【Objective】 This experiment aimed to obtain cluster of differentiation 71 (CD71) gene-edited immortalized porcine intestinal-2I (IPI-2I) cell lines through CRISPR/Cas9 technology，which provided materials for further study of CD71 gene function and its role in Transmissible gastroenteritis virus (TGEV) infection at the cellular level.【Method】 Five sgRNAs were designed for the third exon and its adjacent intron region of CD71 gene,which were ligated into the pX458-GFP vector.The activity of different sgRNA vectors was detected by T7E1 enzymatic cleavage in porcine embryonic fibroblast (PEF) cells.Two sgRNA vector plasmids with higher cleavage efficiency were selected to be electrotransfected into IPI-2I cells,and after 48 h,single cells with green fluorescent protein (GFP) were collected by flow cytometry,and then cultured in 96-well cell culture plates to screen the monoclonal cells.The monoclonal cells were genotyped by PCR amplification and TA cloning to obtain the CD71 gene-edited IPI-2I cell line.【Result】 The results of plasmid sequencing showed that all five sgRNAs were successfully ligated to the pX458-GFP vector.T7E1 enzyme digestion results showed that cleavage occurred in all five transfected sgRNA plasmids.Among them,the cleavage efficiencies of pX458-sgRNA1 and pX458-sgRNA4 were relatively high,which were 35.8% and 44.1%,respectively.The results of flow cytometry sorting showed that the proportion of positive cells transfected with pX458-sgRNA1 and pX458-sgRNA4 were 29.8% and 25.7%,respectively.PCR amplification results showed that among the 65 monoclonal cells obtained,14 cells occurred gene editing,with an editing efficiency of 22%. TA cloning results showed that among the 14 gene-edited cells,5 were monoallelic edited and 9 were biallelic edited.【Conclusion】 In this study,the CD71 gene-edited IPI-2I cell line was successfully constructed using CRISPR/Cas9 technology,and CD71 gene monoallelic and biallelic gene-edited cells were obtained.The results provided a good cell model for clarifying the function of CD71 gene and its mechanism of mediating TGEV infection.

【Objective】 The structure and function of promoters are crucial in gene transcriptional regulation research.This study aimed to analyze the structural characteristics and transcriptional regulatory mechanisms of the insulin-like growth factor 2 (IGF2) gene promoter region in goose,providing a theoretical foundation for understanding the role of IGF2 gene in the growth and development of goose.【Method】 Based on the 2 170 bp sequence of the 5'-flanking region of IGF2 gene (GCA_040182565.1) in goose,the promoter activity region was predicted using tools such as Promoter 2.0,and AnimalTFDB 4.0.The binding sites of transcription factors and regulatory elements were analyzed.The TBtools software was employed to analyze the conservation of the IGF2 gene promoter region in goose through cross-species sequence alignment.Moreover,genomic single nueleotide polymorphism (SNP)genotyping data from Huoyan geese and Zhedong White geese were untlized to compare the genetic variations between both geese breeds.Additionally,the transcriptional expression of IGF2 gene in gonadal axis tissues during the egg-laying period was analyzed for both geese breeds.【Result】 The promoter activity region of IGF2 gene in goose was located 1 020-1932 bp,containing critical elements such as the CAAT-box,GC-box,TATA-box,and E-box,which was associated with cell proliferation and differentiation.The transcription factors Sp1 and Irf3 were identified as potential regulators of gene transcription through binding to these elements.Cross-species sequence alignment revealed a high degree of similarity between the promoter regions of geese and Ferruginous ducks,with a similarity of 92.94%.SNP genotyping analysis showed a significantly higher proportion of CC genotype in Huoyan geese (89%) compared with Zhedong White geese (25%),while the proportions of CT and TT genotypes in Zhedong White geese (both were 38%) were markedly higher than those in Huoyan geese (11%).Furthermore,a significant difference in the expression of IGF2 gene was observed in ovarian tissue between the two goose breeds during the egg-laying period (P＜0.05).【Conclusion】 The promoter region of IGF2 gene in goose was abundant in binding sites for various transcription factors,including Sp1 and Irf3,which played crucial roles in regulating the stable expression of IGF2 gene.Additionally,a SNP variation (C/T) at position 1 450 bp in the promoter region of IGF2 gene had been identified in the germplasms of both Huoyan geese and Zhedong White geese.This genetic variation could affect the growth,development,and egg production performance of geese by influencing the expression of IGF2 gene.

【Objective】 This study aimed to investigate the biological role of the potassium voltage-gated channel subfamily KQT member 1 (KCNQ1) gene and the effects of non-synonymous single nucleotide polymorphisms (nsSNPs) on the renal water reabsorption function of Tarim Red deer.【Method】 Based on the previous sequencing data,genotype frequency alignment was performed to screen and obtain the KCNQ1 gene sequence of Tarim Red deer.Similarity alignment and phylogenetic tree construction were performed,and bioinformatics analysis was performed on the KCNQ1 gene sequence using online software.Whole blood DNA from Northeastern Red deer and Tarim Red deer were utilised as the experimental material to clone and construct the overexpression vectors of the wild-type and mutant of KCNQ1 gene.The vectors were then infected with lentiviruses,and the expression of KCNQ1 protein was detected by Western blotting.【Result】 There was a non-synonymous mutation site c.A835G (p.I279V) associated with drought tolerance in KCNQ1 gene CDS of Tarim Red deer.The results of similarity alignment showed that the similarity was the highest (99.41%) between Tarim Red deer and Cervus canadensis Canadensis,and the similarity with Odocoileus virginianus and Dama dama exceeded 97%.The phylogenetic tree showed that Tarim Red deer had the closest genetic relationship with Cervus canadensis Canadensis,but had a distant genetic relationship with Bos taurus and Capra hircus.KCNQ1 protein of Tarim Red deer was consisted of 340 amino acids,the molecular formula was C₁₆₂₄H₂₆₀₇N₅₃₅O₄₅₆S₆,the protein molecular mass was 37.15 ku,an instability coefficient of 52.12,a theoretical isoelectric point of 11.5,and an average hydrophilicity of ―0.708.KCNQ1 protein was predominantly located in the basolateral membrane,with O-glycosylation and phosphorylation sites,but it lacked N-glycosylation sites,signal peptides,and transmembrane regions.PolyPhen-2 software predicted that the mutation of KCNQ1 protein could lead to a benign change,and this mutant site was on the transcription factor HNF-1β,which promoted the transcriptional efficiency of KCNQ1 gene.The secondary and tertiary structures of KCNQ1 protein were primarily composed of alpha helix and random coil.However,a shift from alpha helix to random coil was observed in the mutant site of Tarim Red deer.Western blotting results revealed that the expression of both wild-type and mutant of KCNQ1 protein in Tarim Red deer were extremely significantly higher than that in control group (P＜0.01).Furthermore,the expression of KCNQ1 protein in mutant was extremely significantly higher than that in wild-type (P＜0.01).【Conclusion】 There was a non-synonymous mutation site c.A835G (p.I279V) in KCNQ1 gene CDS of Tarim Red deer.KCNQ1 protein of Tarim Red deer encoded 340 amino acids and had O-glycosylation and phosphorylation sites.Its mutation site had undergone benign changes and was located on the transcription factor HNF-1β.The experiment successfully cloned and constructed the wild-type and mutant lentivirus overexpression vectors of KCNQ1 gene,and the expression of mutant protein was significantly higher than that of wild-type.The results provided a theoretical basis for the mechanism of renal water reabsorption in Tarim Red deer.

【Objective】 The aim of this study was to isolate preadipocytes during the critical developmental phase of rump adipogenesis in Kazakh sheep,and investigate the developmental mechanism of rump adipose deposition in Kazakh sheep through histological and cytological analysis,so as to lay a foundation for investigating the molecular mechanisms underlying rump adipose deposition in sheep.【Method】 The embryos of Kazakh sheep at 50(E50),60(E60),70(E70),80(E80),94(E94),and 105(E105) days were collected,and the morphological characteristics of rump fat was observed.Oil Red O staining and adipogenic differentiation gene expression analysis were performed on rump adipose from embryos in Kazakh sheep during E70,E80,and E94 periods to determine the crucial phases of rump adipose deposition.Preadipocytes of rump adipose in Kazakh sheep during E70,E80,and E94 periods were isolated using a type Ⅰ collagenase stepwise digestion method.All three stages cells were subjected to 6 days continuous culture for growth curve plotting.Meanwhile,the proliferative capacity of preadipocytes from three embryonic stages was compared.Adipogenic induction and differentiation were performed on rump adipose preadipocytes in Kazakh sheep during E70,E80,and E94 periods.The differentiation potential was evaluated through Oil Red O staining,lipid droplet content quantification,and Real-time quantitative PCR analysis of adipogenic differentiation gene expression,and a comparative analysis of the adipogenic differentiation characteristics of preadipocytes across different embryonic stages was conducted.【Result】 There was obvious rump adipose in Kazakh sheep in E70 period,with visible lipid droplets initially forming rump at E80 period,followed by significant accumulation of lipid droplets in E94 period.Additionally,the expression of genes related to adipogenic differentiation gradually increased with gestation time.Notably,there was a fold increase from E80 to E94 periods.Furthermore，the precursor adipocytes at three developmental periods (E70,E80,and E94) from rump adipose isolated by type Ⅰ collagenase stepwise digestion method exhibited fibroblast-like with robust growth activity.Preadipocytes from all three periods were treated with induced differentiation to produce mature lipid droplets.The expression of adipogenic differentiation genes (CEBPα,PPARγ,LPL,and FABP4) in post-induction were significantly higher than that in pre-induction (P＜0.05).Compared with E70 or E94 periods,preadipocytes in E80 period had a higher capacity for lipid differentiation,earlier lipid droplet formation (3 day),and higher droplet content.【Conclusion】 The proliferation of rump adipose preadipocytes from isolated Kazakh sheep embryos during E70,E80 and E94 periods was well active.The cells could be induced to differentiate to form mature lipid droplets,and the differentiation ability of rump adipose preadipocyte was stronger in E80 period than that in the other two periods.

The ovary serves as a core component of the female reproductive system in livestock,with its structure,function,and role in the reproductive process being of paramount importance.This paper systematically reviews the basic structural and functional characteristics of ovaries in various livestock,including horses,donkeys,cattle/cows,sheep/goats,and pigs,specifically covering aspects such as the location distribution,morphological features,cortex-medulla structure,and unique ovulation fossa characteristics of ovaries.This paper delves into the mechanisms by which the ovarian characteristics influence the estrous cycle,ovulation characteristics,and reproductive performance of livestock.Furthermore,this paper comprehensively introduces the latest application progress of novel technologies in the research field of livestock ovaries,such as CRISPR/Cas9 gene editing,parthenogenesis activation,high-throughput sequencing,and bioinformatics.Additionally,this paper outlines the future research directions in the field of livestock ovaries,with a focus on optimizing reproductive performance through gene editing technology and enhancing reproductive efficiency with the aid of big data and artificial intelligence technologies.This paper aims to provide a solid scientific basis for a deeper understanding of the biological mechanisms of livestock reproduction,the optimization of reproduction strategies,and the improvement of reproductive efficiency,while also offering strong support for the continuous improvement and innovation of livestock reproduction technologies.

【Objective】 The objective of this study was to compare the differences in serum lipid metabolites between healthy and subclinical ketosis diary cows at 14 d after parturition,and to investigate the characteristics of changes in serum lipid metabolism in subclinical ketosis diary cows during the early lactation period and the relationship with subclinical ketosis.【Method】 Blood samples were collected from 70 periparturient cows,blood biochemical indices were measured.According to the concentration of β-hydroxybutyrate (BHBA) in serum,10 healthy dairy cows (H group) and 10 subclinical ketosis diary cows (SCK group) were selected respectively.Liquid chromatography-mass spectrometry (LC-MS) technology was used to perform metabolomic analysis of serum from diary cows in H and SCK groups at 14 d postpartum.Differential metabolites of serum lipids were screened and KEGG pathway enrichment analysis was performed on them，and evaluate its correlation with the blood biochemical indicators related to ketosis.【Result】 Compared with healthy dairy cows,the differential metabolites that showed significant changes in the serum of subclinical ketosis dairy cows were mainly fatty acylates,glycerophospholipids and glyceride.These metabolites were significantly correlated with the blood biochemical indicators related to ketosis,such as non-esterified fatty acids,β-hydroxybutyric acid,direct bilirubin,total bilirubin,glucose,total cholesterol concentrations,and aspartate aminotransferase activity (P＜0.05).The differential metabolites of 14 serum lipids,including lysophosphatidylcholine (18∶0/0∶0),leukotrienes,5,6-dihydroxy-8Z,11Z,14Z-eicosapentaenoic acid,succinic semialdehyde,stearic acid,oleic acid,etc.,were mainly enriched in pathways such as ether lipid metabolism,arachidonic acid metabolism,alanine-aspartic acid and glutamic acid metabolism.【Conclusion】 The composition of serum lipid metabolites in subclinical ketosis dairy cows in the early lactation period had undergone significant changes.Multiple metabolic pathways in cows,such as ether lipid metabolism,arachidonic acid metabolism,unsaturated fatty acid biosynthesis,butyric acid metabolism,fatty acid biosynthesis,and glycerophospholipid metabolism,were affected.The disorder of serum lipid metabolites in lactating cows was closely related to the occurrence of subclinical ketosis.

【Objective】 This study aimed to investigate the effects of different concentrations of sodium selenite and selenomethionine on H₂O₂-induced oxidative damage and mitochondrial function in horse skeletal muscle satellite cells,and provide a reference for selenium as an antioxidant to alleviate oxidative stress during exercise in horses.【Method】 In this study,the oxidative stress model of horse skeletal muscle satellite cells was induced by 600 μmol/L H₂O₂.After 6 h of starvation medium culture,different concentrations of sodium selenite (10,20,50,100,150 and 200 nmol/L) and selenomethionine (10,20,50,100,150 and 200 nmol/L) were used to culture for 24 h.Then,horse skeletal muscle satellite cells were induced with 600 μmol/L H₂O₂ for 6 h.Finally,samples were collected.CCK-8 kit was used to detect the cell viability,antioxidant enzyme kit was used to detect the indices of antioxidant enzyme activity,Real-time quantitative PCR was used to detect the expression of antioxidant genes,and mitochondrial stress kit was used to detect the mitochondrial energy metabolism of skeletal muscle satellite cells to evaluate the mitigation effect of different concentrations of sodium selenite and selenomethionine on oxidative damage.【Result】 The relative cell viability of 100 nmol/L sodium selenite group and 150 nmol/L selenomethionine group was significantly higher than that of injury group (P＜0.05),there was no significant difference compared with control group(P＞0.05),the total antioxidant capacity (T-AOC) and antioxidant enzyme activity were significantly higher than those of injury and control groups (P＜0.05).The content of malondialdehyde (MDA) in cells decreased first and then increased with the increase of sodium selenite concentration,it was the lowest in 100 nmol/L sodium selenite group,which was significantly lower than those in control and injury groups (P＜0.05).The content of MDA in 150 nmol/L selenomethionine group was significantly lower than that in control and injury groups (P＜0.05).The expressions of RELA,glutathione peroxidase 1 (GPX1),superoxide dismutase 1 (SOD1) genes in 100 nmol/L sodium selenite and 150 nmol/L selenomethione groups were extremely significantly higher than those in control and injury groups (P＜0.01),the expression of thioredoxin reductase 1 (TRXR1) gene was extremely significantly higher than that in injury group (P＜0.01),and the expression of TRXR1 gene in 150 nmol/L selenomethione group was extremely significantly higher than that in control group (P＜0.01).The expression of nuclear factor E2-related factor 2 (Nrf2) gene in 150 nmol/L selenomethionine group was significantly higher than that in injury group (P＜0.05).The expression of RELA,GPX1,TRXR1 and SOD1 genes was extremely significantly higher than that in control group (P＜0.01).The basal respiration value,ATP oxygen consumption value and maximum respiration value were 32.28,21.70 and 45.01 pmol/min,respectively,which were significantly higher than those in injury group (P＜0.05),increased by 25.03%,30.22% and 25.59%,respectively.However,there was no significant difference in oxygen consumption rate and mitochondrial function parameters between 100 nmol/L sodium selenite group and injury group (P＞0.05).【Conclusion】 Sodium selenite and selenomethionine could up-regulate the expression of antioxidant genes by activating the downstream signaling pathway of Nrf2,thereby increasing the activities of superoxide dismutase (SOD),catalase (CAT) and glutathione peroxidase (GPX),reducing MDA production and alleviating oxidative damage.Under this test condition,100 nmol/L sodium selenite and 150 nmol/L selenomethionine had the best remission effect.In addition,compared with the inorganic sodium selenite,organic selenomethionine could also improve mitochondrial energy metabolism.

【Objective】 The purpose of this experiment was to explore the effects of different concentrations of alfalfa polysaccharides (APS) to the diet on serum hormone indexes,antioxidant capacity,immune function,reproductive performance and fecal flora of pregnant sows.【Method】 A total of 90 pregnant sows (PIC,parity 3-4) with similar genetics,body condition,and backfat thickness,were randomly assigned to six groups using a single-factor completely randomized design.They were respectively the control group (CON) and the 200,400,600,800 and 1 000 mg/kg APS groups (experimental groups A,B,C,D and E),with 15 replicates in each group and one pig in each replicate.The trial included a 10-day pre-trial (gestation days 45-55) and an 80-day main trial (gestation day 55 to weaning).On the morning of the 55th day of the gestation before feeding,6 pigs in each group were randomly selected for blood collection through the anterior vena cava to determine serum biochemical,antioxidant capacity and immune function indicators.During the experiment,production indicators such as the total litter size of sows,total number born,and the weaning litter weight were recorded.Milk samples were collected on postpartum days 1 and 8 for the routine components analysis.The fecal conditions of sows were observed and the scores were recorded on gestation days 60,80 and 110.From gestation days 111 to 113,6 pigs were randomly selected from each group to collect fresh fecal samples and determine the diversity of fecal microbiota.【Result】 Compared with CON group,①The total antioxidant capacity in the serum of pregnant sows in groups C and D increased significantly (P＜0.5).The serum IgM content of sows in group B increased significantly (P＜0.05).②The total litter size and total number born of pregnant sows in group B,and the average weight and average daily weight gain of weaned piglets in groups C and D were significantly increased (P＜0.5).③The lactose content in the colostrum of sows in groups A,B,C,D and E increased significantly (P＜0.05).The contents of milk protein in normal milk of groups B and C,and the contents of non-fat milk solids and dry matter in normal milk of group C were significantly increased (P＜0.05).④The fecal scores of sows in each experimental group at 110 days of gestation were significantly increased (P＜0.5).⑤At the phylum level,the relative abundance of Firmicutes in fecal of sows in group A increased significantly (P＜0.05).At the genus level,the relative abundances of Escherichia-Shigella in fecal of sows in groups A and B,and Clostridium in groups A,B,C and D were significantly decreased (P＜0.05).The relative abundance of Christensenellaceae_R-7_group and Prevotella in fecal of sows in groups A and B increased significantly (P＜0.05).【Conclusion】 Under this experimental condition,dietary supplementation with 600 mg/kg APS significantly improved antioxidant capacity,immune function,and reproductive performance in gestating sows,while concurrently enhancing milk quality,alleviating constipation,and optimizing fecal microbiota diversity.

The rapid development of large-scale layer farming has positioned China as the world’s largest producer and consumer of eggs,with the layer industry playing a pivotal role in its livestock sector.Phosphorus,an indispensable core nutrient in the diets of laying hens,is critical for maintaining physiological functions,promoting bone development,and ensuring eggshell quality.However,phosphorus in poultry diets primarily exists in the form of phytate and its salts.Due to the endogenous phytase deficiency in poultry,the biological availability of phosphorus is severely limited by the anti-nutritional effects of phytate.Concurrently,challenges such as phosphorus resource scarcity,rising feed costs,suboptimal production efficiency,and inadequate manure management have intensified.Therefore,eliminating the anti-nutritional characteristics of phytic acid,improving the digestibility and utilization rate of feed,and reducing the addition of inorganic phosphorus are critical for the sustainable development of the layer chicken industry.Exogenous addition of phytase to diets can hydrolyze phytic acid and phytates,reducing phosphorus excretion from livestock and poultry and decreasing reliance on mineral phosphorus sources.Although the application of phytase in conventional diets is relatively widespread,systematic research on its use in low-phosphorus diets for layers and the development of corresponding application protocols remain insufficient.The author summarizes the nutritional role of phosphorus in layers and examines the effects of phytase in low-phosphorus diets on performance,egg quality,gut health,and skeletal health,it further explores the extra-phosphoric effects of phytase and the factors influencing its application in low-phosphorus diets,aiming to provide theoretical foundations and practical guidance for the effective use of phytase in layer feed.

【Objective】 The aim of this study was to explore the effects of adding chitosan to drinking water on the serum biochemical indicators,immune function and intestinal microbiota of squeakers in White King pigeons,and evaluate the application effect of chitosan in pigeons breeding，so as to provide theoretical basis for its development and application as an additive for pigeon feed.【Method】 Sixty 2-month-old healthy squeakers were selected and randomly divided into two groups:Control group and chitosan group.Each group was further divided into 5 replicates,with 6 pigeons in each replicate.Squeakers in control group were fed with the basal diet and normal drinking water without chitosan addition.Squeakers in chitosan group were fed with the basal diet and chitosan was added to the drinking water at a concentration of 0.2%.The experiment lasted for 42 days.Before and after the experiment,the squeakers were fasted and weighed.After the experiment,five squeakers were selected from each group to measure the changes in serum biochemical indicators,immune function,slaughter performance,meat quality,and intestinal microflora.【Result】 Compared with control group,the contents of immunoglobulin A (IgA) and IgG in serum,and gizzard index of squeakers in chitosan group were extremely significantly or significantly increased (P＜0.01 or P＜0.05).However,no significant differences were observed in total bilirubin (T-BIL),bile acids (TBA),lactate dehydrogenase (LDH),complement 3 (C3),C4 levels,thymus index,and spleen index (P＞0.05).The initial body weight,final body weight,slaughter rate,half-eviscerated yield,full-eviscerated rate,and breast muscle yield remained unaffected (P＞0.05).The pH,redness (a^*),yellowness (b^*),lightness (L^*),and drip loss rate of breast muscle of squeakers in chitosan group showed no significant changes (P＞0.05).16S rRNA gene sequencing results revealed that chitosan could increase the intestinal microbial diversity in squeakers，and significantly reduced the relative abundance of harmful bacteria such as Campylobacter (P＜0.05),and affected the intestinal adaptive response by regulating the composition of the microbiota.【Conclusion】 Under the conditions of this experiment,the addition of 0.2% chitosan to drinking water enhanced the immune function of squeaker and optimized the intestinal microecological environment,thereby benefiting their gut health.

【Objective】 To investigate the optimal inclusion ratio of a compound Chinese herbal preparation primarily composed of Astragalus membranaceus,Saposhnikovia divaricata,Atractylodes macrocephala,and other ingredients in the early rearing of lambs,providing a reference for the efficient application of Chinese herbal medicine in livestock production.【Method】 120 healthy 8-day-old twin Hu lambs with similar body weights (4.64 kg±0.34 kg) were selected by the trial and randomly divided into four groups:Control group (CK,basal diet),0.2% group (basal diet+0.2% compound Chinese herbal medicine),0.4% group (basal diet+0.4% compound Chinese herbal medicine),and 0.6% group (basal diet+0.6% compound Chinese herbal medicine).The adaptation period lasted for 7 days,followed by a 45-day formal trial period.At the end of the trial,growth performance was measured,and serum samples were collected to determine biochemical parameters,immune indices,antioxidant indicators,and growth hormone levels.【Result】 ①Lambs in 0.2% group exhibited significantly higher body weights at 45 and 60 days of age compared to other groups (P＜0.05),along with significantly greater starter feed intake and average daily gain over the entire trial period (P＜0.05).②Serum urea nitrogen (UREA) levels in 0.6% group were significantly lower than those in CK group (P＜0.05),while no significant differences were observed in total protein (TP),albumin (ALB),total cholesterol (TC),triglycerides (TG),glucose (GLU) levels,or aspartate aminotransferase (AST) activity across groups (P＞0.05).③Lambs in three Chinese herbal medicine supplementation groups exhibited significantly higher serum total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity compared to CK group (P＜0.05). 0.6% group showed significantly elevated glutathione peroxidase (GSH-Px) activity relative to both CK and 0.2% groups (P＜0.05).Additionally,serum malondialdehyde (MDA) levels were significantly lower in three experimental groups than that in CK group (P＜0.05).④No significant differences in serum immunoglobulin A (IgA),IgG or IgM levels were observed among groups (P＞0.05).Herbal-supplemented groups exhibited significantly reduced serum interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) levels compared to CK group (P＜0.05).⑤Serum growth hormone (GH) and insulin-like growth factor-1 (IGF-1) concentrations in 0.2%,0.4%,and 0.6% groups were significantly higher than those in CK group (P＜0.05).【Conclusion】 The addition of a compound Chinese herbal preparation primarily composed of Astragalus membranaceus,Saposhnikovia divaricata,Atractylodis macrocephalae,and other herbs to the diet of lactating Hu lambs could improve growth performance,enhance serum immunity and antioxidant capacity,and stimulate growth hormone secretion.Based on a comprehensive evaluation,it was recommended to add 0.2% of the compound herbal formulation to the diet of lactating lambs for optimal results.

As toxic fungal secondary metabolites,mycotoxins are ubiquitously present in agroecosystems,with their contamination in crops and feed materials has emerged as a global threat to food safety.These compounds exhibit well-characterized hepatotoxic,carcinogenic,teratogenic,immunotoxic,and neurotoxic properties,posing substantial risks to human and animal health.Mycotoxins severely impair the physiological homeostasis of reproductive system in sows through multi-pathway disruptions,leading to infertility,abortions and other reproductive disorders.Therefore,elucidating the mechanisms underlying their reproductive toxicity is critical for mitigating breeding-associated risks and developing targeted prevention strategies.This review systematically summarizes the classification of major mycotoxins and their contamination profiles in Chinese raw materials and feed.Mycotoxins enter sows through contaminated feed,inducing multi-level impairments including ovarian dysfunction,uterine developmental anomalies,and dysregulation of reproductive endocrine homeostasis.The review provides a molecular-level analysis of mycotoxin-induced damage mechanisms associated with follicular developmental disorders,oocyte maturation defects,and regulation of granulosa cell apoptosis through specific signaling pathways.Regarding mitigation technologies,the article integrates diversified detoxification strategies including physical adsorption,chemical degradation,and microbial biotransformation,and proposes a systematic framework.By elucidating the reproductive toxicological effects of mycotoxins and their intervention nodes from multi-faceted analysis,so as to provide a critical theoretical foundation for establishing a comprehensive prevention system against sow reproductive disorders.

【Objective】 This study aimed to investigate the effects of curcumin and its primary metabolite,tetrahydrocurcumin,on growth performance,backfat thickness,and transcriptomic gene expression in finishing pigs,and to compare their regulatory mechanisms in backfat deposition.【Method】 Thirty Duroc×Landrace×Yorkshire fattening pigs were selected and divided into the control group (basal diet),the curcumin group (with 300 mg/kg curcumin added to the basal diet),and the tetrahydrocurcumin group (with 300 mg/kg tetrahydrocurcumin added to the basal diet) based on the principle of consistent body weight.There were 10 pigs in each treatment group,and the experimental period was 28 d.After the experiment,the initial body weight,final body weight and feed intake of pigs in each group were counted.Blood was collected through the anterior vena cava to detect blood lipid levels,the backfat thickness of carcasses was measured,and backfat samples were collected for morphological analysis and counting the size of dorsal adipocyte area.Western blotting was used to analyze the expression of fat metabolis-related proteins.High-abundance genes and DEGs were analyzed by transcriptome sequencing,and the molecular mechanism of back fat deposition was explored by combining GO functional enrichment and protein-protein interaction analysis.【Result】 Compared with the control group,the addition of tetrahydrocurcumin in the diet significantly reduced the average daily feed intake,backfat thickness,and the size of back adipocytes of fattening pigs (P＜0.05).Western blotting analysis showed that the expression of FASN in the back fat of fattening pigs in both the curcumin group and the tetrahydrocurcumin group decreased significantly (P＜0.05).The transcriptomic results showed that the DEGs of dorsal fat in the curcumin group were mainly enriched in regulating metabolic pathways such as hydrogen peroxide reaction and cell migration. The DEGs of dorsal fat genes in the tetrahydrocurcumin group were mainly enriched in regulating pathways such as protein catabolism and cholesterol excretion.The genes significantly upregulated in the tetrahydrocurcumin group were mainly enriched in fat metabolism-related pathways such as mitochondrial electron transport-cytochrome C to oxygen,and proton-driven ATP synthesis.Further GO functional enrichment and protein-protein interaction network analysis were conducted on the DEGs，the genes that were significantly elevated in the dorsal fat of the tetrahydrocurcumin group were mainly enriched in fat metabolic-related pathways such as oxidative phosphorylation and thermogenesis.The top-ranked genes in the protein-protein interaction network nodes were the mitochondrial electron transport chain-related genes：COX5B,COX7A1,COX6C,NDUFA7,and NDUFA1.【Conclusion】 At the same dose,tetrahydrocurcumin was more effective than curcumin in reducing fat deposition in fattening pigs.Adding 300 mg/kg of tetrahydrocurcumin to the diet of fattening pigs significantly reduced backfat thickness by enhancing the oxidative phosphorylation ability of dorsal fat mitochondria in fattening pigs,reducing the size of adipocytes.

Guanidinoacetic acid (GAA) is a key precursor for endogenous creatine synthesis and has attracted much attention in recent years due to its unique role in regulating animal energy metabolism.With the increasing demand for efficient and safe nutritional regulators in animal husbandry,the potential application of GAA is gradually becoming prominent.However,its large-scale application still faces scientific problems such as unclear dose-response relationships,unresolved metabolic molecular mechanisms,and insufficient long-term safety assessments.By regulating the phosphocreatine/creatine kinase system,GAA significantly improves the efficiency of skeletal muscle adenosine triphosphate (ATP) regeneration,enhances animal growth performance,and improves meat quality.GAA can enhance the body’s antioxidant capacity and insulin secretion function,optimize rumen fermentation efficiency.Compared with exogenous creatine,GAA has higher bioavailability (small intestine absorption rate ≥90%),rumen stability (degradation rate ≤ 10%,which can be further reduced to ≤5% if coated),and cost-effectiveness.The author provides theoretical basis for the scientific application of GAA in animal husbandry production from three dimensions:Metabolic regulation,industrial application,and mechanism of action,it also proposes innovative paths for the development of new nutritional regulators,which has important practical value in promoting the improvement and efficiency of animal husbandry.

【Objective】 This study aimed to investigate the effects of different dietary ammonium chloride levels on the hepatic histomorphology and metabolism of Tibetan sheep,and expected to provide a scientific basis for determining the optimal dietary ammonium chloride level for Tibetan sheep.【Method】 A total of 80 healthy,2-month-old weaned female Tibetan lambs with similar body weight (16.22 kg±1.01 kg) were selected and randomly assigned to four groups (20 lambs per group),with dietary ammonium chloride levels set at 0 (CON group),1.49% (NH1 group),2.24% (NH2 group),and 3.01% (NH3 group),respectively.The experiment lasted for 105 days,including a 15-day preliminary period and a 90-day formal period.After the trial,five lambs were randomly selected from each group for slaughter.Hepatic lobules were collected for histomorphological observation.The remaining hepatic samples were stored for subsequent metabolomics analysis.【Result】 ①The histomorphological results indicated that the hepatic cells in the CON and NH1 groups exhibited normal structure,radiating from the central vein,well-arranged,with large,round nuclei centrally located,and regular,closely-packed hepatic sinusoids.In contrast,in the NH2 and NH3 groups,inflammatory cells infiltrated the portal areas,and the hepatic sinusoids and hepatic cords were irregularly arranged and indistinct.②There were 308 differential metabolites between the CON group and the NH1,NH2 and NH3 groups,which were significantly enriched in purine metabolism,β-alanine metabolism,and the interconversion of pentoses and glucuronates (P＜0.05).When the dietary ammonium chloride level was 1.49%,the differential metabolites were significantly enriched in purine metabolism and other metabolic pathways (P＜0.05).When the dietary ammonium chloride level was 2.24%,the differential metabolites were significantly enriched in the biosynthesis of secondary bile acids and other metabolic pathways (P＜0.05).When the dietary ammonium chloride level was 3.01%,the differential metabolites were significantly enriched in the pathways of microbial metabolism in diverse environments (P＜0.05).【Conclusion】 Under the conditions of this experiment，dietary ammonium chloride primarily affected nucleotide metabolism,glucose metabolism,and lipid metabolism in the hepatic of Tibetan sheep.Notably,when the dietary ammonium chloride level exceeds 2.24%,it could trigger an inflammatory response in the hepatic of Tibetan sheep,which might adversely impact the normal physiological functions of the hepatic.

【Objective】 This experiment aimed to investigate the optimal supplementation dosage of N-carbamylglututamate(NCG) in laying hens during the late laying period,thereby providing theoretical foundations and practical guidance for the scientific application of NCG in the production of local specialty laying hens.【Method】 A total of 360 Huainan patridge chickens at 70 weeks of age were randomly divided into four groups,each group has 6 replicates,with 15 individuals per replicate.The chickens in experimental groups were fed diets supplemented with 0.5,1.0,and 1.5 g/kg NCG in the basal diet,respectively.The test period was 42 days.Egg production,egg weight,and daily feed intake were recorded throughout the experiment.During the experiment,egg production,egg weight,and daily feed intake were recorded.On days 21 and 42 of the experiment,all eggs were collected to measure egg quality.At the end of the experiment,3 hens were selected per replicate to collect serum and liver samples for biochemical and antioxidant analysis.【Result】 Compared with the control group,when NCG was added at 1.0 g/kg,the egg production rate of Huainan patridge chickens was significantly increased (P＜0.05),the yolk’s triglyceride (TG) and total cholesterol (T-CHO) contents,as well as superoxide dismutase (SOD) activity,were all significantly increased (P＜0.05),while malondialdehyde (MDA) content was significantly decreased (P＜0.05).The activity of nitric oxide synthase (NOS) in serum was significantly increased (P＜0.05),and the activity of SOD and the content of nitric oxide (NO) in liver were significantly increased also (P＜0.05).When NCG was added at 1.5 g/kg,the egg production rate,average egg weight,yolk color and eggshell thickness of laying hens were significantly increased (P＜0.05),the activity of SOD in egg yolk was significantly increased,while the content of MDA was significantly decreased (P＜0.05).The content of NO and the activity of NOS in serum were significantly increased (P＜0.05).【Conclusion】 Supplementing the basal diet with NCG significantly improved the production performance,egg quality and antioxidant capacity of Huainan patridge chickens in the late laying period,with 1.0 g/kg identified as the optimal dosage.

With the rapid development of metagenomics,binning technology has gradually become an indispensable tool for quantifying microbial communities.This technology clusters sequences from the same microorganism based on shared features using clustering algorithms,thereby separating the genomes of different microorganisms in high-throughput sequencing data,enabing single-strain genome reconstruction and species-specific association analysis.Due to its ability to provide high-resolution species annotation at the strain level,binning technology is gradually replacing traditional assemble-free metagenomic analysis methods.Currently,binning technology has been increasingly applied in veterinary and animal science,particularly in studies of the composition and dynamics of gut microbiota.As pigs are one of the most economically important livestock animals,and their gut microbiota significantly influences nutrition,health,disease resistance,and immune capacity,the authors introduce recent advances in the application of metagenomic binning technology in pig gut microbiome research,including its contributions to constructing reference catalogs for the pig gut microbiome and capturing microbial community shifts driven by breed,growth stage,diet,and environmental factors.These advancements not only enhance our understanding of the composition and function of the pig gut microbiota but also provide valuable technical references and theoretical support for future microbiome research in the livestock industry.

【Objective】 The aim of this study was to investigate the effects of supplementary feeding of trace elements (Cu,Se,Zn and I) on the growth performance,serum biochemical, immune function and rumen fermentation of grazing ewes.【Method】 Forty lactating Bashibai ewes with similar body weight (42.79 kg±5.72 kg) and similar parity were selected and randomly divided into 5 groups,with 8 replicates in each group and 1 ewe in each replicate.The experiment was set up with pure grazing group (CON),blank control group (CK),and low (L),medium (M),and high (H) level groups of trace elements.The pre-test period was 10 days,and the experimental period was 35 days.At the beginning and end of the experimental period,the ewes were weighed fasting in the morning.Blood was collected by vein puncture,and serum biochemical,antioxidant and immune indicators were measured.Rumen fluid was collected to determine pH,ammonia nitrogen and volatile fatty acid contents.【Result】 Supplementary feeding of trace elements increased the average daily gain of ewes,but there was no significant difference among the groups (P＞0.05).Compared with CK group, the activity of alanine aminotransferase (ALT) in the serum of ewes in group L was significantly decreased (P＜0.05), while the total antioxidant capacity (T-AOC) and immunoglobulin G (IgG) content in the serum were significantly increased (P＜0.05), and the content of tumor necrosis factor-α (TNF-α)was significantly decreased (P＜0.05). The contents of acetate and total volatile fatty acids in the rumen of ewes in group L were significantly higher than that in group H (P＜0.05)，the contents of propionate and isobutyrate were significantly higher than those in groups M and H (P＜0.05).【Conclusion】 Trace element supplementation could improve the antioxidant capacity,immune performance and rumen fermentation in lactating grazing ewes.Under this experimental conditions,the supplementary feeding effect was the best when the addition amounts of Cu,Se,Zn and I were 5.71,12.46,0.14 and 1.47 mg/kg, respectively.

Intramuscular fat (IMF) is a key indicator affecting muscle quality,but due to the timing of fat deposition.IMF deposition requires a long feeding cycle and high production cost.It is a hot and difficult point in the industry to study the use of nutrition regulation means to control more fat deposition as IMF.This review briefly describes the formation and composition of IMF,the key factors regulating IMF deposition (including peroxisome proliferator-activated receptors,fatty acid-binding proteins and sterol regulatory element-binding proteins,etc.),and analyzes the effects of dietary energy level and nutritional factors such as starch type,vitamin A,amino acids,natural plants and their extracts on IMF deposition in livestock.The authors suggest using multi-omics technology to analyze the differences of lipid metabolism in different parts of adipose tissue,combined with nutritional regulation and omics technology to further clarify the molecular mechanism of various nutritional factors,and study the effect of the synergy of multiple nutritional factors,so as to provide innovative nutritional regulation and control scheme for IMF deposition in livestock,and help the production of high-quality livestock products.

【Objective】 The aim of this study was to conduct a thorough analysis of the genetic diversity and population genetic structure of Taihang goat ram population through whole genome resequencing (WGS) technology,so as to provide scientific foundations for the breeding and conservation of Taihang goats.【Method】 A total of 35 healthy adult Taihang goat rams without diseases were selected,venous blood samples were collected to extract DNA for constructing a sequencing library,and the single nucleotide polymorphism (SNP) data in Taihang goat rams was subjected to quality control using Plink 1.9 software.The genetic parameters of Taihang goat ram population were calculated,including expected heterozygosity (He),observed heterozygosity (Ho),minor allele frequency (MAF),and polymorphic information content (PIC).Additionally,analysis were performed on linkage disequilibrium,nucleotide diversity,runs of homozygosity (ROH),principal component analysis (PCA),phylogenetic tree construction,and population genetic structure.【Result】 The MAF of Taihang goat ram population was 0.1889,and the average PIC was 0.2760,indicating moderate polymorphism.The He of Taihang goat ram population was 0.2764,and the Ho was 0.1929.A total of 7 499 ROH were identified in 35 rams,with the largest number of ROH ranging from 0 to 2 Mb.The average inbreeding coefficient was 0.0334.PCA and phylogenetic tree analysis revealed that 35 rams were divided into four main clades,while population genetic structure analysis showed distinct stratification when K=2.【Conclusion】 Taihang goat ram population showed relatively high genetic diversity with moderate polymorphism,demonstrating significant potential for breeding improvement.There was low-to-moderate inbreeding in the population,and inbreeding should be avoided in breeding. The population had a complex genetic structure,showing obvious stratification (most significant at K=2),and could be divided into multiple familial lineages.The results provided a theoretical basis for conservation strategies and breeding directions of Taihang goats.

【Objective】 Dolang sheep,Luobu sheep,Altay sheep,Bashibai sheep,and Turpan Black sheep are representative local sheep breeds in Xinjiang.This study focused on five major local sheep breeds in Xinjiang,aiming to characterize their genetic features and develop a rapid identification strategy,thereby providing a scientific basis for precise breed identification and the conservation of genetic resources.【Method】 This study focused on the analysis of population structure and breed identification of local sheep breeds in Xinjiang.A total of 206 samples from 27 representative sheep populations were selected,encompassing five major local breeds (Luobu sheep,Altay sheep,Bashibai sheep,Dolang sheep,and Turpan Black sheep),as well as several cultivated and introduced breeds.The sample design considered geographic distribution,breeding background,and economic type,providing a comprehensive reflection of the current genetic structure of sheep resources.Whole-genome single nucleotide polymorphism (SNP) variation detection and population genetic analysis were performed to assess the genetic independence of Xinjiang local sheep breeds.Using a case-control approach,the fixation index (Fst) and allele frequency difference (ΔAF) were calculated to preliminarily screen for breed-specific variants.A random forest (RF) algorithm was subsequently employed to identify the optimal set of breed-specific SNP markers for the five local sheep breeds.Principal component analysis (PCA) and Neighbor-Joining (NJ) phylogenetic tree construction were used to validate the accuracy of the identification results.【Result】 This study conducted a targeted selection of specific markers for five indigenous sheep breeds in Xinjiang.By integrating Fst,ΔAF,and RF algorithms for comprehensive screening and optimization,and validating the selected markers through PCA and NJ phylogenetic tree construction,a set of highly discriminative SNP was successfully identified.Specifically,30 SNPs were selected for Dolang sheep,47 SNPs for Bashibai sheep,46 SNPs for Luobu sheep,50 SNPs for Altay sheep,and 46 SNPs for Turpan Black sheep.All selected SNPs met stringent selection criteria,achieving an identification accuracy of approximately 98%,and demonstrated strong capability for efficient and precise breed differentiation.【Conclusion】 Leveraging whole-genome data,this study developed a SNP-based breed-specific genetic labeling system for five indigenous sheep breeds in Xinjiang.This system enabled rapid and accurate molecular-level identification,providing a robust theoretical basis for the future development of breed-specific molecular diagnostic kits.The approach offered an effective tool for the precise identification of local sheep breeds in Xinjiang,thereby supporting genetic resource management and advancing precision breeding initiatives.

Gene editing,as an emerging genetic engineering technology,can precisely modify the specific gene sequences of an organism’s genome.In recent years,gene editing technology has evolved from the first-generation zinc finger nuclease (ZFN),the second-generation transcriptional activator-like effector nuclease (TALEN),to the current third-generation CRISPR-Cas9 technology,and has been successfully applied in the fields of crops and livestock.With the rapid development of science and technology and the poultry industry,the application of gene editing technology in the field of poultry disease-resistant breeding has become the focus of attention for breeders.This article systematically introduces gene editing technology and its development and application in poultry disease-resistant breeding,briefly describes the development process of three generations of gene editing technology,and expands other types of CRISPR/Cas systems.The application of new gene editing technologies represented by CRISPR-Cas9 technology in disease-resistant breeding of poultry such as chickens and ducks was analyzed,aiming to provide references for the research and application of disease-resistant breeding of poultry.Although gene editing technology shows great potential,it still faces challenges such as off-target effects,the safety of delivery systems and public acceptance.In the future,the precision of the technology can be further enhanced by developing high-fidelity Cas variants,optimizing delivery vectors (such as nanomaterials),and establishing multi-gene collaborative editing strategies.Gene editing technology will achieve industrial breakthroughs in areas such as disease-resistant breeding and medicinal protein production,and promote the dual improvement of independent innovation in the poultry breeding industry and food safety guarantee.

【Objective】 This study was conducted to perform a genetic evaluation and estimate the genomic breeding values of growth traits and wool traits in the core population of Chinese Merino sheep,so as to provide a foundation for breeding new ultra-fine wool sheep strains.【Method】 The core population of Chinese Merino sheep at the Xinjiang Gongnaisi Stud Farm was used as the research subject.Pedigree data,phenotypic data (including birth weight,yearling weight,daily weight gain,gross from length,shearing amount,and weight after shearing),and systematic environmental effect data were collected from individuals born between 2013 and 2022.In addition,a reference population consisting of 999 individuals from the 2023 cohort was subjected to low-depth (1×) sequencing to obtain their genotypic data.The least squares method in SAS 9.2 software was used to analyze systematic environmental effects and determine fixed effects in the model.The likelihood ratio test was conducted to compare model 1 and model 2 to assess whether including maternal genetic effects significantly impacted the model.The optimal model under these study conditions was determined and then applied using ASREML software.Genetic parameters and genomic breeding values for various traits were estimated using the ABLUP,GBLUP,and ssGBLUP methods,with validation of accuracy.【Result】 The estimated heritabilities of birth weight,yearling weight,daily weight gain,gross from length,shearing amount,and weight after shearing in the core population of Chinese Merino sheep were found to be 0.1215-0.1647,0.1434-0.3631,0.1954-0.2545,0.1398-0.1781,0.1129-0.2076,and 0.2434-0.3017,respectively,all were low to medium heritability traits.Among the different estimation methods,the highest accuracy for genomic estimated breeding values (GEBVs) was obtained using model 2 with ssGBLUP method.Compared with ABLUP method,the overall accuracy of ssGBLUP method improved by 1.48%-27.02%.【Conclusion】 Under the conditions of this study,the most accurate estimation of genetic parameters and genomic breeding values for growth and wool traits in the core population of Chinese Merino sheep was achieved using a model that incorporated both individual additive and maternal genetic effects in combination with ssGBLUP method.

【Objective】 The aim of this study was to analyze the polymorphism of anti-Müllerian hormone (AMH) and cytochrome P450,family 19,subfamily A,polypeptide 1 (CYP19A1) genes in Guizhou Black goats,and screen out the single nucleotide polymorphism (SNP) that had significant effects on the litter size of goats.【Method】 All the exons of AMH and CYP19A1 genes of Guizhou Black goats were screened by direct sequencing of PCR products,the secondary and tertiary structures of AMH and CYP19A1 proteins and signal peptides were predicted by online software,and the correlation between SNP and litter size was analyzed.【Result】 One SNP (g.2612 A＞G) was identified in the fifth exon of AMH gene in Guizhou Black goats.One SNP (g.37836 T＞C) was found in the fifth exon of CYP19A1 gene,and two SNPs (g.48986 C＞T and g.49046 A＞G) were discovered in the tenth exon of CYP19A1 gene.The secondary and tertiary structure prediction results revealed that the stability of the secondary structure of AMH and CYP19A1 proteins decreased after mutation,with the highest proportion of random coil,indicating higher complexity.Moreover,AMH and CYP19A1 proteins lacked signal peptides. χ² test results showed that all four SNPs deviated from the Hardy-Weinberg equilibrium (P＞0.05),and the polymorphism was moderate (0.25＜PIC＜0.5).The correlation analysis results showed that the litter size of GG genotype at g.2612 A＞G of AMH gene in Guizhou Black goats was extremely significantly or significantly higher than that of AG and AA genotypes (P＜0.01 or P＜0.05).The litter size of GG genotype at g.49046 A＞G of CYP19A1 gene in Guizhou Black goats was significantly higher than that of AA and AG genotypes (P＜0.05),while there was no significant difference in litter size among different genotypes of the other two SNPs (P＞0.05).【Conclusion】 g.2612 A＞G and g.49046 A＞G of AMH and CYP19A1 genes had significant association with the litter size of Guizhou Black goats,and could be used as molecular markers for the litter size of goats.The results provided a theoretical basis for the study of the multiple litter traits of Guizhou Black goats.

【Objective】 This study aimed to investigate the gene expression differences and associated signaling pathways in white adipocytes and beige adipocytes of Tibetan pigs before and after thermogenic activation,providing a theoretical basis for understanding adipocyte thermogenesis mechanisms and improving cold resistance in piglets.【Method】 Stromal vascular fraction (SVF) cells were isolated from the adipose tissue of Tibetan pigs and induced to differentiate into white adipocytes and beige adipocytes.Thermogenesis was activated by treating with 5 μmol/L forskolin (FSK) for 24 hours.The experiment comprised five groups:Untreated SVF cells group (C), white adipocytes group (W),FSK-treated white adipocytes group (WF), beige adipocytes group (B),and FSK-treated beige adipocytes group (BF).RNA-Seq was performed using the Illumina HiSeq platform.Differentially expressed genes were identified using DESeq2.Functional and metabolic pathway analyses of differentially expressed genes were conducted using GO and KEGG,and the expression of key genes was validated by Real-time quantitative PCR.Additionally,data from cold-stimulated Tibetan pigs were integrated with the in vitro beige adipocyte thermogenesis model (BF vs B) to identify co-expressed thermogenesis-related genes and transcription factors.【Result】 The transcriptome sequencing results demonstrated that FSK treatment significantly altered the gene expression profiles of both white adipocytes and beige adipocytes.Principal component analysis (PCA) and heatmaps revealed a marked separation in gene expression patterns before and after FSK treatment.Among them,beige adipocytes exhibited a stronger response to FSK treatment compared to white adipocytes.After treatment with FSK on beige adipocytes,a total of 1 514 genes were up-regulated and 1 544 genes were down-regulated.After treatment with FSK,a total of 815 genes were up-regulated and 1 096 genes were down-regulated in white adipocytes.GO and KEGG analyses indicated that the upregulated genes in beige adipocytes were significantly enriched in lipid metabolism pathways (fatty acid oxidation),cAMP signaling pathways,and immune-related pathways,and the enrichment score was higher than that of white adipocytes.Compared with the results before treatment after FSK treatment,there were a total of 485 up-regulated and 525 down-regulated differentially expressed genes in the two types of adipocytes.The up-regulated genes were enriched in thermogenic pathways such as cAMP,AMPK and PPAR,while the down-regulated genes were enriched in adiposynthetic pathways (such as unsaturated fatty acid biosynthesis).The results of Real-time quantitative PCR showed that the expression levels of KLF11 and PDK4 genes in white adipocytes after FSK treatment were extremely significantly upregulated (P＜0.01).By integrating the transcriptome sequencing data of cold-stimulated adipose tissue in Tibetan pigs and beige adipocytes,81 up-regulated co-expressed differentially expressed gene both in vivo and in vitro . There were 7 transcription factors such as PPARGC1A and NCOA3，and these transcription factors were related to processes such as mitochondrial biosynthesis and lipid metabolism reprogramming,suggesting their potential role in thermogenesis regulation.【Conclusion】 In this experiment,the transcriptome data of Tibetan pigs adipocytes were processed by FSK,revealing the key signaling pathways and genes in the thermogenesis process of adipocytes.Genes such as KLF11 and PDK4 that potentially regulated thermogenesis were screened out from the in vitro transcriptome data.Combined with the in vivo transcriptome data,7 transcription factors that potentially regulated thermogenesis in adipose tissue were screened out.The results provided potential targets for improving the cold resistance and fat deposition characteristics of pigs.

【Objective】 The genetic parameters of wool fiber diameter and bending traits were estimated using genomic information in Chinese Merino sheep from the Xinjiang Gongnaisi sheep farm.【Method】 Whole genome resequencing was performed for 1 520 yearling sheep and 836 adult ewes from 2022 to 2023.Pedigree data were collected,and 8 traits,including mean fiber diameter (MFD),fiber diameter standard deviation (FDSD),fiber diameter coefficient of variation (FDCV),percentage of fiber content with diameter less than 30 μm (CF),standard deviation of average fiber diameter along profile (APSD),curvature number (CN),mean fiber curvature (MFC),and fiber curvature standard deviation (FCSD),were measured using OFDA and other instruments to yield a total of 18 496 data points.Kinship matrices were constructed based on pedigree and genome information respectively,and multi-trait animal models were established to estimate genetic parameters using ASREML software with the average information maximum likelihood method.【Result】 The heritability ranges for wool traits based on pedigree information were 0.066-0.859 and 0.088-0.782 in yearlings and adults respectively,and the heritabilities of all traits based on genomic information were high for yearlings (0.349-0.684),and medium to high for adult ewes (0.125-0.941).The four traits of CF,APSD,CD,and CDSD,investigated for the first time in Chinese Merino sheep,had the higher heritability values of 0.445,0.403,0.684 and 0.635 respectively in yearlings,and moderate to high heritability of 0.776,0.125,0.471,and 0.624 respectively in adult ewes.The MFD estimated based on genomic information in yearlings and adult ewes showed a high positive genetic correlation with FDSD (P＜0.01),and a high negative genetic correlation with CF (P＜0.01);MFC and FCSD showed a high positive genetic correlation (P＜0.01).【Conclusion】 Based on genomic information,it was estimated that the wool fiber diameter and bending traits of Chinese Merino sheep were highly heritable in yearlings ewes,and moderately to highly heritable in adult ewes.There was a high negative genetic correlation between CF and MFD in yearlings and adult ewes,while the genetic and phenotypic correlations between MFC and FCSD were the highest.The estimation of genetic parameters based on genomic information had improved accuracy,providing a reference for understanding the variation in fiber diameter and bending traits in the Chinese Merino sheep population and further improving breeding programs.

【Objective】 Candidate genes associated with average daily gain (ADG) in Duroc pigs were identified using a genome-wide association study (GWAS),and colocalization analysis was performed by integrating gene expression regulatory data to gain deeper insights into the genetic regulatory mechanisms underlying ADG.【Method】 A GWAS was conducted based on ADG phenotypic records from 1 346 Duroc pigs and genotypic data generated using the Zhongxin No.1 50K SNP chip to identify quantitative trait locus (QTL) and associated candidate genes for ADG.GO and KEGG enrichment analyses were employed to explore the biological functions of the candidate genes,and colocalization analysis was performed by integrating expression quantitative trait locus (eQTL) data from 34 tissues provided by the pig genotype-tissue expression (PigGTEx) project,to identify potential causal genes and functionally relevant tissues contributing to the genetic regulation of ADG.【Result】 A total of 1 530 significant single nucleotide polymorphisms (SNPs) (P＜1×10^-5) associated with ADG were identified through GWAS and were mapped to 50 QTL, distributed across chromosomes 1,3-8,10-13,and 15-18.The QTL regions were annotated through the Ensembl database,resulting in the identification of 513 candidate genes.GO and KEGG enrichment analyses revealed that ALPK2,TULP3,and TMOD4 genes were significantly enriched in pathways related to limb development and muscle cell development.Colocalization analysis further revealed that three potential causal variants influence ADG by regulating the expression of MVP,PRR14,and DDX11 genes in different tissues.【Conclusion】 In this study,key QTL regions and potential causal genes associated with ADG in Duroc pigs were identified through the integration of GWAS and colocalization analysis.These findings provided important reference information for the genetic improvement of ADG in Duroc pigs.

【Objective】 This study aimed to investigate the differences in disease resistance among different populations of Nile tilapia and establish a general disease resistance evaluation model,so as to lay a foundation for screening new resistant phenotypes of tilapia with overall tolerance to common pathogens and adverse environments.【Method】 Five mixed families of Nile tilapia (populations A-E) that produced seedlings at the same time during the breeding process of anti streptococcal disease were used as the research objects.A total of 150 fish from each group were tested and compared for the changes of serum immune indicators,including superoxide dismutase (SOD),acid phosphatase (ACP),alkaline phosphatase (AKP),complement 3 (C3),tumor necrosis factor-α (TNF-α),interferon-γ(IFN-γ),immunoglobulin M (IgM),as well as gill immunoglobulin T (IgT).At the same time,the general disease resistance of each population was modeled and evaluated by principal component analysis and cluster analysis based on the immune index data.【Result】 The results showed that the coefficients of variation for the immune indicators across different tilapia populations ranged from 10.18% to 78.17%. Significant or extremely significant positive correlations were observed in the following pairwise comparisons: ACP with AKP, C3, IFN-γ and IgT, AKP with C3, IFN-γ and IgT, C3 with TNF-α, IFN-γ and IgT, TNF-α with IFN-γ and IgT, IFN-γ with IgT, and IgM with SOD (P＜0.05 or P＜0.01). According to the ranking of variance contribution rate,the top five principal components were selected for analysis,and the results showed that the cumulative variance contribution rate reached 85.422%.Based on these findings,a comprehensive disease-resistance assessment model for Nile tilapia was constructed:f_i=0.006Zx₁+0.056Zx₂+0.054Zx₃+0.221Zx₄+0.282Zx₅+0.232Zx₆+0.259Zx₇+0.082Zx₈.According to the model,the general disease-resistance scores of the five populations were ranked as follows:E(0.694)＞D(0.537)＞C(－0.052)＞A(－0.475)＞B(－0.704).Cluster analysis indicated that populations D and E clustered together,while populations A,B and C formed another group. The challenge with Streptococcus agalactiae and spleen histopathological sections both revealed that the disease resistance of each population was basically consistent with the results of the model assessment.In other words, the higher the population score from model evaluation, the stronger the disease resistance.【Conclusion】 There were population differences in the general disease resistance of Nile tilapia,and it was feasible to construct a disease-resistance assessment model based on these differences.These results provided references for the development and utilization of tilapia germplasm resources and the genetic breeding of disease-resistant tilapia.

【Objective】 The purpose of this study was to understand the biological characteristics and pathogenicity of the isolates of Streptococcus suis serotype 9 in Guangdong,and provide a reference for the prevention and treatment of the disease caused by it.【Method】 In this experiment,bacterial isolation and culture were carried out with spleen samples from a pig with porcine reproductive and respiratory syndrome (PRRS) in a pig farm in Guangdong,and the isolated strain was identified by morphological observation,Gram staining microscopy,biochemical tests,16S rRNA gene sequencing and serotype identification.The biological characteristics and pathogenicity of the isolated strain were analysed by virulence gene detection,drug sensitivity test and mouse infection test.【Result】 The isolated strain showed rounded colonies of greyish-white,moist,smooth,and neat edges on blood plate,characteristic of alpha-haemolysis.Gram staining microscopic examination showed purple and was arranged in a circular chain pattern.Sequencing results of the 16S rRNA gene of the isolate showed more than 99% similarity with the published strains of Streptococcus suis on NCBI,and serotyping results showed that the isolate was Streptococcus suis serotype 9.The virulence gene phenotype of the isolated strain was luxS^-/ccpA⁺/srtA⁺/mrp^-/gdh⁺/gapdh⁺/fbps⁺/epf^-/orf2⁺/sly^-.The isolated strain was resistant to five antibiotics:Macrolide,lincomycin,doxycycline,tetracycline and sulfadimethoxazole.The results of pathogenicity test showed that the LD₅₀ of the isolate to mice was 1×10¹⁰ CFU,and the infected mice exhibited different degrees of lesions in organs,such as liver,kidney,spleen,lung,heart and brain.【Conclusion】 In this study,a strain of Streptococcus suis serotype 9,which carried multiple virulence genes was isolated,it was resistant to a variety of antibiotics,and could cause different degrees of tissue damage in infected mice.The results of this study could provide a reference basis for the prevalence analysis and prevention and control of Streptococcus suis infection.

【Objective】 The aim of this study was to prepare the S1 recombinant protein of Porcine epidemic diarrhea virus (PEDV) and its neutralizing monoclonal antibodies (mAbs),and provide critical materials for the development of PEDV diagnostic reagents and novel vaccines.【Method】 A recombinant plasmid that expressed S1 protein was constructed and transfected into HEK293F cells.Nickel affinity chromatography and gel filtration chromatography were used to purify the expressed protein. PEDV CH/Hubei/2016 strain was cultured in Vero cells for propagation.The viral suspension was subsequently concentrated and purified via gel filtration chromatography.The purified PEDV was characterized by SDS-PAGE and Western blotting.BALB/c mice were then immunized with the purified antigen to generate hybridoma cells.Positive hybridoma cells were screened by indirect ELISA and immunoperoxidase monolayel assay (IPMA).The neutralizing monoclonal antibodies against PEDV S1 protein were screened and identified using virus neutralization assay.【Result】 SDS-PAGE and Western blotting analysis demonstrated that S1 recombinant protein was efficiently expressed in HEK293F cell culture supernatant.Purified PEDV with TCID₅₀ of 1 ×10^-6.5/0.1 mL was obtained.Five hybridoma cell lines stably secreting anti-S1 mAbs were obtained and the IPMA and ELISA titers of the ascitics ranged from 6.4×10^-4 to 1.28×10^-5 and 6.4×10^-5 to 1.024×10^-7,respectively.Virus neutralization assay revealed that mAbs 2E1E10 and 4A8C8 exhibited high neutralizing activity,of which the inhibitory concentration 50% (IC₅₀) values were calculated as 1.212 and 1.203 μg/mL for strain DR13,and 0.948 and 1.712 μg/mL for strain CHHB,respectively.【Conclusion】 PEDV S1 recombinant protein with high antigenic activity was obtained,and neutralizing monoclonal antibodies against S1 protein were produced,which providing a good research foundation for the development of PEDV diagnostic reagents and vaccines.

【Objective】 This experiment aimed to investigate the immunopotentiating effect of FliC protein as a mucosal adjuvant on the Pseudorabies virus (PRV) Bartha-K61 attenuated vaccine strain,so as to lay a foundation for developing PRV-targeted mucosal vaccines.【Method】 FliC gene was cloned from Salmonella Typhimurium and linked to the prokaryotic expression vector pET-28a(+)，the linked product was transformed into Escherichia coli (E.coli) Trans10 competent cells,and the plasmid was extracted for double enzyme digestion identification and sequencing.FliC-pET-28a was transformed into E.coli BL21(DE3) competent cells to induce the expression of FliC protein,and the purified FliC protein was identified by Coomassie brilliant blue staining and Western blotting.Bartha-K61 virus suspension group and the commercial Bartha-K61 vaccine group were set up as control.FliC protein as mucosal adjuvant was mixed with Bartha-K61 virus solution,BALB/c mice were intranasally immunized twice.The levels of IgG and IgA in the serum of mice before immunization (day 0) and at post-immunization days 7,14,21,28 and 35 were measured.Mice in each group were challenged with PRV GD-WH of 10^4.5 TCID₅₀ on the 35th day after immunization and the survival was observed and recorded daily after challenge.【Result】 The recombinant plasmid FliC-pET-28a was successfully obtained,the soluble expression of FliC was realized,and the size of the purified FliC protein was approximately 58 ku.The results of mouse immune test showed that the serum IgG and IgA antibodies of Bartha-K61 virus solution+FliC protein group were significantly higher than those of Bartha-K61 virus solution group and commercial Bartha-K61 vaccine group (P＜0.05).The results of challenge protection test showed that Bartha-K61 virus solution+FliC protein could provide 100% protection to mice against the lethal attack of PRV GD-WH,which was higher than that of Bartha-K61 virus solution alone.【Conclusion】 FliC protein adjuvant could enhance mucosal immunity of PRV Bartha-K61 attenuated vaccine strain,and enable mice to obtain certain immune protection.

【Objective】 The purpose of the experiment was to investigate the virulence gene profile of the goat-derived Klebsiella pneumoniae strain KPHN001 with the K57 capsule serotype,and examine the distribution of virulence genes and sequence types (STs) among K57 capsule serotype Klebsiella pneumoniae strains from various sources,in order to assess their genetic diversity and potential evolutionary features.【Method】 After resuscitation and cultivation,the virulence phenotype of KPHN001 strain was determined by string test.The serotype and virulence genes of the strain were examined by PCR method.According to multilocus sequence typing (MLST) and phylogenetic tree reconstruction of single copy homologous genes,KPHN001 strain was compared with 30 strains of K57 capsule serotype Klebsiella pneumoniae from different sources in NCBI database.【Result】 String test result showed that KPHN001 strain was unable to produce a mucoviscous string ≥5 mm in length.PCR results showed that KPHN001 strain was K57 capsule serotype,containing several virulence genes including khe,fimH,uge,kfuBC,wabG,ureA,entB,mrkD,repA and sopB,while none of the important genetic marker genes for high-virulence Klebsiella pneumoniae (hvKP),such as rmpA2,iucA and iroB,were detected.Based on the negative string test and the absence of hvKP marker genes,the KPHN001 strain was identified as a classical Klebsiella pneumoniae (cKP) strain.The MLST results showed that the ST type distribution of K57 capsule serotype Klebsiella pneumoniae strains from bovine,goat and environment exhibited diversity,while the strains from human mainly concentrated on ST412 and ST218.KPHN001 strain was classified as ST528.The combined analysis of phylogenetic tree with virulence gene heatmap indicated that strains from human exhibited significant differences in uge gene expression compared to strains from other sources,and strains from human formed independent branches,indicating that these strains might have accumulated unique genetic variation during evolution.【Conclusion】 This study elucidated the virulence gene profile of a goat-derived K57 capsule serotype Klebsiella pneumoniae strain and highlighted the genetic diversity of K57 capsule serotype strains,providing a theoretical basis for understanding their virulence mechanisms and the diagnosis and prevention of Klebsiella pneumoniae infection.

【Objective】 The aim of this study was to investigate the species and genetic diversity of hard ticks from cattle and sheep in the China-Myanmar border region of Tengchong city,Yunnan province,and provide a basis for the prevention and control of diseases caused by them.【Method】 The present study conducted a survey on tick infection in cattle and sheep in four towns in Tengchong city from June to July 2024,the ticks were collected and submitted to morphological identification.The mitochondrial cox1 gene and ribosomal ITS2 gene sequences of 16 ticks (8 from cattle and 8 from sheep) were amplified by PCR,their genetic diversity,haplotype polymorphism and phylogenetic relationships were analyzed using DNAStar,Mega 7.0 and DnaSP software.【Result】 The farm positive rate of tick infection in cattle and sheep in the four investigated towns was 100% (24/24).The infection rates of ticks in cattle and sheep were 94.37% (64/71) and 91.74% (111/121),respectively.A total of 362 ticks were collected,all of which were identified as Rhipicephalus microplus through morphological analysis.The intraspecific differences in cox1 (762 bp) and ITS2 (1 477 bp) gene sequences of 16 R.microplus isolates were 0-0.5% and 0-1.3%,respectively.The similarity between cox1 and ITS2 gene sequences of R.microplus (branch C) in GenBank exceeded 99.5% and 99.0%,respectively.The haplotype diversity of cox1 and ITS2 genes was 0.858 and 0.932,respectively,while the nucleotide diversity was 0.00395 and 0.00514,respectively.The results of phylogenetic tree analysis revealed that all 16 R.microplus belonged to the same branch as known R.microplus,the isolates from different host sources were randomly distributed in this branch,but they were far away from the branches of other ticks.【Conclusion】 The prevalence of ticks on the surface of cattle and sheep in Tengchong city,Yunnan province was severe,and all ticks were R.microplus belonging to branch C.The R.microplus strains from different hosts had certain genetic variations,but the haplotype diversity and nucleotide diversity were low,and there was no genetic differentiation phenomenon.

【Objective】 The purpose of this study was to construct and express the fusion protein of Escherichia coli (E.coli) FliC protein D0/D1 domain and Pal protein,provide a basis for developing a novel avian enteropathogenic E.coli (APEC) vaccine.【Method】 The amino acid sequence characteristics of FliC and Pal proteins of E.coli O1 serotype strains in GenBank database were analyzed.The D0/D1 domain of FliC protein (N-terminal 5-140 amino acids and C-terminal 499-583 amino acids) and the main structure of Pal protein (21-173 amino acids) were selected to construct the "sandwich-type" fusion protein.The physicochemical properties,secondary structure,tertiary structure,antigenicity,sensitization,linear and discontinuous epitopes of the fusion protein were predicted.The binding ability and immune effect of the fusion protein with immune receptors were evaluated through molecular docking and immune simulation.The base sequence of the fusion protein was synthesized and the vector was constructed using pET-28a(+).The fusion protein was expressed using E.coli BL21(DE3) competent cells.The fusion protein was purified using nickel-agarose gel 6FF and renatured.The reactivity of the fusion protein was verified by Western blotting.【Result】 The molecular mass of the fusion protein was precisely determined to be 42.72 ku,and it was identified as a hydrophilic protein.The composition of the protein’s secondary structure was precisely quantified,with the alpha helix,extended strand,beta turn and random coil accounting for 48.79%,14.25%,6.76% and 30.19%,respectively.Notably,the alpha helix was mainly located within the D0/D1 domain of the FliC protein.The Ramachandran plot of the tertiary structure revealed that the advantage region encompassed an impressive 96.9% of the residues.The fusion protein had outstanding antigenicity,no sensitization,and contained 8 linear epitopes and 5 discontinuous epitopes.Additionally,the fusion protein demonstrated the ability to molecularly dock with Toll-like receptors 2,4 and 5,and the immune simulation results exhibited promising and significant outcomes.The chimeric gene FliCN-Pal-FliCC positive monoclonal was identified by PCR and sequencing and transformed into E.coli BL21 (DE3) competent cells.The fusion protein FliCN-Pal-FliCC was successfully expressed and purified.The fusion protein could react with the serum of E.coli O1 serotype-positive mice.【Conclusion】 In this study,"sandwich" fusion protein FliCN-Pal-FliCC based on FliC and Pal proteins of APEC O1 serotype was successfully constructed and expressed,which provided a research basis for the research and development of candidate antigens of APEC vaccine.

【Objective】 This experiment aimed to express the capsid protein (Cap) of Porcine circovirus type 4 (PCV4) through the prokaryotic expression system and prepare its polyclonal antibody,providing technical support for the prevention and control of PCV4.【Method】 In this study,a pair of specific primers was designed based on the immunogenic region of the PCV4 open reading frame 2(ORF2) gene.The PCV4 ORF2 gene sequence was obtained through PCR amplification and ligated to the pET-28a(+) vector to construct the recombinant expression plasmid pET-28a-ORF2. The recombinant plasmid pET-28a-ORF2 identified by PCR amplification and sequencing was transformed into E.coli BL21(DE3) competent cells,and 1 mmol/L IPTG was added to induce the expression of recombinant Cap protein.Then the induced recombinant protein products were analyzed by SDS-PAGE.The recombinant protein was purified using urea and identified by Western blotting.The purified recombinant Cap protein was used to immunize New Zealand White rabbits to prepare polyclonal antibodies and Western blotting was performed for identification.The titer of rabbit serum antibodies was detected by indirect ELISA.【Result】 PCV4 ORF2 gene fragment with a size of 414 bp was amplified by PCR,and the recombinant expression plasmid pET-28a-ORF2 was successfully constructed.SDS-PAGE analysis results showed that the recombinant Cap protein induced for expression was 16 ku.The results of Western blotting indicated that the prepared rabbit-derived polyclonal antibodies had good immunogenicity.Indirect ELISA detection showed that the titer of the prepared rabbit serum antibody reached 1∶12 800.【Conclusion】 In this study,the PCV4 Cap protein was successfully expressed using the prokaryotic expression system and polyclonal antibodies were prepared.The results provided a material basis for the in-depth study of PCV4 Cap protein and the subsequent development of PCV4 genetic engineering subunit vaccines,serological diagnostic reagents,etc.

【Objective】 This study aimed to explore the effect of FnrS gene on the pathogenicity of Salmonella Typhimurium by constructing non-coding small RNA (sRNA) FnrS gene deletion strain.【Method】 The λ-Red homologous recombination technology was used to construct the FnrS gene deletion strain ΔFnrS of Salmonella Typhimurium,and its genetic stability,growth curve and biochemical characteristics were determined.The effect of FnrS gene on the pathogenicity of Salmonella Typhimurium was analyzed by detecting the median lethal dose (LD₅₀) of the strain on mice,the bacterial load of the tissue and the pathological tissue sections.【Result】 The FnrS gene deletion strain ΔFnrS was successfully constructed and it could be stably inherited.The growth curve and biochemical test results showed that the deletion of FnrS gene did not affect the growth ability and biochemical characteristics of Salmonella Typhimurium.The results of the pathogenicity test in mice showed that the LD₅₀ of deletion strain ΔFnrS for mice was 1.06×10⁵ CFU，which was significantly higher than that of parental and complement strains (P＜0.05).There was no significant difference in the survival rate of mice between deletion strain ΔFnrS and parental strain (P＞0.05).At 7 and 9 days after infection with deletion strain ΔFnrS,the bacterial load in the liver of mice was significantly lower than that of parental and complement strains (P＜0.05).At 5，7 and 9 days after infection，the bacterial load in the spleen of mice was significantly lower than that of parental and complement strains (P＜0.05).【Conclusion】 The deletion of FnrS gene reduced the pathogenicity of Salmonella Typhimurium.This results laid a foundation for the further research of the pathogenic mechanism of Salmonella Typhimurium.

【Objective】 This experiment aimed to establish an indirect enzyme-linked immunosorbent assay (ELISA) method applicable to detecting antibodies against Chlamydia psittaci (Cps) in pigeons.【Method】 The codons of the gene encoding the major outer membrane protein (MOMP) of Cps were optimized.After adding trigger factor (TF) at the N-terminus,it was ligated to the pET-30a(+) vector to construct the plasmid pET-30a-M.After induction with IPTG,expression and purification were carried out.Solubility analysis and identification were performed using SDS-PAGE and Western blotting.A checkerboard titration was used to determine the optimal antigen coating concentration and serum dilution,thereby optimizing the indirect ELISA method for detecting pigeon Cps antibodies.The sensitivity of the indirect ELISA method established in this study were compared with that of the indirect hemagglutination assay (IHA).The specificity of this method was evaluated by detecting the positive sera of other common diseases in pigeons.Three positive and three negative sera for Cps were detected respectively to assess its repeatability.The coincidence rate of this indirect ELISA method and the IHA method was evaluated by using 253 pigeon-derived sera (174 clinical serum samples and 79 serum samples after immunization with Cps inactivated vaccine).【Result】 SDS-PAGE and Western blotting results showed that the MOMP protein with a size of approximately 100 ku was successfully expressed in the supernatant after induction and sonication,indicating that this protein was soluble.The optimized conditions of the indirect ELISA method were as follows:Coating with 2 μg/mL antigen at 4 ℃ overnight,blocking with 5% skimmed milk at 37 ℃ for 2 h,serum dilution of 1∶200,serum incubation time of 1 h,HRP-labeled rabbit anti-pigeon IgY incubation for 30 min,and substrate reaction time of 10 min.When the dilution of the positive serum of pigeon Cps was 1∶16,the detection result of the indirect ELISA method was still positive,and the sensitivity was higher than that of the IHA method.There was no cross-reaction between the indirect ELISA method and the positive sera of other common pigeon diseases.Both intra- and inter-assay test results showed that the coefficients of variation of the indirect ELISA method were all less than 10%,indicating that this method had good repeatability.The coincidence rates of the indirect ELISA method established in this study and the IHA method for detecting clinical samples and serum samples after immunization were 80.72% and 85.71% respectively,and the Kappa values were 0.60 and 0.69 respectively.【Conclusion】 The indirect ELISA method established in this experiment exhibited high sensitivity,strong specificity,and excellent repeatability,and was suitable for the detection of serum antibodies against Cps in pigeons.

【Objective】 Bovine rotavirus (BRV) type A is an important pathogen causing severe enteritis in newborn calves.The experiment aimed to understand the genotypes and molecular characteristics of the prevalent BRV strains in large-scale cattle farms in Yunnan province,providing a theoretical basis for the prevention and control of viral diarrhea in cattle farms.【Method】 Pathogen detection was carried out on 7 fecal samples of Simmental calf diarrhea from a large-scale beef cattle farm in Yunnan by RT-PCR method.Samples positive for BRV nucleic acid were subjected to continuous passage and isolation of the virus through the MA-104 cell line.The virus proliferation was detected by indirect immunofluorescence assay (IFA),and the virus growth curve was plotted.The whole genomes of the isolated strain were further sequenced.The genotypes were analyzed using online comparison software.The genetic evolutionary trees of VP4 and VP7 genes were drawn using Mega 7.0 software, respectively,and the amino acid mutations were analyzed using GeneDoc software.【Result】 7 diarrhea samples were all positive for BRV,from which a BRV strain was isolated and named YNLL-2023,with being genotyped as G10-P[11]-I2-R2-C2-M2-A3-N2-T6-E2-H2.MA-104 cells infected with the isolated strain showed obvious cytopathic effect (CPE) for 12 h.The characteristic fragment of approximately 300 bp of the VP6 gene of the isolated strain was obtained by RT-PCR amplification.IFA showed that specific green fluorescence appeared in the cytoplasm of MA-104 cells after the isolated strain infected.The growth curve showed that the virus titer continuously increased from 6 to 54 h after inoculation,reaching a peak of 10^5.57 TCID₅₀/100 μL.Genetic evolution analysis indicated that the nucleotide sequence of VP4 gene of the isolated strain was in the same branch as the RVA/Yak-tc/CHN/HB-3/2021/G10P[11] strain (accession No.:ON711387.1).The nucleotide sequence of VP7 gene was in the same branch as the RVA/Bovine-wt/CHN/JS31/2020/G10 strain (accession No.:PQ332932.1) and the RVA/Bovine-wt/CHN/SD1/2023/G10 strain (accession No.：PQ332943.1).Amino acid sequence analysis demonstrated that there were 7 amino acid mutations in VP4 between the isolate and the P[11] type sheep vaccine strain,and 2 amino acid mutations in VP7 between the isolate and the G10 type human-bovine recombinant vaccine strain in neutralizing epitope region.【Conclusion】 In this experiment,one strain of type G10P[11] BRV was successfully isolated from MA-104 cells.This strain was highly similar to several strains of bovine origin in China，but there were differences in neutralizing epitopes with vaccine strains of the same genotype.

【Objective】 This study was conducted to explore the prevalence and drug resistance risk of Klebsiella in fresh camel milk in Xinjiang,and provide a theoretical basis for monitoring Klebsiella in fresh camel milk in this area.【Method】 Bacteria were isolated and purified from fresh camel milk in Xinjiang by plate scribing method.The isolates were identified by morphological observation,Gram staining microscopy,biochemical identification and PCR technology.The drug resistance of the isolates was analyzed by broth microdilution method.The isolated bacteria were tested for 17 key resistance genes by PCR.【Result】 A total of 58 strains of Klebsiella were isolated from 95 fresh camel milk samples collected in Xinjiang,and the isolation rate was 61.05%.All isolates were raised pink round colonies,and under Gram-staining microscopy,they showed red,short-stemmed Gram-negative bacteria.The biochemical identification results showed that the isolated bacteria were positive for urease,citrate,glucose,lactose,glycine,ornithine and lysine.The results of hydrogen sulfide test,methyl red test and V-P test were all negative.PCR amplification results showed that 428 and 1 476 bp bands of khe specific gene and 16S rDNA were amplified respectively.The sequencing results showed that the 16S rDNA sequence of the isolated bacteria was ≥99.0% similar to the reference sequence of Klebsiella in the NCBI database,and it was comprehensively determined that all the isolated bacteria were Klebsiella.The results of drug sensitivity test showed that the drug resistance rates of the isolates to ampicillin and sulfamethoxazole were 27.59% and 15.52%,respectively.The sensitivity rates of the isolates to amoxicillin/clavulanic acid,meropenem,streptomycin,gentamicin,tetracycline,dotetracycline,florfenicol,ciprofloxacin,compound sulfamethoxazole were 100%,and the sensitivity rates of the isolates to kanamycin was 98.28%.One strain of Klebsiella was multidrug-resistant.The results of drug resistance gene detection showed that the positive rates of bla_CTX-M-14,bla_SHV,bla_CTX-M-123 and bla_CTX-M were 28.0%,24.0%,24.0% and 4.0%,respectively.No polypeptide resistance genes mcr-1, mcr-2 and mcr-3, and sulfonamide resistance genes sul1,sul2 and sul3 were detected.【Conclusion】 The prevalence of Klebsiella in fresh camel milk in Xinjiang was serious,and there were some drug resistance phenomena.Therefore,it was of great significance to monitor the drug resistance of Klebsiella in fresh camel milk.

【Objective】 This study aimed to obtain antimicrobial peptides with broad-spectrum antimicrobial activity through molecular design and antibacterial performance evaluation,and provide a new antibiotic alternatives for clinical production.【Method】 A polypeptide named Loong-3 was designed using online bioinformatics tools.The antimicrobial activity,minimal inhibitory concentration (MIC),minimum bactericidal concentration (MBC) and hemolysis rate of polypeptide Loong-3 were determined by agarose diffusion method,double dilution method,colony culture method and double dilution method,respectvely.And the effects of various physicochemical factors (heat,UV,repeated freeze-thaw,salt ions,acid-base,serum,organic reagents,enzymes) on the antimicrobial activity of polypeptide Loong-3 were analyzed.【Result】 The results showed that the polypeptide Loong-3 was a new cationic hydrophobic heat-resistant spiral peptide with a broad antimicrobial spectrum.Its MIC and MBC against Gram-negative bacteria Escherichia coli,Salmonella and Pseudomonas aeruginosa were 0.976-500 and 1.953-31.25 μg/mL,respectively,the MIC and MBC against Gram-positive bacteria Staphylococcus aureus were 31.25 and 62.5 μg/mL,respectively,and the MIC and MBC against fungi Candida albicans were 125 and 500 μg/mL,respectively.Peptide Loong-3 had a certain degree of hemolysis,and when the concentration of polypeptide Loong-3 increased from 0.976 to 250 μg/mL,the hemolysis rate also increased from 5.78% to 76.68%.Except for trypsin,the polypeptide Loong-3 still had antimicrobial activity under various physical and chemical factors (heat,UV,repeated freeze-thaw,salt ions,acid-base,serum,organic reagents,enzymes) though the MIC was fluctuated.【Conclusion】 The designed polypeptide Loong-3 was a new cationic broad-spectrum antimicrobial peptide with certain hemolytic properties.Heat,UV,repeated freeze-thaw cycles,salt ions,acid-base,serum,organic reagents,and enzymes had no significant effect on the antimicrobial activity of polypeptide Loong-3.

【Objective】 Niclosamide (NIC),originally developed as an anthelmintic agent,has recently been recognized for its potent inhibitory activity against a broad spectrum of viruses.This study aimed to evaluate the in vitro antiviral activity of NIC against Porcine epidemic diarrhea virus (PEDV),develop and assess a novel oral NIC nanomedicine for antiviral efficacy in vivo,and preliminarily explore the molecular mechanisms underlying its anti-PEDV action.【Method】 The antiviral effect of NIC against PEDV was first evaluated in vitro using cytotoxicity and plaque reduction assays.An enhanced niclosamide-delivery nanoparticle (eNDN) was formulated based on a previously established bile salt-based delivery system,and its physicochemical properties,including particle size,water solubility,and pharmacokinetics,were characterized.The antiviral efficacy of orally administered eNDN was then evaluated in APN-transgenic mice challenged with PEDV.Additionally,literature mining and integrative bioinformatics analyses were performed to identify potential antiviral targets and signaling pathways,and molecular docking was used to predict key targets involved in its anti-PEDV activity.【Result】 The results of in vitro antiviral test showed that,NIC exhibited potent antiviral activity against PEDV in vitro,with a half-maximal inhibitory concentration (IC₅₀) of 226.4 nmol/L.Compared with niclosamide-delivery nanoparticles (NDN),the optimized nanomedicine eNDN exhibited a reduced particle size by approximately 30 nm (average diameter was about 140 nm),a 20% improvement in water solubility (reaching 5 mg/mL),and an approximately 10% increase in oral bioavailability (up to 47.54%).Toxicological assessment showed no apparent clinical symptoms or hepatic pathology following oral administration of eNDN,indicating good biosafety.Real-time quantitative PCR analysis revealed an extremely significant reduction of PEDV titer in intestines of eNDN-treated mice (P＜0.01),compared with control group.Bioinformatics analysis identified 159 NIC-related targets and 1 678 PEDV-associated targets,with 22 overlapping targets.Molecular docking suggested MAPK14,NAIP,NLRC4,SERPINE1,ATP2A2,CLTC,MCL1,TMPRSS2,APN and STAT3 as core targets mediating NIC’s antiviral effects.【Conclusion】 Both NIC and its novel oral nanomedicine eNDN exhibited robust antiviral activity anti-PEDV in vitro and in vivo.Bioinformatics predicted that NIC played an anti-PEDV role through multiple targets and pathways such as anti-inflammatory response and inhibition of virus invasion.The results of this study provided a theoretical and experimental basis for exploring the molecular mechanism of NIC against PEDV infection and the clinical transformation of nanomedicine eNDN.

Porcine epidemic diarrhea (PED),which is caused by Porcine epidemic diarrhea virus (PEDV),is a highly contagious gastrointestinal infection in pigs.This disease is characterized by a rapid course,susceptibility of pigs of all ages,and a high mortality rate,causing significant economic losses to the pig industry.PEDV can infect and damage small intestinal epithelial cells,leading to a reduction in enzyme activity in small intestine,resulting in the accumulation of water and electrolytes and metabolic disorders in small intestine,especially the accumulation of undigested lactose in intestine,increasing intestinal osmotic pressure and causing the retention of body fluids in intestine,ultimately leading to diarrhea and dehydration.Currently,vaccination is the primary approach for preventing PED.However,the existing commercial vaccines have limitations in terms of immune effect and broad-spectrum protection and cannot completely prevent the infection of PEDV.Additionally,there is still a scarcity of effective therapeutic drugs.Chinese herbal medicine offers a novel perspective for the prevention and control of PED due to its natural origin,diverse biological activities,high safety,low toxicity,and low resistance.Some studies have confirmed that some Chinese medicine or Chinese medicine extracts have the potential to inhibit PEDV,and domestic and foreign scholars have made positive progress in the research on the anti-PEDV effect of Chinese medicine and its mechanism.This paper reviewed the pathogenic mechanism of PEDV,TCM syndrome differentiation methods,TCM compounds for PED treatment,and the mechanism of action of TCM active components in PED treatment,aiming to provide new ideas and research references for the prevention and treatment of PED.

【Objective】 The aim of this study was to understand the molecular epidemiological characteristics and drug resistance of avian pathogenic Escherichia coli (APEC) from geese,so as to provide reference for the prevention and control of colibacillosis in waterfowl.【Method】 A total of 240 diseased goose samples were collected from different regions in Jiangsu province.The strains were isolated and purified using the plate streaking method.Positive strains were identified through 16S rDNA PCR amplification，and O antigen serotype,phylogenetic cluster and virulence gene carrying of the isolates were detected.Drug resistance was detected by drug sensitivity test,and multi-drug resistance analysis was performed.【Result】 A total of 31 strains of APEC were isolated from 240 clinical samples.The serotype detection results showed that the detection rate of O145 serotype was 19.35%,followed by O1 (6.45%),O78 (6.45%) and O2 (3.23%) serotypes. Phylogenetic cluster analysis showed that the proportion of the isolates belonging to A,B1,B2,and D evolutionary groups were 38.71%,22.58%,9.68%,and 29.03%,respectively.Virulence gene detection results showed that the detection rates of fimA and hlyF genes were both above 90%,the detection rates of iroN,ompT,iucD,ECs3737,ECs3703 genes were 60%-90%,the detection rates of iss,tsh,irp2,cvaC,papC and hlyE genes were below 50%，and eaeA gene was not detected.All isolates contained at least 4 virulence genes.The drug susceptibility test results showed the resistance rates of the isolates to doxycycline,lincomycin,ampicillin and amoxicillin were more than 80%,and the resistance rates of the isolates to neomycin,flufenicol and enoxacin were 50%-70%,while the sensitivity rates of the isolates to daviomycin (74.19%) and gentamicin (54.84%) were higher.The proportion of 14 drug-resistant strains was the highest (19.35%),and the proportion of more than 7 drug-resistant strains was 64.52%.【Conclusion】 The results indicated that the serotype of the APEC isolates from geese in Jiangsu province was complex,carrying multiple virulence genes,and exhibiting severe multidrug resistance.The results provided data support for the prevention and control of colibacillosis in waterfowl in this area.

【Objective】 The purpose of this test was to develop an immunocolloidal gold test strip for the rapid detection of zilpaterol (ZIL) residues.【Method】 A polyclonal antibody against ZIL was purified from rabbit serum using ammonium persulfate precipitation.The optimal concentration of IgG for colloidal gold labeling was determined through a combination of IgG gradient dilution and NaCl-induced aggregation stability testing.Gold-labeled antibody conjugates were then prepared accordingly.A nitrocellulose membrane was employed as the solid-phase carrier,with ZIL-BSA conjugates immobilized as the test line (T line) and goat anti-rabbit IgG antibodies as the control line (C line),to construct a immunocolloidal gold test strip for the detection of ZIL residues.The developed strip was evaluated for its sensitivity,specificity,and applicability in spiked bovine urine samples.【Result】 The results of antibody purification showed that the purified polyclonal antibody concentrations before and after purification were 5.17 and 2.55 mg/mL,respectively.SDS-PAGE results showed that no specific bands were produced after serum purification,and obvious IgG heavy chain (56 ku) and light chain (23 ku) bands appeared,which was consistent with expectations.The optimal IgG concentration for gold conjugation was determined to be 1.36 μg/mL (0.17 μg/well).Sensitivity and specificity assessments showed that the test strip had a visual cut-off value (COV) of 1 ng/mL,with a detection time of 8-10 min.There was no cross-reaction with 8 drugs such as clenbuterol (CLB),salbutamol (SAL),simaterol (CIM),and terbutaline (TBL).The results of the spiked test of bovine urine showed that its COV value was 1 ng/mL,demonstrating good tolerance to matrix interference.【Conclusion】 In this study,a ZIL rapid detection test strip based on immunocolloidal gold technique was successfully prepared,which had high sensitivity,high specificity and good matrix tolerance.It could be used for on-site screening of ZIL residues in livestock products,providing a rapid,convenient and cost-effective tool for food safety supervision.

【Objective】 This study was conducted to diagnose the type and nature of spontaneous tumors in a Tupaia belangeri subsp.yaoshanensis,clarify the biological characteristics of the strains isolated from the tumor,and provide new evidence for the correlation between bacteria and tumors.【Method】 The tumor was resected surgically and subjected to pathological and immunohistochemical examination.Bacteria within the tumor were isolated using the streak plate method.The isolated bacteria underwent morphological observation,biochemical tests,and 16S rRNA sequence analysis.The median lethal dose (LD₅₀) of the isolated strain to Kunming mice was determined.The drug sensitivity of the isolated strain to commonly used drugs was analyzed using the drug sensitivity paper disc method.【Result】 The tumor was located in the subcutaneous tissue adjacent to fat.It exhibited a lobular structure with basal-like cells peripherally and mature sebaceous cells centrally,showing no significant atypia except for occasional nuclear division in basal-like cells.The surrounding fat tissue showed liquefaction necrosis,forming cystic structures of varying sizes.Reactive multinucleated giant cells and inflammatory cell infiltration were observed.Immunohistochemistry revealed diffuse strong positivity for CK14 in tumor cell cytoplasm.CK-P was positive in some tumor cells,P63 was positive in nucleus,and Ki-67 was positive in individual nucleus with a positivity rate of approximately 20%.The isolated bacteria were Gram-negative rods.Biochemical tests showed that H₂S,phenylalanine,urea,indole and citrate of the isolated strain were positive,and side calendula alcohol,sorbierite,malonate,lysine,ornithine and V-P tests were all negative,which was consistent with the biochemical characteristics of Proteus vulgaris.16S rRNA sequencing identified the isolate as Proteus,and the similarity to human Proteus hauseri (NR_104767.1) was ≥99%.The LD₅₀ of the isolated strain for Kunming mice was 3.54×10⁶ CFU/0.025 kg.Drug sensitivity tests showed that the isolate was high sensitivity to 14 drugs,including cephalosporins,aminoglycosides and quinolones,moderate sensitivity to azithromycin,and varying degrees of resistance to 13 drugs,including penicillins and lincosamides.【Conclusion】 This study diagnosed a spontaneous tumor in Tupaia belangeri subsp.yaoshanensis as a sebaceous adenoma and isolated Proteus vulgaris from the tumor tissue.The findings challenged conventional understanding by providing direct evidence for the role of opportunistic pathogens in the tumor microenvironment.The observed multidrug resistance and high pathogenicity of this bacterial strain suggested its potential involvement in remodeling the tumor microenvironment through chronic inflammation and immune evasion mechanisms.However,current evidence remained insufficient to establish a direct causal relationship between this bacterium and tumor initiation or progression.