shikimic acid,rubusoside,chlorogenic acid and salidroside to canine E.coli were 0.48,15.625,62.5,31.25 and 125 mg/mL respectively,while their MBCs were 0.48,31.25,125,62.5 and 250 mg/mL,respectively.Growth curve measurements indicated that all plant extracts at MIC doses inhibited the growth of E.coli.Analysis of the absorbance at D_{260 nm} and D_{280 nm} values revealed that chlorogenic acid,salidroside,rubusoside and phloretin significantly damaged the cell membrane of E.coli.AKP activity in the supernatant under the influence of rubusoside and salidroside was significantly higher than that of control group (P<0.05),indicating that they could destroy the cell wall of E.coli and exert antibacterial effects.LDH activity in bacteria treated with shikimic acid,rubusoside,chlorogenic acid,phloretin and salidroside was significantly lower than that of control group (P<0.05),suggesting that all five plant extracts could inhibit intracellular LDH activity and affect bacterial metabolism. 【Conclusion】 Shikimic acid,rubusoside,chlorogenic acid,phloretin and salidroside had significant inhibitory effects on canine E.coli.

【Objective】 The purpose of the experiment was to elucidate the cross-regulatory mechanism of ERK signaling pathway in the inflammatory response process of avian macrophages. 【Method】 The chicken macrophage cells (HD11) were divided into four groups:Control group (CON),lipopolysaccharide group (LPS),inhibitor＋lipopolysaccharide group (AST-Ⅳ＋LPS),and inhibitor group (AST-Ⅳ).HD11 cells in LPS group were treated with 1 μg/mL lipopolysaccharide,HD11 cells in AST-Ⅳ group waere treated with 5 μg/mL ERK inhibitor astragaloside Ⅳ (AST-Ⅳ),HD11 cells in AST-Ⅳ＋LPS group were treated with 5 μg/mL ERK inhibitor for 12 h and then treated with 1 μg/mL LPS for 6 h,and HD11 cells in control group were treated with an equal volume of DMEM culture medium.Each group was set up with three biological replicates.Real-time quantitative PCR and Western blotting were used to detect the expression of classical Ras-MEK-ERK signaling pathway-related factors and inflammatory factor genes and proteins,while hypoxia inducible factor-1α (HIF-1α) and nuclear factor kappa-B (NF-κB) protein expression were also detected.The changes of intracellular reactive oxygen species (ROS) were detected by fluorescence probe method,and the mitochondrial membrane potential was detected by flow cytometry. 【Result】 Compared with control group,when LPS induced an inflammatory response in cells,the expression of key classical ERK pathway factors MAP3K1,MEK and ERK,as well as downstream transcription factors p-c-Jun,p-c-Fos,and inflammatory factors interleukin-6 (IL-6),IL-8,and tumor necrosis factor-α (TNF-α) were significantly increased (P＜0.05).Mitochondrial membrane potential was decreased,ROS generation was increased (P＜0.05),and the expression of transcription factor HIF-1α and NF-κB protein were also increased significantly (P＜0.05).However,the expression of IL-6,IL-8,TNF-α, ROS,HIF-1α and NF-κB produced by HD11 cells could be significantly reduced by pre-addition of ERK inhibitors (P＜0.05),and mitochondrial membrane potential was increased (P＜0.05). 【Conclusion】 When macrophages underwent inflammatory damage,they could upregulate inflammatory factor expression through the classical ERK pathway,damaging the integrity of the mitochondrial inner membrane and promoting the generation of ROS.ROS-HIF-1α/NF-κB further promoted inflammatory factor expression in a vicious cycle,exacerbating the cellular inflammatory response.This study laid a foundation for elucidating the crosstalk between the ERK pathway and other inflammation pathways and the mechanisms of inflammation development.

【Objective】 The purpose of this experiment was to explore the effect of store operated Ca^2＋ entry (SOCE) on the phagocytic and adhesion functions of mononuclear macrophages in dairy cows. 【Method】 Peripheral blood of healthy (control，Con)and subclinical hypocalcemia dairy cows (hypocalcemia,Hyp) was collected,and CD14^＋ mononuclear macrophages in the blood were isolated and cultured in vitro using magnetic beads.The mononuclear macrophages of subclinical hypocalcemia dairy cows were divided into three groups: Hyp, thapsigarsin (TG) treatment group (Hyp+TG) and 2-aminoethyl diphenylborinate (2-APB) treatment group (Hyp＋2-APB).The intracellular Ca^2＋ level was detected by flow cytometry.Real-time quantitative PCR and Western blotting were used to detect the mRNA and protein expression of SOCE-related factors.The phagocytosis and adhesion of mononuclear macrophages were detected by laser confocal and immunofluorescence,and the expression of phagocytosis and adhesion factors of mononuclear macrophages was detected. 【Result】 Flow cytometry analysis showed that,compared with Con group,the intracellular Ca^2＋ level of mononuclear macrophages in Hyp group was extremely significantly decreased (P＜0.01).Real-time quantitative PCR results showed that,compared with Con group,the expression of calcium release-activated calcium modulator 1 (ORAI1)，stromal interaction molecule 1 (STIM1),STIM2 and selectin L (SELL) genes of mononuclear macrophages in Hyp group were extremely significantly decreased (P＜0.01)，expression of ORAI2,ORAI3 and myeloperoxidase (MPO) genes were significantly decreased (P＜0.05).Western blotting results showed that,compared with Con group,the expression of ORAI2,ORAI3,STIM2,ORAI1 and STIM1 proteins of mononuclear macrophages in Hyp group were extremely significant or significantly decreased (P＜0.01 or P＜0.05).Meanwhile,compared with Con group,the phagocytic and adhesion abilities of mononuclear macrophages in Hyp group were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).After TG treatment,compared with Hyp group,the expression of ORAI1,ORAI2,MPO,ORAI3,STIM1,STIM2 and SELL genes in mononuclear macrophages of subclinical hypocalcemia dairy cows were significantly or extremely significantly increased (P＜0.05 or P＜0.01)，the expression of ORAI1,ORAI3,STIM1,STIM2 and ORAI2 proteins were significantly or extremely significantly upregulated (P＜0.05 or P＜0.01).The phagocytic and adhesion abilities of cells were significantly or extremely significantly enhanced (P＜0.05 or P＜0.01).After 2-APB treatment,compared with Hyp group,the expression of ORAI1,ORAI2,MPO,ORAI3,STIM1,STIM2 and SELL genes in mononuclear macrophages of subclinical hypocalcemia dairy cows were significantly or extremely significantly decreased (P＜0.05 or P＜0.01)，the expression of ORAI2,STIM2,ORAI1,ORAI3 and STIM1 proteins were significantly or extremely significantly decreased (P＜0.05 or P＜0.01).The phagocytic and adhesion abilities of cells were extremely significant or significantly weakened (P＜0.01 or P＜0.05). 【Conclusion】 After SOCE was activated by TG,the Ca^2＋ level in mononuclear macrophages of subclinical hypocalcemia dairy cows increased,and the phagocytic and adhesion abilities enhanced.After SOCE was inhibited by 2-APB,the intracellular Ca^2＋ level in mononuclear macrophages of subclinical hypocalcemia dairy cows decreased,and the phagocytic and adhesion functions weakened，which indicated that SOCE was involved in regulating the phagocytosis and adhesion of mononuclear macrophages.

【Objective】 The purpose of this experiment was to construct Salmonella Pullorum expressing red fluorescent protein (mCherry) to explore its localization and tracing effect in macrophages. 【Method】 The exogenous gene mCherry was inserted into the prokaryotic expression vector pUC19 to obtain the pUC19-mCherry plasmid.Using the genome of Salmonella Pullorum as a template,the endogenous virulence gene virK promoter was amplified by PCR and inserted into the pUC19-mCherry plasmid to drive the expression of mCherry.The recombinant plasmid PvirK-mCherry was constructed and transformed into the Salmonella Pullorum CVCC 533.Single colonies were selected,and fluorescence expression was confirmed using upright fluorescence microscopy.The recombinant fluorescent strain with an multiplicity of infection (MOI) of 200 was co-cultured with RAW264.7 and HD11 cells.The phagocytic effects of the two types of macrophages on the fluorescent strain were detected by DAPI staining and flow cytometry. 【Result】 The double enzyme digestion results of the pUC19-mCherry plasmid showed a 720 bp mCherry gene fragment and a 2 600 bp vector fragment.Red fluorescence was observed after transforming the Escherichia coli DH5α competent cells.The sequencing results of the recombinant plasmid PvirK-mCherry showed that the promoter was successfully inserted into the pUC19-mCherry plasmid.Double enzyme digestion revealed a 990 bp virK gene promoter fragment and a 3 300 bp vector fragment.Fluorescence microscopy of Salmonella Pullorum transformed with PvirK-mCherry plasmid showed red fluorescence.The results of the macrophage phagocytosis test showed that after DAPI staining,fluorescence microscopy revealed that the red fluorescent Salmonella Pullorum strains were aggregated around the blue nuclei.Flow cytometry results showed that the phagocytic efficiency of RAW264.7 cells was 88.06%,and that of HD11 cells was 34.18%. 【Conclusion】 This study successfully constructed the plasmid PvirK-mCherry，after transfection with Salmonella Pullorum,it expressed red fluorescence,and the red fluorescent strain could be phagocytosed by macrophages.This results not only helped to precisely observe the localization of bacteria within host cells,but also provided an economical and effective tool for studying the interaction between Salmonella and host cells,which was of great significance to the health of poultry and the development of breeding industry.

【Objective】 This experiment was to investigate the prevalence of infectious pathogenic microorganisms causing diarrhea in calves from large-scale cattle farms in Gansu and to understand the resistance status of Clostridium perfringens (Cp) to common antibacterial drugs. 【Method】 5-combined nucleic acid detection kit for pathogenic microorganisms of calf diarrhea was used to test 108 fecal samples of calf diarrhea collected from 10 cattle farms in 8 regions including Lanzhou,Zhangye and Wuwei，which mainly included Cp,Bovine rotavirus (BRV),Bovine coronavirus (BCoV),enterotoxigenic Escherichia coli K99 (K99) and Cryptosporidium (COWP). Cp nucleic acid positive bovine fecal samples were subject to bacterial isolation and culture,and identified using staining microscopy and biochemical tests.At the same time,PCR amplification was used to type the isolated strains.The drug sensitivity test and drug resistance gene detection of the isolated strains were conducted by disk diffusion method (K-B) and PCR，respectively. 【Result】 Among the 108 fecal samples collected from calves with diarrhea,the positive rate of Cp nucleic acid was 78.70% (85/108),the positive rate of individual infection was 14.81% (16/108),and the total mixed infection rate was 63.89% (69/108).The mixed infection rates of Cp with BRV and BCoV was 44.44% (48/108) and 24.07% (26/108),respectively.A total of 13 Cp were isolated,and all of them were type A.The results of drug sensitivity test showed that 84.62% (11/13) of the isolated strains were resistant to gentamicin,and 76.92% (10/13) were resistant to kanamycin.The results of the resistance gene detection showed that 84.62% (11/13) of the isolated strains were detected with Acc(6')/ph(2″) gene,53.85% (7/13) of the isolated strains were detected with ermC gene,and none of the seven resistance genes such as Sul1,qnrS,and ermB were detected. 【Conclusion】 The mixed infection of calf diarrhea in Gansu region was relatively serious.The main prevalent Cp strain was type A，and most Cp isolated strains were severely resistant to aminoglycoside and carried multiple resistance genes.

【Objective】 The purpose of this study was to investigate the effect and mechanism of total flavonoids of Melastoma dodecandrum Lour.(TFMD) on nonalcoholic fatty liver disease (NAFLD) in rats. 【Method】 In vitro,HepG2 cells were divided into 6 groups:Control group,NAFLD model group,TFMD high,medium and low dose groups (25,50 and 100 μg/mL TFMD),iron death inhibitor group (Fer-1,2 μmol/L Fer-1),6 duplicate wells per group.HepG2 cells were treated with 0.25 mmol/L palmitic acid for 24 h to establish cell model in all groups except for control group,and HepG2 cells in TFMD low,medium and high dose groups and the iron death inhibitor group were treated with corresponding concentrations of TFMD and Fer-1 for 24 h,respectively.The cell viability was determined by cytotoxicity assay.The contents of total cholesterol (TC) and triglyceride (TG) in HepG2 cells were detected by related kits,and the effects of TFMD on the lipid peroxidation level of NAFLD in vitro model were evaluated by detecting the activities of superoxide dismutase (SOD),contents of glutathione (GSH) and malondialdehyde (MAD).The level of reactive oxygen species (ROS) in HepG2 cells was detected by DCFH-DA fluorescence probe method.In vivo,SD rats were fed with high fat diet to establish NAFLD model,and were randomly divided into 6 groups:Control group,NAFLD model group,TMFD high,middle and low dose groups,and polyene phosphatidylcholine (PPC) group,with 10 rats in each group.After the success of modeling was judged,the corresponding drug intervention was given immediately.Rats in PPC group were given intragastric administration of PPC (0.07 g/kg BW),and the rats in TMFD low,medium and high dose groups were given intragastric administration of TMFD (0.33,0.50 and 0.75 g/kg BW) for 6 weeks.Rats in control group and NAFLD model group were given the same volume of saline.At the end of the treatment period,the contents of TG,TC,high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol (LDL-C),the activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum of NAFLD rats, the contents of MDA and Fe²⁺,and the total antioxidant capacity (T-AOC) and the activity of catalase (CAT) in liver of NAFLD rats were detected by related kits.The hepatic pathological changes of NAFLD rats were observed by hematoxylin-eosin staining and oil red O staining.The expression of NF-E2-related factor 2 (Nrf2),peroxisome proliferator-activated receptor γ (PPARγ),peroxisome proliferative receptor gamma coactivator 1α (PGC-1α),NAD(P)H quinone dehydrogenase 1 (NQO1),RelA (NF-κB P65),glutathione peroxidase 4 (GPX4) and Kelch-like ECH-associated protein 1 (Keap1) were detected by Western blotting. 【Result】 In vitro test results showed that compared with control group,the activity of HepG2 cells in NAFLD model group was extremely significantly decreased (P＜0.01),the contents of TG,TC and MDA were extremely significantly increased (P＜0.01),and the content of GSH and activity of SOD were extremely significantly decreased (P＜0.01).The level of ROS in HepG2 cells was extremely significantly increased (P＜0.01),and the fluorescence intensity was significantly enhanced.Compared with NAFLD model group,the activity of HepG2 cells in TFMD groups and Fer-1 group was extremely significantly increased (P＜0.01),the contents of TG,TC and MDA were extremely significantly decreased (P＜0.01),and the content of GSH and activity of SOD were extremely significantly increased (P＜0.01).The level of ROS in HepG2 cells was extremely significantly decreased (P＜0.01),and the fluorescence intensity was significantly decreased.The results of in vivo study showed that compared with control group,the content of HDL-C in serum of rats in NAFLD model group was extremely significantly decreased (P＜0.01),while the contents of TC,TG and LDL-C were extremely significantly increased (P＜0.01).Compared with NAFLD model group,the content of HDL-C in serum of rats in TFMD groups and PPC group was extremely significantly or significantly increased (P＜0.01 or P＜0.05),the content of LDL-C was extremely significantly or significantly decreased (P＜0.01 or P＜0.05),and the contents of TG and TC were extremely significantly decreased (P＜0.01).Pathological observation showed that compared with control group,the hepatocytes of the NAFLD model group had a large number of fat vacuoles,disorderly and uneven arrangement,severe fatty degeneration in the hepatocytes,a large number of red lipid droplets in the hepatocyte,and the volume of lipid droplets became larger and had a certain tendency to aggregate.The morphology of hepatocytes was neat,the degree of fatty degeneration was significantly decreased,the number of red lipid droplets in the liver was reduced,and the volume became smaller in TMFD groups and PPC group.Compared with control group,the activities of AST and ALT in serum of rats in NAFLD model group were extremely significantly increased (P＜0.01),the activity of CAT and T-AOC in the liver of rats in NAFLD model group were extremely significantly decreased (P＜0.01),and the contents of MDA and Fe²⁺ were extremely significantly increased (P＜0.01).Compared with NAFLD model group,the activities of AST and ALT in serum of rats in TFMD groups and PPC group were extremely significantly or significantly decreased (P＜0.01 or P＜0.05),the content of MDA in liver of rats in TFMD groups and PPC group was extremely significantly or significantly decreased (P＜0.01 or P＜0.05), T-AOC was extremely significantly or significantly increased (P＜0.01 or P＜0.05),the activity of CAT was extremely significantly increased (P＜0.01),and the content of Fe²⁺ was extremely significantly decreased (P＜0.01).Western blotting results showed that the expressions of PPARγ,PGC-1α,Nrf2,NQO1 and GPX4 proteins in liver of rats in NAFLD model group were extremely significantly lower than those in control group (P＜0.01),the expressions of Keap1 and NF-κB P65 proteins were extremely significantly increased (P＜0.01).Compared with NAFLD model group,the expressions of PPARγ,PGC-1α,Nrf2,NQO1 and GPX4 proteins were extremely significantly increased (P＜0.01) in TFMD groups and PPC group,the expressions of Keap1 and NF-κB P65 proteins were extremely significantly decreased (P＜0.01). 【Conclusion】 The mechanism of TFMD intervention on NAFLD was related to the regulation of iron metabolism and lipid peroxidation imbalance in the body,and TFMD inhibit the iron death of related cells in the liver by regulating PPARγ/PGC-1α/Nrf2 signaling pathway.

Aflatoxins are mainly toxic secondary metabolites produced by fungi such as Aflatus and Aspergillus parasiticus under suitable environmental conditions.They are widely present in food and feed,posing a great threat to human and animal health,and have become a major problem in the field of food and feed safety.Aflatoxin B₁(AFB₁) is one of the most harmful mycotoxins,which has high toxicity and strong carcinogenicity for humans and animals.Its toxicity mainly includes liver toxicity,neurotoxicity,intestinal toxicity,immunotoxicity and reproductive toxicity.How to effectively detect and detoxification aflatoxin B₁ has been a research hotspot at home and abroad.Traditional detection methods such as chromatography or immunoassay have been widely used for a long time,and with the progress of science and technology,some new detection methods have been developed with higher accuracy and sensitivity.In terms of detoxification,compared with physical and chemical detoxification,biological detoxification has become a current research hotspot due to its unique advantages and has better application potential.In this paper,the toxicity mechanism,detection methods and detoxification methods of aflatoxin B₁ will be comprehensively reviewed based on the latest scientific research results,the advantages and limitations of different detection and detoxification methods will be discussed,the feasibility of their practical application will be evaluated,and the future development direction will be looked forward to in order to provide a more solid theoretical basis for the research of aflatoxin B₁.Therefore,it can effectively reduce the threat of aflatoxins to human and animal health,and ensure food safety and public health.

【Objective】 The purpose of this experiment was to isolate and identify Salmonella from goat,detect its resistance to common antibacterial drugs and the bacteriostati effect of Chinese herbal medicines,aiming to provide a reference basis for the prevention and treatment of Salmonella-related diseases. 【Method】 A total of 33 anal swab samples and 17 nasal swab samples of Leizhou goats with symptoms of diarrhea and rhinorrhea were collected from a goat farm in Zhanjiang city,and bacterial isolation and purification were carried out.The pathogenic bacteria were identified by Gram staining,PCR amplification and sequencing.The minimum inhibitory concentration (MIC) of 15 commonly used antibacterial drugs against the isolated strains was determined by microbroth dilution method.The sensitivity of the isolates to 12 kinds of Chinese herbal medicines was determined by the double dilution method in test tubes.Subsequently,the in vitro bacteriostati effect of the combined application of different Chinese herbal medicines on the isolates was further explored.The biofilm formation amount of the isolates was determined by crystal violet staining. 【Result】 The isolated bacteria presented as round,neatly edged,slightly raised,colorless and transparent single colonies on Maclonkey agar medium.White,round and neatly edged single colonies appeared in the nutrient agar.Gram staining showed Gram-negative bacilli with blunt and round ends,no spores and no capsules.The PCR amplification results of 16S rRNA gene showed that a band with a fragment size of 1 500 bp.BLAST comparison results showed that the similarity between the isolated strains and Salmonella Enteritis (accession number:AE016841) was more than 97%.A total of 15 goat-derived Salmonella were isolated from 50 collected samples,with an isolation rate of 30%.The results of the drug susceptibility test showed that the resistance rates of the isolates to penicillin and ceftazidime were 90% and 70% respectively,the resistance rates to azithromycin and sulfadiazine sodium were both 40%,and the resistance rates to cefazolin sodium,oxytetracycline,streptomycin and acetoquinine were all 10%.The isolated strains were sensitive to amikacin,doxycycline,kanamycin,sulfamonomethoxil,piperacillin,minocythromycin and enrofloxacin.The results of the antibacterial test of Chinese herbal medicines showed that the MIC of Prunus mume and Schisandra chinensis against the isolates was 15.62 mg/mL,and the MIC of Coptis chinensis against the isolates was 62.5 mg/mL.The results of the combined medication test of Chinese herbal medicines showed that the MIC of the Chinese herbal medicine compound prescriptions composed of Coptis chinensis＋Atractylodes lancea,Schisandra chinensis＋Terminalia chebula,and Schisandra chinensis＋Pogosteom cablin was 15.62,7.81 and 7.81 mg/mL respectively.The results of growth curve and biofilm detection showed that the 2MIC Coptis chinensis,Prunus mume and Schisandra chinensis had the good antibacterial effect,among which Schisandra chinensis had the best effect in inhibiting the formation of Salmonella biofilms. 【Conclusion】 In this study,15 goat-derived Salmonella were isolated and obtained.The extracts of Chinese herbal medicines had a significant in vitro antibacterial effect,and when some Chinese herbal medicines were used in combination,the antibacterial activity could be enhanced.This results had significant guiding significance for Chinese herbal medicine in the prevention and treatment of Salmonella infections,and also provided data support for further exploration of comprehensive prevention and treatment strategies for Salmonella diseases in the future.

Against the backdrop of the rapid development of modern animal husbandry,the breeding industry of laying hens still faces many challenges,among which intestinal diseases are particularly prominent.Although traditional antibiotic treatment can control the disease to a certain extent,the ensuing problems of increased bacterial resistance,imbalance of intestinal flora,and increased treatment costs should not be ignored.Therefore,it is crucial to explore new products that are safe and effective and can replace antibiotics for the sustainable development of the breeding industry of laying hens.Bacillus subtilis is a Gram-positive non-pathogenic Bacillus with strong resistance.It can be used as a green and residue-free probiotic additive in the breeding industry of laying hens.The bacteria can effectively colonise the intestinal tract of laying hens,inhibiting the growth of pathogenic bacteria and promoting the growth of beneficial bacteria.It can maintain the balance of intestinal flora in laying hens.At the same time,it can enhance the immunity of laying hens by increasing the level of antibody and maintain the intestinal health.In addition,Bacillus subtilis can maintain the intestinal morphology and structure of laying hens by increasing the villi height and decreasing the crypts depth.In the practice of laying hens,the application of Bacillus subtilis can significantly enhance the performance,including the improvement of egg quality,the increase of egg production,and the reduction of the feed-to-egg ratio.The author reviewed the biological characteristics of Bacillus subtilis,its mechanism of action in the intestinal tract and the application effects of different strains of Bacillus subtilis in laying hens，to provide a reference basis for the subsequent development and utilisation of Bacillus subtilis for feeding.

【Objective】 Bacillus subtilis (B.subtilis) ZJ20 is a strain with the ability to degrade aflatoxin B₁(AFB₁).The aim of this experiment was to evaluate the in vitro safety of B.subtilis ZJ20 with a view to provide a basis for the application of this strain in the degradation of AFB₁ in feed and food. 【Method】 The genome-wide sequence of B.subtilis ZJ20 was used to predict virulence genes,drug resistance genes,and genes related to biofilm formation,and drug sensitivity and biofilm formation ability were experimentally verified based on the genome prediction results.In addition,the surface hydrophobicity,self-aggregation,and copolymerization of B.subtilis ZJ20 were examined.Using leghorn male hepatoma (LMH) cells as a model,B.subtilis ZJ20 cell-free supernatant (CFS) was administered to LMH cells at different concentrations.The effects of B.subtilis ZJ20 on LMH cell viability,apoptosis-related factors (Caspase-3,Caspase-9,BAX,and Bcl-2),and inflammatory cytokines (IL-1β,IL-6,TNF-α,and IFN-γ) were assessed to determine the potential cytotoxicity of B.subtilis ZJ20. 【Result】 There were 26 genes coding for virulence factors annotated in the genome of B.subtilis ZJ20,most of which were related to immune evasion and defense,and there were no genes coding for enterotoxins,and 11 antibiotic-resistant genes were identified,the results of the drug sensitivity test showed that B.subtilis ZJ20 was sensitive to a variety of antibiotics.Fourteen genes for the regulation of the bioepidermis were predicted,and the bioepidermal formation capacity of the strain reached its highest value at 24 h.The surface hydrophobicity was 47.842%,and the auto-aggregation rate was 59.334%.The co-aggregation rate with Salmonella Typhimurium ATCC 14028 was 60.905%,and with Escherichia coli CVCC 1527 was 47.309%.The results of cell viability assay showed that B.subtilis ZJ20 CFS added at 1% increased the cell viability to 107.34% at 36 h of interaction with LMH cells.The results of apoptosis and inflammation-related cytokine assays showed that compared with control group,the expression of Caspase-3 was significantly up-regulated with increasing dose (P＜0.05),but there was no significant change of Caspase-9 and BAX/Bcl-2 value (P＞0.05),and the expression of IL-6,IL-1β,TNF-α,and IFN-γ were all significantly down-regulated (P＜0.05). 【Conclusion】 The whole genome sequence analysis and its detection of the effects on the expression of LMH cell viability,apoptotic factors,and inflammation-related cytokines indicated that B.subtilis ZJ20 had a certain degree of safety,which provided a reference for the safety of its application in the development as a functional probiotic.

Embryonic death and abortion are among the major issues affecting reproductive efficiency and economic benefits in beef cattle production in China.The causes of embryonic death and abortion include genetic defects in embryos,maternal endocrine imbalances,pathogenic infections,and stress.Abnormal gene expression and chromosomal abnormalities in genetic factors can disrupt the normal development of embryos during critical developmental stages,leading to stasis or abnormalities.Maternal endocrine imbalances may interfere with embryo implantation and the maintenance of pregnancy,resulting in pregnancy loss.Pathogenic infections can cause embryonic death or abortion through mechanisms like inducing placental inflammation and disrupting the host’s innate immune response.Additionally,environmental stress can affect the metabolism and division of embryonic cells,triggering apoptosis and increasing the risk of early embryo death.Current research on embryonic death and abortion in domestic beef cattle mainly consists of case reports or epidemiological surveys,with relatively few studies on the mechanisms leading to these issues.This article systematically reviewed the causes of embryonic death and abortion in beef cattle,covering research advancements in genetic,maternal,pathogenic,environmental,and management factors,aiming to provide references for reducing the incidence of embryonic death and abortion in domestic beef cattle breeding.