Oocyte cryopreservation is a vital technology for preserving germplasm resources of genetically superior livestock and endangered species， and also serving as a critical tool for breed improvement and commercial embryo production. As essential agents in vitrification， cryoprotectant agents protect cells by lowering the freezing point， increasing viscosity， preventing ice crystallization， and mitigating osmotic damage. However， the cytotoxicity of cryoprotectant agents remains one of the critical challenges to be addressed in vitrification technology. The development of novel cryoprotectant agents with low toxicity and high efficacy has become a key research direction for improving the efficiency of livestock oocyte vitrification. This review systematically analyzes the cytotoxic mechanisms of conventional cryoprotectant agents in oocytes preservation， with particular emphasis on recent advances in novel cryoprotective agents， including small-molecule permeable cryoprotectants， lipid modulators， antioxidants， and cytoskeleton stabilizers. It will provide both theoretical basis and technical references for optimizing the vitrification systems of livestock oocytes.

Preantral follicles represent a critical stage in the transition from follicular quiescence to active growth and are abundant in the ovaries of ruminants， possessing high developmental potential and significant application value. With the advancement of reproductive biotechnology， in vitro culture of preantral follicles has become an important focus in ruminant reproductive biology as an assisted reproductive approach. This technique not only contributes to improve livestock reproductive efficiency but also provides new strategies for the preservation of genetic resources and population restoration of endangered species. However， the in vitro development of ruminant preantral follicles is still constrained by multiple factors， such as the structural complexity of follicles， instability of culture systems， and low oocyte developmental competence， which limit its further application. Ruminant preantral follicles can achieve a certain degree of growth and development under in vitro conditions， and some studies have obtained early embryos such as morulae， but the overall maturation rate and developmental potential remain limited. This review systematically summarizes the isolation methods and in vitro culture systems of ruminant preantral follicles， including mechanical and enzymatic isolation， in situ culture， two-dimensional culture， and three-dimensional culture， and compares the differences among these methods and systems in terms of follicular integrity， applicability， and developmental performance. Future studies should focus on the standardization of isolation methods， optimization of culture systems， and elucidation of the mechanisms underlying long-term follicle survival and in vitro maturation， with the goal of establishing more efficient and stable culture systems and promoting the application of this technology in livestock production and the conservation of endangered species.

Microorganisms are the most abundant biological groups on the earth， exerting crucial functions in diverse aspects of human existence， spanning production， healthcare， and daily routines. Nevertheless， the traditional methodologies employed for microbial cultivation and exploration have remained relatively primitive， which means that merely an infinitesimal proportion of microorganisms can be successfully cultured under such circumstances. In recent time， propelled by the remarkable advancements in high throughput sequencing， culturomics has progressively captured the attention of researchers and revealed remarkable potential for application in the pursuit of microbial germplasm resources. For example， by combining multiple culture conditions with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry （MALDI-TOF MS）， new strains have been successfully screened from environments like the intestines of livestock and poultry， as well as forest or marine sediments， providing novel approach for the exploration of microbial resources. Meanwhile， synthetic biology offers fresh ideas for the directed modification and functional enhancement of microorganisms. It achieves this by constructing microbial cell factories， analyzing the metabolic networks of non-model strains， and designing and synthesizing microbial communities. For instance， the construction of microbial cell factories has enabled the efficient biosynthesis of products such as artemisinic acid and polyhydroxyalkanoates （PHAs）. New chassis cells developed from non-model strains （e.g.， thermophiles and halotolerant actinomycetes） have overcome the metabolic limitations of traditional model bacteria. Additionally， synthetic microbial communities have optimized biosynthetic pathways for complex products and improved the efficiency of microbial screening and cultivation. This review summarizes recent advances in the exploration and breeding of microbial germplasm resources， with a focus on the combined application strategies of culturomics and other technical approaches. It integrates the research progress in constructing microbial cell factories， modifying non-model strains， and designing synthetic microbial communities， along with their applications and challenges in diverse fields， so as to provide theoretical foundation and technical reference for the efficient exploration and utilization of microbial germplasm resources in China.

Cystic echinococcosis （CE）， a severe zoonotic parasitic disease caused by the larval stage of Echinococcus granulosus （Eg）， primarily affects human and livestock liver and lung tissues. Current treatment primarily involves surgical removal of cystic lesions combined with chemotherapy. However， traditional medications such as mebendazole（MBZ） and albendazole（ABZ） only inhibit parasite growth and reproduction without complete eradication， requiring long-term high-dose administration which can lead to adverse reactions including hepatotoxicity. Recent research has focused on novel ABZ formulations， delivery systems， and combination therapy strategies. Novel formulations and delivery methods aim to enhance efficacy by improving bioavailability and reducing toxicity： Emulsions and salt preparations optimize drug solubility， while ABZ combined with nanoparticles or carriers （liposomes and chitosan） creates targeted delivery systems that increase local drug concentration while minimizing systemic toxicity. Combination therapies demonstrate synergistic effects through multi-mechanism interference with parasite metabolism， combining ABZ with drugs such as praziquantel， flubendazole， and α-interferon. Despite these advances， no breakthrough therapeutic alternatives have emerged. This review summarizes the research progress of ABZ in treating CE， analyzing the advantages and limitations of various treatment approaches to provide a scientific basis for future drug development and clinical treatment optimization.

Zearalenone （ZEA）， also known as F-2 toxin， is a type of mycotoxin with estrogenic toxicity produced by Fusarium species. It is commonly found in corn， wheat， soybean meal， and other grains and their by-products. After animals consume feed contaminated with ZEA， it can cause various toxic reactions. Among them， the liver， as the main metabolic and transformation organ of ZEA， is particularly vulnerable to damage. ZEA can easily induce oxidative stress， alterations in the expression of inflammation-related factors， and apoptosis of liver cells in the liver， thereby seriously endangering human and animal health. Polyphenols， as a class of plant chemical substances with strong antioxidant， antibacterial， anti-inflammatory and other biological activities， mainly include two categories： Flavonoids and non-flavonoids. The author selected rutin （RUT）， a flavonoid， and curcumin （CUR）， a non-flavonoid， as representative compounds to explain the mechanism by which polyphenols alleviate the liver toxicity induced by ZEA in animals. Both RUT and CUR can counteract the liver toxicity of ZEA through multi-target synergistic effects， mainly through antioxidant stress， anti-inflammatory and anti-apoptotic actions. The authors mainly reviewed the toxic effects of ZEA on animal livers and the mechanisms by which RUT and CUR alleviated ZEA induced animal liver toxicity， in order to provide a certain theoretical basis for subsequent research on polyphenols alleviating ZEA liver toxicity in animals and promoting the healthy development of animal husbandry.

Heat stress （HS） triggers complex pathological responses in animals， posing a serious threat to health and even leading to death， thereby causing substantial economic losses. As a key target during HS， vascular endothelial cells （VECs） sustain injury through both direct and indirect injuries. Direct injury includes disruption of cell membranes and cytoskeletal structures， DNA damage， and induction of apoptosis-related gene expression， resulting in structural and functional abnormalities of VECs and excessive apoptosis. Indirect injury is mediated by systemic inflammatory responses， oxidative stress， and dysregulation of vascular tone. Damaged VECs also release multiple cytokines， further exacerbating the adverse effects of HS on the organism.Traditional Chinese medicine （TCM）， by virtue of its abundant sources， diverse constituents， target specificity， and favorable safety profile， has become a research hotspot for anti-HS veterinary therapeutics. Active ingredients of TCM， such as terpenoids （paeoniflorin， ginsenoside Rg1， etc.）， phenolics （ferulic acid， resveratrol， etc.）， and flavonoids （quercetin， puerarin， etc.）， as well as compound preparations and extracts （Shenfu injection， Ginkgo biloba extract， etc.） exhibit notable antioxidant， anti-inflammatory， and anti-apoptotic activities and enhance cellular thermotolerance， primarily through coordinated actions on multiple signaling pathways. For antioxidation， these agents activate the Keap1/Nrf2/HO-1 pathway to upregulate antioxidant enzymes and thereby alleviate oxidative stress. For anti-inflammatory effects， they inhibit the PI3K/Akt， TLR2/NF-κB and TLR4/NF-κB pathways， reducing the release of inflammatory cytokines and adhesion molecules. With respect to anti-apoptosis， they increase the Bcl-2/Bax ratio， suppress activation of the caspase family， and protect mitochondrial function. To improve thermotolerance， they promote the expression of heat shock proteins （HSPs）， thereby mitigating heat-induced damage.In this review， the authors summarize the major mechanisms by which HS induces VEC injury and systematically collate current research on TCM monomers and extracts that protect VECs against HS-induced damage， with the aim of providing a theoretical reference for further investigation of active ingredients of TCM and their application in livestock and poultry production.

Oxidative injury of testes and ovaries， as well as excessive cellular apoptosis induced by animal reproductive system diseases， are profoundly associated with dysregulation of signaling pathways. Among these， iron metabolism exerts a critical role in the pathogenesis of animal reproductive diseases. As a regulated form of cell death driven by iron-dependent lipid peroxidation， ferroptosis is characterized by intracellular iron homeostasis imbalance， accumulation of lipid peroxides， and functional defects in the antioxidant system mediated by glutathione peroxidase 4 （GPX4）. It is synergistically regulated primarily through the SystemXc^--GSH-GPX4 axis， iron metabolism pathways （regulated by transferrin receptor 1， ferritin heavy chain， etc.）， and lipid metabolism pathways （mediated by acyl-CoA synthetase long-chain family member 4， etc.）. In the field of animal reproduction， ferroptosis exerts a significant impact on the reproductive functions of mammals （human， rodent， rabbit， etc.） and poultry. In males， it can induce testicular oxidative stress， damage to spermatogenic cells， and a decline in sperm quality. In females， it is closely associated with ovarian hypofunction， follicular atresia， oocyte maturation disorders， and abnormal embryonic development. Environmental pollutants （such as PM2.5 and microplastics）， toxins （such as cadmium and zearalenone）， and pathological factors can exacerbate reproductive damage by activating ferroptosis pathways. In contrast， ferroptosis inhibitors （Ferrostatin-1）， iron chelators， and natural products （melatonin， Lycium barbarum polysaccharides， etc.） can alleviate related damage by regulating signaling pathways such as Nrf2/GPX4. This review systematically sort out the molecular regulatory mechanisms of ferroptosis， comprehensively summarize its roles in the reproductive systems of different types of animals and the related research progress， and in-depth analyze the mechanism of ferroptosis as well as its mitigation approaches. It is expected to provide a novel target for deciphering the pathological mechanisms of animal reproductive diseases and improving reproductive performance， and lay a theoretical foundation for the research and development of prevention and control strategies for reproductive disorders in animal husbandry.

Autophagy is a highly conserved self-degradation process in eukaryotic cells that maintains intracellular homeostasis by removing damaged organelles， aberrant proteins， and invading pathogens. Under stress conditions， it provides cells with energy and material support， enabling the recycling and renewal of intracellular substances. Autophagy-related proteins （ATGs） are the core executors of this process. As a central protein in autophagy， ATG5 interacts with proteins such as ATG12 and ATG16L1 through two ubiquitin-like domains （UblA and UblB） and one helix-rich domain to form complexes， driving the formation and extension of autophagosomes. During autophagy， ATG5 mediates autophagosome membrane extension by promoting LC3 lipidation. Post-translational modifications of ATG5 （such as phosphorylation， ubiquitination and SUMOylation） further refine its function， affecting autophagosome maturation and cellular localization. ATG5 not only participates in autophagy regulation but also plays a crucial role in non-autophagic pathways such as apoptosis， inflammatory responses， DNA damage repair and metabolic regulation. Additionally， abnormal expression or dysfunction of ATG5 is closely associated with various diseases， including tumors， neurodegenerative diseases， autoimmune diseases and metabolic disorders. This article provides a systematic review of the structural characteristics， biological functions， and disease-related mechanisms of ATG5， aiming to offer insights and references for the treatment of related diseases.

Trans-10，cis-12conjugated linoleic acid （t10，c12-CLA） is a type of isomer of conjugated linoleic acid （CLA）， and it possesses various biological functions， such as anti-obesity， anti-inflammation， and anti-diabetes. The biological functions and signaling pathways of t10， c12-CLA have shown extensive application prospects and significant value in multiple different fields. However， the mechanism of action of t10， c12-CLA has not yet been fully elucidated. The author has comprehensively reviewed the research results of t10， c12-CLA in livestock and poultry， with a particular focus on the effects on key aspects such as oocyte development， function of mammary epithelial cells， muscle growth， regulation of milk fat content， regulation of fat metabolism， immune function， and lipid regulation. Furthermore， the molecular mechanism of t10， c12-CLA was explored， aiming to provide a reference basis for in-depth research on related issues in the livestock industry and to promote the development of the livestock sector.

Meloxicam is a highly selective cyclooxygenase-2 inhibitor and is a commonly used nonsteroidal anti-inflammatory drug in clinical practice. It exerts significant anti-inflammatory and analgesic effects by effectively inhibiting the synthesis of prostaglandins. Meanwhile， meloxicam has a relatively weak inhibitory activity against cyclooxygenase-1， which can significantly reduce the risk of adverse reactions in the gastrointestinal tract and kidneys. These properties establish meloxicam as a safe and efficacious therapeutic cornerstone for pain and inflammation management in canine and feline practice. In recent years， with the continuous growth of pet healthcare demands， the clinical value of meloxicam continues to rise. The authors summarized the physicochemical properties and mechanism of action of meloxicam， then systematically compared the advantages and disadvantages of the commonly used formulations in clinical practice， and further discussed the potential of novel formulations such as oral semi-solid preparations and microneedle patches in improving administration convenience and animal compliance. Subsequently， a detailed analysis was conducted on the research progress of meloxicam’s application in areas such as joint diseases in dogs and cats， adjuvant treatment for tumors， pancreatic and intestinal protection and perioperative analgesia. Finally， the challenges and future research directions of current clinical application of meloxicam were analyzed. The aim of this review was to provide a scientific reference for the safe， rational， and efficient use of meloxicam in the clinical treatment of pet dogs and cats， while also offering new insights for its innovative development and translational research.

Camel milk fat exists in the form of fat globules. The milk fat globule membrane （MFGM） is a three-layer membrane structure wrapped around the exterior of fat globules. This three-layer membrane with biologically active functions is mainly composed of proteins， enzymes， triacylglycerol and polar lipids （cholesterol， sphingolipids， etc.）. MFGM protein has received extensive attention due to its excellent biological activity functions and nutritional value. The author mainly reviewed the composition distribution and core functional characteristics of MFGM protein in camel milk， including its antibacterial， immunomodulatory， emulsifying stabilizing and tumor intervention effects. And from the perspective of proteomics， the differences in the composition and functional characteristics of the main MFGM proteins between camel milk and other milk sources such as cow milk and goat milk were briefly described. The influence of different processing techniques on the MFGM protein in camel milk was analyzed.An in-depth analysis of the functional properties and differential mechanisms of camel milk MFGM proteins will not only help elucidate the nutritional value of camel milk but also provide a theoretical foundation for the development of camel milk MFGM protein-based products.

Objective The aim of this study was to investigate the effects of quercetagetin（QG） supplementation in the diet on the reproductive performance of female rabbits and the growth performance of offspring， as well as to screen optimal dosage and gestational stage of QG supplementation for rabbits. Method In experiment 1， 460 Hyla female rabbits with similar parity（2-3 parity） and body weight were randomly divided into five groups. The rabbits in control group were fed a basal diet， while in the QG100， QG200， QG400， and QG600 groups were respectively fed basal diets supplemented with 100， 200， 400， and 600 mg/kg of QG. The test diets were administered from 6 d before female rabbits mating until 35 d postpartum （weaning of suckling rabbits）. In experiment 2， 276 Hyla female rabbits with similar parity and body weight were randomly divided into three groups， including the control group （basal diet）， the pre-gestation group （0-12 d of gestation）， and the mid-late gestation group （13-30 d of gestation）， and female rabbits in pre-gestation group and the mid-late gestation group were fed with diet supplemented with the appropriate additive dosage selected from experiment 1. Conception rate， farrowing rate， litter size， number of live born kits per Litter， stillbirth size， litter weight， and live litter weight， as well as the litter size and litter weight at 7， 14， 21， and 35 d were recorded and analyzed. Result The results of experiment 1 showed that QG supplementation significantly enhanced the average daily feed intake compared with control group （P<0.05）. Furthermore， 200 and 400 mg/kg QG supplementation markedly improved litter size， total number of kits born alive， number of live born kits per Litter， total litter weight， litter weight born alive， average litter size at 7 d， 14 d， and 21 d， and litter weight of rabbits at 7 d compared with control group （P<0.05）. In addition， 600 mg/kg QG supplementation elevated conception rate and live litter size （P<0.05）. The results of experiment 2 showed that QG supplementation during mid-late gestation significantly improved combined litter size， total litter size at birth，total number of kits born alive， and total litter weight， litter weight at 7 d， and average litter weight of rabbits， as well as litter weight at 14 d compared with the control group （P<0.05）. Moreover， QG supplementation during pre-gestation significantly enhanced litter weight and average litter weight at 7 d （P<0.05）. Conclusion Adding 400 mg/kg QG to the diet significantly improved the reproductive performance of female rabbits and the growth performance of their offspring，and it was better when supplementation occurred during mid-late gestation.

Objective This study aimed to systematically analyze the differences of protein composition and free amino acid profiles in beef between Huaxi cattle and Pingliang Red cattle based on proteomics techniques， aiming to reveal the relationship between proteins composition in beef and meat quality. Method Liquid chromatography-mass spectrometry was employed to separate and identify proteins and free amino acids of beef in Huaxi cattle and Pingliang Red cattle. Principal component analysis （PCA）， differentially expressed protein screening and functional enrichment analysis were conducted to characterize the protein composition in beef from different breeds and analyze the differences of free amino acid contents between Huaxi cattle and Pingliang Red cattle. Result There was significant difference of meat quality trait between Huaxi cattle and Pingliang Red cattle. A total of 190 differentially expressed proteins were identified in beef of both cattle. Specifically， the relative abundances of proteins such as MYBPC2， MYL1 and MYL11 in beef were higher in Huaxi cattle compared with Pingliang Red cattle， whereas the relative abundances of proteins such as MYH3， MYOZ2 and PDLIM3 in Pingliang Red cattle were higher. The differentially expressed proteins were predominantly localized in energy-producing compartments such as mitochondria， ribosomes and filamentous actin， and participate in biological processes such as muscle contraction， actin cytoskeleton organization， and mitochondrial respiratory chain complex Ⅰ assembly. The molecular functions including actin binding， nucleotide binding， aldehyde dehydrogenase （NAD⁺） activity， muscle alpha-actinin binding， etc. which were intimately associated with meat quality formation. Moreover， significant differences were observed in the contents of various free amino acids in beef between Huaxi cattle and Pingliang Red cattle， including glycine， isoleucine， leucine， methionine， phenylalanine， serine， tyrosine， taurine and tryptophan. Conclusion The disparities in the levels of myocyte cytoskeletal proteins， mitochondrial functional proteins， metabolic enzymes and free amino acids in beef were crucial factors influencing the meat quality in Huaxi cattle and Pingliang Red cattle.

Objective This study aimed to investigate the optimal dosage of lipopolysaccharide （LPS） for establishing an inflammatory model in Mahuang chickens via intraperitoneal injection. Method A total of 120 one-day-old male Mahuang chickens （39.11 g ± 3.13 g） were randomly assigned to 4 groups with 6 replicates per group and 5 chickens per replicate. The trial lasted 21 days. The control group （CON） received intraperitoneal injections of normal saline at 15， 17， 19， and 21 days of age， while the treatment groups （LL， LM， and LH） received LPS at 0.25， 0.5， and 1 mg/kg body weight， respectively. Blood collection and slaughter were performed on days 15 and 21 to calculate growth performance and immune organ indices， serum biochemical parameters， corticosterone， inflammatory cytokines， antioxidant indicators， and related gene expression. Result On day 15， compared with CON group， the final body weight and average daily gain of Mahuang chickens in all LPS-treated groups showed no significant changes （P>0.05）. The jejunal villus height （VH） and villus-to-crypt ratio （V/C） of Mahuang chickens in LH group decreased significantly （P<0.05）. The expression of Occludin and nuclear factor-κB （NF-κB） in the jejunum of Mahuang chickens in LM and LH groups was significantly down-regulated （P<0.05）. The serum corticosterone content of Mahuang chickens in LH group increased （P<0.05）. The serum triglyceride （TG） content of Mahuang chickens in LL， LM， and LH groups decreased significantly （P<0.05）. The serum interleukin-6 （IL-6） content of Mahuang chickens in LH group increased significantly （P<0.05）. The serum total superoxide dismutase （T-SOD） activity of Mahuang chickens in LH group decreased significantly （P<0.05）， while the malondialdehyde （MDA） content increased significantly （P<0.05）.On day 21， compared with CON group， the spleen index and thymus index of Mahuang chickens in LM and LH groups showed an increasing trend （P>0.05）. The jejunal crypt depth （CD） of Mahuang chickens in LH group increased extremely significantly （P<0.01）， while the V/C decreased significantly （P<0.05）. The expression of Claudin-1 in the jejunum of Mahuang chickens in LH group was down-regulated extremely significantly （P<0.01）， while the expression of IL-1β， tumor necrosis factor-α （TNF-α）， and nuclear NF-κB was up-regulated extremely significantly （P<0.01）. The T-SOD activity of Mahuang chickens in all LPS-treated groups decreased significantly （P<0.05）. Conclusion LPS injection significantly affected immune organ development， intestinal morphology， inflammatory cytokine expression， and antioxidant function in Mahuang chickens， successfully inducing inflammatory stress. Considering all indicators， 1.0 mg/kg LPS was the optimal dose for establishing an inflammatory model in Mahuang chickens.

Objective To investigate the mechanism by which liver injury in sheep was alleviated and the key pathways of glucose metabolism were regulated through the addition of total alkaloids from Sophora alopecuroides to high-concentrate diets based on transcriptome sequencing. Method Eighteen 3-month-old Dumont male sheep were randomly divided into three groups： G1 （concentrate to roughage ratio was 50∶50）， G2 （concentrate to roughage ratio was 70∶30）， and S3 （concentrate to roughage ratio was 70∶30， added with 121 mg/kg total alkaloids of Sophora alopecuroides）. After a 15-day pre-feeding period and a 60-day experimental period， three sheep were randomly selected from each group for slaughter. Liver tissue was collected for transcriptomic sequencing， with differentially expressed genes （DEGs） identified and subjected to GO functional and KEGG pathway enrichment analyses. The fold changes in gene expression levels for glucose metabolism were further analyzed to evaluate expression differences in key enzyme genes， with four genes selected for validation via Real-time quantitative PCR. Result In the G2 vs G1 group，411 DEGs were identified （233 up-regulated，178 down-regulated）. In the S3 vs G2 group，3 664 DEGs were identified （1 799 up-regulated，1 865 down-regulated）.GO functional enrichment analysis revealed that DEGs in the G2 vs G1 group were primarily enriched in entries such as extracellular matrix and inflammatory and immune responses. Whereas the DEGs in the S3 vs G2 group were mainly enriched in entries related to immune system regulation and positive regulation of biological processes. KEGG pathway enrichment analysis indicated that no pathways related to glucose metabolism were significant enriched in the G2 vs G1 group. Whereas the S3 vs G2 group exhibited significant enrichment of DEGs across six glucose metabolism-related pathways， including glycolysis/gluconeogenesis， pyruvate metabolism， fructose and mannose metabolism， ascorbate and aldarate metabolism， pentose and glucuronate interconversions， and inositol phosphate metabolism. In the glycolysis pathway， the expression levels of HK1， HK2， HK3， and PFKP genes were significantly downregulated， while the expression level of PKLR gene was significantly upregulated （P<0.05）. In the citrate cycle pathway， the expression level of MDH2 gene was significantly increased （P<0.05）. The results of Real-time quantitative PCR validation for PCK1， G6PC1， IDH3B， and SDHC genes were consistent with those of transcriptomic sequencing. Conclusion The total alkaloids of Sophora alopecuroides could alleviate high-concentrate diet induced inflammation and fibrosis in sheep liver by enriching functions related to immune system regulation and positive regulation of biological processes. The expression of key gluconeogenesis enzyme genes was increased， the expression of upstream glycolysis enzyme genes was inhibited， and the expression of downstream glycolysis enzyme genes was promoted， while the liver citrate cycle was enhanced. Through these effects， the glucose metabolism disorders induced by high-concentrate diet were ameliorated， and systemic glucose metabolic homeostasis was maintained.

Objective This study aimed to investigate the effects of Lactobacillus plantarum selenium on the ovarian and oviduct structure， as well as the antioxidant function and inflammatory indices of laying hens，in order to provide a new reference for selenium sources in maintaining the reproductive health of laying hens. Method A total of 96 healthy 22-week-old Hy-Line Brown laying hens were randomly divided into 2 treatment groups， with 16 replicates per group and 3 hens per replicate. Hens in control group （CON） was fed a conventional corn-soybean meal-based diet for laying hens， with a measured selenium content of 0.38 mg/kg. The Lactobacillus plantarum selenium group （SEL） was supplemented with 500 mg/kg of Lactobacillus plantarum selenium product on the basis of the basal diet， and the measured selenium content in the diet was 1.58 mg/kg. The experiment lasted for 188 days. At the end of the experiment， 8 laying hens were randomly selected from each group for slaughter. Oviduct and ovary tissue samples were collected to analyze the effects of Lactobacillus plantarum selenium on the production performance of laying hens. The tissue structure of ovaries and oviducts was observed by HE staining， and the effect of Lactobacillus plantarum selenium on the ovarian hormone levels of laying hens was further examined. Real-time quantitative PCR was performed to determine the relative expression of antioxidant and inflammation-related genes in ovarian and oviduct. Result ①There were no significant differences in the main production performance indicators such as daily feed intake， egg production rate， average daily egg weight， and feed-egg ratio between the two groups of laying hens （P>0.05）. ② Compared with CON group， the ovarian of SEL group was more intact， and the number of large yellow follicles significantly increased （P<0.05）. Moreover， the levels of follicle-stimulating hormone （FSH） and estradiol （E₂） in ovarian were significantly increased （P<0.05）.③ Compared with CON group， the tissue structure and morphology of the magnum of oviduct in SEL group were more complete. The expression of antioxidant-related genes GPX1， GPX4， CAT， TXNRD2， TXNRD3 and SOD1， as well as inflammatory factor-related genes IL-6 and TNFAIP3 in the magnum tissue of oviduct were significantly increased （P<0.05）. Conclusion The supplementation of feed with Lactobacillus plantarum selenium could improve the morphological structure of ovaries and oviduct tissues in laying hens， and enhance the antioxidant function of oviduct tissues. The results of this experiment indicated that Lactobacillus plantarum selenium has significant application value in improving the health of oviducts and enhancing the overall production efficiency of laying hens.

Objective This study aimed to compare the differences in growth performance， slaughter performance and plasma biochemical indicators between meat ducks with high and low feed efficiency， and analyze the correlation among these indicators. Method Seventy-five 1-day-old male Pekin ducks with similar body weights and in good health， which had high and low feed efficiency respectively， were selected and divided into high feed efficiency group （HFE） and low feed efficiency group （LFE）. Five repetitions in each group， with 15 ducks in each repetition. The experimental ducks were fed the same commercial feed， and the experimental period was 35 days. At 14 and 35 days of age， the weight of each cage of ducks and the weight of the remaining feed were measured. The average weight， average daily gain （ADG）， average daily feed intake （ADFI） and feed-to-gain ratio （F/G） of the experimental ducks at each stage were calculated， and the residual feed intake （RFI） was also counted. At 35 days of age， blood was collected from the jugular vein and then the ducks were slaughtered.The breast muscle rate， leg muscle rate， liver index， abdominal fat rate， skin fat rate and total eviscerated rate were statistically analyzed， and the plasma biochemical indicators were also measured. The correlations among the various indicators were further analyzed. Result ① The body weight of meat ducks at 14 and 35 days of age and ADG at 1-14， 15-35， and 1-35 days of age in HFE group were significantly higher than those in LFE group （P<0.05）. The F/G and RFI of meat ducks in HFE group at 1-14， 15-35 and 1-35 days of age were significantly lower than those in LFE group （P<0.05）.② The breast muscle rate and leg muscle rate of meat ducks in HFE group were significantly higher than those in LFE group （P<0.05）， while the abdominal fat rate and skin fat rate were significantly lower than those in LFE group （P<0.05）.③ Compared with LFE group， the contents of triglycerides （TG）， total cholesterol （TC）， high-density lipoprotein （HDL）， low-density lipoprotein （LDL）， uric acid （UA）， direct bilirubin （DBIL）， as well as the activities of aspartate aminotransferase （AST） and lactate dehydrogenase （LDH） in the plasma of meat ducks in HFE group were significantly decreased （P<0.05）， while the activity of γ-glutamyl transpeptidase （GGT） was significantly increased （P<0.05）.④ The F/G of meat duck was significantly negatively correlated with ADG and breast muscle rate （P<0.05）， and significantly positively correlated with skin fat rate， RFI， liver index and plasma HDL and LDL contents （P<0.05）. Conclusion Meat ducks with high feed efficiency had higher daily weight gain， breast muscle rate and leg muscle rate， and possessed stronger protein deposition ability， while meat ducks with low feed efficiency had higher skin fat rate and abdominal fat rate， and had stronger fat deposition ability.

Objective This study aimed to explore the effects of adding 1% oils from different sources （soybean oil， coconut oil， palm oil， and linseed oil） to diets on the laying performance and egg quality of laying hens， thereby providing a scientific basis for the rational selection of oils in laying hen diets. Method A total of 700 32-week-old Hy-line Brown laying hens with similar egg production rates and body weights were selected and randomly divided into 5 groups， with 5 replicates per group and 28 hens per replicate. The hens in control group were fed the basal diet， while in experimental groups were fed the basal diet supplemented with 1% soybean oil， coconut oil， palm oil， and linseed oil， respectively. The experimental period lasted for 28 days. During the experiment， the laying performance of each replicate was evaluated based on statistical data. On the 28th day， 5 eggs were randomly selected from each replicate for egg quality analysis， and the fatty acid composition of egg yolk was determined using a gas chromatograph. Result Compared with control group， dietary supplementation with 1% soybean oil， palm oil， linseed oil， or coconut oil significantly reduced the average daily feed intake， broken egg rate， and feed-to-egg ratio of laying hens （P<0.05）. The addition of coconut oil significantly increased the egg production rate， average egg weight， and eggshell strength compared with control group （P<0.05）. The results of egg fatty acid analysis indicated that supplementation with 1% soybean oil significantly increased the linoleic acid content in eggs （P<0.05）， the content of ω-6 polyunsaturated fatty acids （ω-6 PUFA） in soybean oil group was 37.41%， 36.42%， 30.00%， and 37.98% higher than that in control， palm oil， linseed oil， and coconut oil groups， respectively （P<0.05）. Supplementation with 1% linseed oil significantly increased the contents of linolenic acid （C18∶3）， eicosapentaenoic acid （EPA， C20∶5）， and docosahexaenoic acid （DHA， C22∶6） in eggs （P<0.05）， and the content of ω-3 polyunsaturated fatty acids （ω-3 PUFA） in linseed oil group was 77.82%， 63.53%， 73.31%， and 73.68% higher than that in control， soybean oil， palm oil， and coconut oil groups， respectively （P<0.05）. In addition， dietary supplementation with 1% soybean oil or linseed oil significantly increased the content of unsaturated fatty acids （UFA） in eggs （P<0.05）. Conclusion The addition of 1% vegetable oils of different types could effectively optimize the production performance of laying hens： Coconut oil was the best choice for improving laying performance， soybean oil and linseed oil were ideal sources for enhancing ω-6 PUFA and ω-3 PUFA， respectively， and palm oil was a safe and cost-effective option.

Objective This study aimed to analyze the differences in rumen microbiota composition， function， and hepatic gene expression between Duolang sheep and Hu sheep in the Kashgar region of Xinjiang， revealing mechanisms of environmental adaptation. Method Utilizing metagenomic sequencing to analyze differences in taxonomic composition and functional enrichment of rumen microbiota between the both breeds， and employing transcriptomic sequencing to detect differentially expressed genes in liver for GO function， KEGG pathway， and protein-protein interaction （PPI） network analysis， followed by integrated analysis to investigate associations between rumen microorganisms and hepatic genes. Result Metagenomic analysis results revealed that Duolang sheep exhibited greater microbial gene diversity compared with Hu sheep. At the taxonomic level， the dominant bacterial phyla in both breeds were Bacteroidetes and Firmicutes， yet the abundance of Clostridiales bacterium was significantly higher in Duolang sheep at the species level. Functional annotation results indicated that rumen microbial functions were primarily enriched in metabolic pathways related to carbohydrates and amino acids. Specifically， Duolang sheep showed prominent enrichment in amino acid and fatty acid metabolism pathways， whereas Hu sheep exhibited enrichment in reproduction-related pathways. Transcriptomic analysis of liver identified 498 differentially expressed genes （DEGs） （191 upregulated and 307 downregulated） between the both breeds， including key genes such as ADIPOQ （regulating lipid metabolism）， ATP2A1 （modulating calcium signaling and stress responses）， and MX1 （pathogen defense）. GO functional enrichment analysis results demonstrated that DEGs were primarily involved in biological processes including muscular system processes and glucose import. KEGG pathway enrichment results revealed that DEGs were significantly enriched in eight signaling pathways， including the muscle cell cytoskeleton and AMPK signaling pathway. PPI network analysis results identified that ATP2A1 as a hub gene interacting with multiple proteins. Integrated analysis results revealed a significant linear correlation between the rumen Clostridiales bacterium and MX1 gene in liver （P<0.05）， suggesting potential host-microbiome interactions regulating metabolic and immune pathways. Conclusion Duolang sheep and Hu sheep exhibited significant differences in rumen microbiota composition， functional capacity， and hepatic gene expression. These distinctions， along with the identified associations between rumen microorganisms and liver genes， collectively constitute their mechanisms of environmental adaptation.

Objective This study aimed to investigate the effects of dietary supplementation with pomegranate peel powder （PPP） on the growth performance， intestinal morphology， serum biochemical and antioxidant indices， and cecal microbiota structure of White-feathered broilers. Method A total of 288 one-day-old Arbor Acres （AA） broilers were randomly divided into 4 groups with 3 replicates per group and 24 broilers per replicate. The control group （CON） was fed a basal diet， while groups 1， 2 and 3 received the basal diet supplemented with 0.75%， 1.5% and 3.0% PPP， respectively. The trial lasted for 42 days. Body weight and feed intake were recorded at the beginning and end of the trial to calculate average daily gain （ADG）， average daily feed intake （ADFI）， and feed-to-gain ratio （F/G）. At the end of the experiment， 12 chickens were randomly selected. Blood was collected and serum was sparated. Collect jejunal and ileal tissues as well as cecal contents and determine serum biochemical and antioxidant indices， intestinal morphology， and cecal microbial composition （analyzed via 16S rDNA sequencing） were determined. Result Compared with CON group， the growth performance of broilers in group 1 showed an improving trend， but the difference was not significant （P>0.05）. Compared with CON group， the activity of aspartate aminotransferase （AST） in the serum of broilers in group 1 was significantly decreased （P<0.05）， while the activity of superoxide dismutase （SOD） and total antioxidant capacity （T-AOC） were significantly increased （P<0.05）. Compared with CON group， the villus height （VH） and villus/crypt ratio （V/C） of the jejunum in group 1 broilers were significantly increased （P<0.05）. The results of cecal microbiota sequencing showed that 0.75% PPP could significantly increase the richness and diversity of the microbiota， promote the proliferation of beneficial bacteria such as Bacteroides， Bifidobacterium and Christensenella （P<0.05）， and significantly reduce the abundance of the potential pathogen Alistipes （P<0.05）. Correlation analysis indicated that Bacteroides and Bifidobacterium were positively correlated with serum antioxidant indicators and intestinal morphology parameters. Conclusion Under the present experimental conditions， dietary supplementation with 0.75% PPP could significantly enhance antioxidant capacity， improve intestinal mucosal morphology， and increase the richness of beneficial cecal microbiota in broilers.

Objective This study aimed to investigate the effects of annual seasonal differential feeding on the growth performance， serum biochemistry， and feeding economics of yaks. Method Fifty healthy male yaks with similar body weight （147.49 kg±19.72 kg） were selected and randomly divided into a control group （CON） and an experimental group （SF）， with 25 replicates per group and one yak per replicate. Yaks in CON group grazed year-round on natural pasture. The SF group received different feeding models according to seasonal variations： During the warm season supplementation period （April to October），“rotational grazing+supplementation” was implemented； During the cold season supplementation period （October to January of the following year）， “rotational grazing+supplementation” was used； And during the cold season indoor feeding period （January to April of the following year）， “warm shed+indoor feeding” was applied. The trial lasted 365 days. During the experiment， yak production performance was measured in April， October， January of the following year， and April of the following year. Serum samples were collected in October and April of the following year for serum biochemical analysis， and an economic benefit analysis was conducted. Result The average daily gain （ADG） of yaks in SF group was significantly higher than that of CON group in October and from January to April of the following year （P<0.05）. The annual ADG was significantly increased from 115.56 g in CON group to 430.63 g in SF group （P<0.05）. Serum biochemical indicators： In October， serum albumin （ALB）， globulin （GLB）， and total protein （TP） levels in SF group were significantly higher than those in CON group （P<0.05）. In April of the following year， glucose （GLU）， ALB， and TP concentrations in SF group were significantly higher than those in CON group （P<0.05）. Within the SF group， serum ALB and TP concentrations in October were significantly higher than those in April of the following year （P<0.05）.Regarding lipid metabolites： In October， high-density lipoprotein cholesterol （HDL-C） and very-low-density lipoprotein （VLDL） concentrations in SF group were significantly higher than those in CON group （P<0.05）. In April of the following year， serum low-density lipoprotein cholesterol （LDL-C）， VLDL， non-esterified fatty acid （NEFA）， and β-hydroxybutyrate （BHBA） concentrations in SF group were significantly lower than those in CON group （P<0.05）. In CON group， VLDL， NEFA， and BHBA concentrations in October were significantly lower than those in April of the following year （P<0.05）. Similarly， in SF group， serum VLDL， NEFA， and BHBA concentrations in October were also significantly lower than those in April of the following year （P<0.05）.Among energy metabolism-related hormones： In October， serum insulin （INS） concentration in SF group was significantly higher than that in CON group （P<0.05）. In April of the following year， serum glucagon （GLN） concentration in SF group was significantly lower than that in CON group （P<0.05）. In CON group， serum adiponectin （APN） and GLN concentrations in October were significantly lower than those in April of the following year （P<0.05）， while INS concentration was significantly higher in October than in April of the following year （P<0.05）. In SF group， GLN concentration in October was significantly lower than that in April of the following year， while INS concentration was significantly higher in October than in April of the following year （P<0.05）.In October， serum growth hormone （GH） and insulin-like growth factor Ⅰ （IGF-Ⅰ） concentrations in SF group were significantly higher than those in CON group （P<0.05）. In April of the following year， GH and IGF-Ⅰ concentrations in SF group remained significantly higher than those in CON group （P<0.05）. Within the SF group， serum IGF-Ⅰ concentration in October was significantly higher than that in April of the following year （P<0.05）.The economic benefit analysis showed that the profit per yak in SF group increased by 884.14 yuan compared to CON group. Conclusion In summary， compared with traditional grazing， year-round seasonal differentiated feeding， which supplements nutrient intake based on grazing， significantly improved the weight gain performance of yaks during the warm season， elevated serum levels of glucose and nitrogen metabolites， reduced fat mobilization and weight loss during the cold season， and promoted growth hormone secretion， ultimately enhancing the economic benefits of yak farming.

Objective This experiment aimed to investigate the effects of dietary supplementation with Suaeda salsa extract （SSE） on rumen fermentation parameters and gastrointestinal （ruminal and intestinal） microbial community structure in Hu sheep. Method Thirty-six healthy male Hu sheep lambs with an average body weight of （30.27±1.18） kg were randomly divided into a control group （CON） and an experimental group （SSE）， with 6 replicates per group and 3 lambs per replicate. The sheep in CON group were received a total mixed ration （TMR）， while in SSE group were fed TMR supplemented with 0.6% SSE. The pre-test period lasted for 15 days， followed by a 30-day formal trial period. On the 30th day of the formal trial， rumen fluid and rectal faecal samples were collected from fasted Hu sheep to determine rumen fermentation parameters and ruminal faecal microbial communities. Result ①Compared with CON group， the ruminal total volatile fatty acid （TVFA） content of sheep in SSE group was extremely significantly increased （P<0.01）， and ruminal pH， acetate， propionate， isobutyrate， isovalerate and valerate contents were significantly increased （P<0.05）. ②No significant differences were found in ruminal microbial composition at either the phylum or genus level， nor in Alpha and Beta diversity indices between the two groups （P>0.05）. However， the relative abundance of the genus Enteromonas was significantly higher in SSE group （P<0.05）. Correlation analysis between ruminal microbes and fermentation parameters showed that the relative abundance of Limivicinus was significantly positively correlated with ruminal butyrate concentration （P<0.05）， while the relative abundance of Succiniclasticum was significantly negatively correlated with ruminal butyrate content （P<0.05）.③No significant differences in rectal faecal microbial composition at the phylum level， or in the Alpha and Beta diversity indices was observed between the two groups （P>0.05）. Compared with control group，the relative abundance of Prevotella， Agathobacter， Butyrivibrio_A， Eubacterium_J， and Methylobacterium in the rectum of SSE group were significantly increased （P<0.05）. Conclusion Dietary supplementation with 0.6% SSE to the diet could optimize rumen fermentation parameters， improve the structure and composition of ruminal microbial communities， and increase the genera Agathobacter， Butyricimonas， and Eubacterium in the intestine of Hu sheep.

Objective This study aimed to investigate the effects of different dietary fiber （DF） feeding regimens on slaughter performance， tibia development， gut microbiota， and antibiotic resistance genes （ARGs） in Yellow-feathered broilers. Method A total of 250 one-day-old healthy male Yellow-feathered broilers with similar initial body weight （35 g±5 g） were randomly divided into 5 groups， with 5 replicates per group and 10 broilers per replicate. The control group （CON） was fed a fiber-free basal diet （FF）. Group 1 （T1） received a low-fiber diet （LF， containing 1.5% DF）. Group 2 （T2） was fed a high-fiber diet （HF， containing 3% DF）. Group 3 （T3） was fed FF during weeks 0-4 and LF during weeks 5-8. Group 4 （T4） received LF during weeks 0-4 and HF during weeks 5-8. The experiment lasted for 56 days. At the end of the trial， blood was collected from the wing vein to determine serum calcium and phosphorus levels. Slaughter performance and tibia traits were measured， along with tibia ash， calcium， and phosphorus content. Fresh fecal samples were collected from the CON and T2 groups for metagenomic analysis. Result ①Slaughter performance： No significant differences were observed among groups in dressing percentage， semi-eviscerated yield， eviscerated yield， breast muscle yield， or leg muscle yield （P>0.05）. ②Tibia traits： There were no significant differences in tibia length， tibia weight， medullary cavity diameter， volume， or density among the groups （P>0.05）. However， compared with group T1， tibia diameter was significantly greater in groups T2 and T3 （P<0.05）， and cortical bone thickness in group T2 was significantly higher than in groups T3 and T4 （P<0.05）. No significant differences were found in tibia ash， calcium， or phosphorus content between the experimental groups and CON group （P>0.05）. ③Serum parameters： Serum calcium in groups T1， T3 and T4 was significantly higher than in CON group （P<0.05）， while serum phosphorus was significantly lower in all trial groups （P<0.05）.④Metagenomic findings： The relative abundances of fecal microbiota including Parabacteroides， Bacteroides， Clostridium， Faecalibacterium， Streptococcus， Enterococcus， Corynebacterium and Rothia in group T2 were significantly higher than in CON group （P<0.05）. Meanwhile， the relative abundances of ARGs poxtA and vatE were significantly lower in group T2 （P<0.05）. Conclusion Under the conditions of this study， the inclusion of 3% DF throughout the feeding period could improve tibia traits and gut microbiota composition， and reduce the abundance of specific antibiotic resistance genes in the feces of Yellow-feathered broilers.

Objective This study aimed to investigate the effects of stevia extract， honeysuckle （Lonicera japonica） extract， Astragalus （Astragalus membranaceus） extract， grape seed proanthocyanidin extract， licorice （Glycyrrhiza uralensis） extract， and capsaicin extract on immune function in cyclophosphamide-induced immunosuppressed mice， so as to provide a scientific basis for the application of plant extracts in the prevention of immunosuppressive diseases. Method A total of 64 male Kunming mice （6 weeks old， SPF grade， body weight 20 g±2 g） were randomly divided into eight groups （8 mice per group）： Control， model （cyclophosphamide） and six plant extract-treated groups. The experiment lasted 31 days. From days 1 to 28， mice in control and model groups were given 0.2 mL of normal saline daily by gavage， while mice in treatment groups received the corresponding plant extracts at doses of 500， 180， 400， 200， 250 and 15 mg/（kg·d）， respectively. On days 29 to 31， mice in control group was intraperitoneally injected with 0.2 mL/d of normal saline， while mice in cyclophosphamide and plant extract groups received cyclophosphamide at 80 mg/（kg·d）. Body weights were recorded on days 0， 28 and 32. On day 32， liver and spleen samples were collected for organ index calculation and histopathological examination. Serum levels of immunoglobulins （IgA， IgG and IgM） and cytokines （TNF-α， IL-6 and IL-10） were measured. Result ①On days 32， the final body weight of mice in cyclophosphamide group was significantly lower than that of control group （P<0.05）， while mice in honeysuckle extract group showed a significant increase compared with cyclophosphamide group （P<0.05）. ②The liver index did not differ significantly between cyclophosphamide and control groups （P>0.05）， but hepatocellular vacuolation was observed in cyclophosphamide group. All plant extracts alleviated the vacuolation， indicating a protective effect. ③The spleen index in cyclophosphamide group was 38.71% lower than that in control group， with structural damage such as blurred red-white pulp boundaries and loss of central arteries. Most plant extracts ameliorated splenic injury， with honeysuckle extract showing the strongest protective effect. ④Serum immunoglobulin and cytokine contents were generally decreased in cyclophosphamide group （P>0.05）. Honeysuckle， Astragalus， and grape seed proanthocyanidin extracts significantly increased IgA contents （P<0.05）， while capsaicin extract significantly elevated IgG （P<0.05）. All extracts except stevia significantly increased TNF-α contents， and honeysuckle extract also significantly enhanced IL-6 and IL-10 contents （P<0.05）. Conclusion Under the experimental conditions， all plant extracts provided varying degrees of protection against cyclophosphamide-induced immunosuppression. Honeysuckle extract exhibited the strongest effect， followed by Astragalus， grape seed proanthocyanidins， capsaicin， licorice， and stevia extracts， indicating their potential application in preventing immunosuppressive diseases.

Objective This study aimed to investigate the effects of dietary supplementation with rare earth-chitosan chelate （RECC） on growth performance， nutrient digestibility， serum parameters， and fecal microbiota in growing-finishing pigs. Method Eighty healthy “Landrace×Rongchang” crossbred pigs with similar body weight （56.17 kg±0.05 kg） were randomly divided into 2 groups with 5 replicates per group and 8 pigs per replicate （half male and half female）. The pigs in control group were received a basal diet， while in the experimental group were fed the basal diet supplemented with 200 mg/kg RECC. The trial period included a 3‑day adaptation period and a 34‑day experimental period. Pigs were weighted on an empty stomach at the beginning and end of the experiment， and daily feed intake was recorded. On the final day， feed， fecal， and blood samples were collected to determine growth performance， nutrient digestibility， serum biochemical indices， antioxidant capacity， hormone levels， and fecal microbial composition. Result Compared with control group， ①The final body weight and average daily gain of growing-finishing pigs in experimental group showed a tendency to increase， and the feed-to-gain ratio showed a tendency to reduce （0.05≤P<0.10）； ②Apparent digestibility of crude protein of growing-finishing pigs in experimental group was extremely significantly increased （P<0.01）， while apparent digestibility of total phosphorus was tended to increase （0.05≤P<0.10）； ③Serum biochemical indices of growing-finishing pigs in experimental group had no significant effects （P>0.05）， but the serum total antioxidant capacity （T-AOC） and glutathione peroxidase （GSH-Px） activity were significantly increased （P<0.05）， and the content of malondialdehyde（MDA）was significantly reduced （P<0.05）； ④The levels of serum insulin-like growth factor-1 （IGF-1） and growth hormone（GH） of growing-finishing pigs in experimental group were extremely significantly increased （P<0.01）， and the level of tetriodothyronine （T₄） was significantly increased （P<0.05）； ⑤Shannon index of fecal microbiota of growing-finishing pigs in experimental group was tended to increase （0.05≤P<0.10）， Beta diversity showed obvious spatial separation in two treatment groups， and the relative abundance of Spirochaetota at the phylum level was significantly reduced （P<0.05）. Conclusion Under the conditions of this study， dietary supplementation with 200 mg/kg RECC improved nutrient digestibility， enhanced serum antioxidant capacity and hormone levels， and modulated the diversity and composition of gut microbiota， thereby promoting the growth and development of growing-finishing pigs. These findings provide a theoretical basis and practical reference for the application of RECC in pig production.

Objective This study aimed to investigate the effects of green tea aqueous extract on slaughter performance， meat quality， and serum biochemical indices of Cyan-shank partridge chickens. Method A total of 400 one-day-old healthy Cyan-shank partridge chicks with similar body weight （40 g±5 g） were randomly divided into four groups， with five replicates per group and 20 chicks per replicate. All chicks were fed the same basal diet. Chicks in control group received cooled boiled water at room temperature， while chicks in experimental groups Ⅰ， Ⅱ and Ⅲ were provided with 0.3%， 0.6% and 1.2% green tea aqueous extract for free intake， and the experiment period was 42 days. At the end of the experiment， two chicks from each replicate were randomly selected for blood collection from the wing vein， followed by slaughter. Bilateral breast and thigh muscles were collected to determine slaughter performance and meat quality. Result Regarding slaughter performance， compared with control group， the Cyan-shank partridge chickens in all green tea aqueous extract-treated groups showed a significant increase in slaughter rate and semi-eviscerated yield （P<0.05）. Chickens in groups Ⅰ and Ⅱ also exhibited a significantly higher eviscerated yield （P<0.05）. For meat quality parameters， shear force of the breast muscle was significantly decreased in all treatment groups （P<0.05）. The lightness （L^*） value of breast muscle was significantly reduced in groups Ⅱ and Ⅲ （P<0.05）. Furthermore， the Cyan-shank partridge chickens in group Ⅱ showed a significant increase in thigh muscle crude fat content （P<0.05）， while the redness （a^*） value of thigh muscle in group Ⅰ was significantly increased （P<0.05）. Analysis of serum biochemical indices revealed that chickens in group Ⅱ had significantly higher serum levels of total protein （TP）， albumin （ALB）， and high-density lipoprotein cholesterol （HDL-C） （P<0.05）， along with significantly lower alkaline phosphatase （ALP） activity （P<0.05）. In groups Ⅰ and Ⅲ， chickens exhibited significantly decreased serum TP and globulin （GLB） contents （P<0.05）， but an increased ALB content （P<0.05）. Additionally， chickens in group Ⅲ showed significantly reduced serum triglyceride （TG） content and ALP activity （P<0.05）. Serum urea nitrogen （BUN） content in group Ⅱ was significantly lower than that in groups Ⅰ and Ⅲ （P<0.05）. Conclusion Dietary supplementation with green tea aqueous extract improved the slaughter performance and meat quality of Cyan-shank partridge chickens， enhanced fat deposition in thigh muscle， and positively regulated protein and lipid metabolism. The most effective concentration was 0.6%.

Objective The aim of this study was to investigate the persistent effects of yeast culture （YC） supplementation during mid-to-late gestation on the productive performance， postpartum serum biochemical indices and rumen fermentation parameters of primiparous ewes. Method Thirty-six healthy Hu sheep weighing （37.72±1.91） kg on the 51st day of gestation were selected for the experiment， and were randomly divided into 2 groups according to the principle of similarity in body weight， with 6 replicates in each group and 3 sheep in each replicate. After 9 days of pre-feeding， the experimental feeding was conducted from gestation day 60 to 150. The ewes in control group （CK group） were fed a total mixed ration， while in experimental group （YC group） were fed the same ration supplemented with 5% YC. After lambing， both groups were fed the same diet as the CK group. On postpartum day 20， blood and rumen fluid samples were collected from the ewes for determination of serum biochemical， immune， and antioxidant indices， as well as rumen fermentation parameters. The production performance of the ewes and lambs was also measured to assess the sustained effects of YC. Result Compared with CK group， ① The birth weight of lambs in YC group was significantly increased （P<0.05）， and the weight of lambs on day 20 was also tended to increase （0.05<P<0.10）； ② The serum cortisol （COR） content of ewes in YC group was extremely significantly increased （P<0.01）， the total protein （TP） content was significantly increased （P<0.05）， and the albumin （ALB） content tended to increase （0.05<P<0.10）； ③ The serum interleukin-2 （IL-2） and tumor necrosis factor-α （TNF-α） contents of ewes in YC group were extremely significantly increased （P<0.01）， and the immunoglobulin G （IgG） content was significantly increased （P<0.05）； ④ In the serum of ewes in YC group， the activity of glutathione peroxidase （GSH-Px） was extremely significantly increased （P<0.01）， the total antioxidant capacity （T-AOC） was significantly increased （P<0.05）， the activity of superoxide dismutase （SOD） showed an increasing trend （0.05<P<0.10）， and the content of malondialdehyde （MDA） showed a decreasing trend （0.05<P<0.10）； ⑤ In the rumen fluid of YC group， the content of microbial protein （MCP） and the concentration of propionic acid showed an increasing trend （0.05<P<0.10）. Conclusion Adding YC in the middle and late stages of pregnancy could improve the production performance of primiparous ewes and improve their immune function and antioxidant capacity.

Objective This study aimed to precisely edit myostatin （MSTN） gene in Huaxi cattle to enhance meat production performance， and establish an efficient gene editing technology system to provide a theoretical basis and technical support for the biotechnological breeding of beef cattle in China. Method Six sgRNAs targeting exons 1 and 2 of MSTN gene were designed using CRISPR/Cas9 system. Cas9 knockout vector was constructed and transfected into Huaxi cattle fetal fibroblast cells via electroporation. Fluorescence-activated cell sorting （FACS） was employed to screen monoclonal cell lines with mutations， and the editing outcomes were validated by Sanger sequencing. Western blotting was used to detect MSTN protein expression in both wild-type and edited Huaxi cattle fetal fibroblast cells to evaluate the gene editing efficiency. Potential off-target sites were predicted using the Cas-OFFinder software and verified by sequencing in the monoclonal cell lines. Additionally， in vitro fertilization （IVF） zygotes were utilized to deliver Cas9 ribonucleoprotein （RNP） complexes under different electroporation parameters. Cleavage and blastocyst formation rates were assessed， and the editing efficiency in embryos was verified by nested PCR and sequencing. Result Six types of Cas9 knockout vectors were successfully constructed， with transfection efficiency consistently ranging from 12.7% to 18.6%. A total of 35 monoclonal cell lines with MSTN gene knockout were obtained through FACS. Among these， the E2-3 single-vector and E2（2+3） dual-vector strategies achieved the highest editing efficiencies， reaching 57.9% and 55.6%， respectively. Western blotting results showed that MSTN protein expression in the edited groups were extrenely significantly reduced compared with wild-type group （P<0.01）. No significant off-target effects were detected， confirming the high specificity of the editing system. In the embryonic editing experiments， the optimized electroporation parameters （25 V， 6 pulses， and 2.5 ms） resulted in a blastocyst formation rate of 18.7%， which was significantly higher than that of other parameter-treated groups （P<0.05）. Conclusion The experiment successfully established a gene editing technology system for MSTN gene in Huaxi cattle， achieving highly efficient gene knockout and embryonic editing， thereby providing a reliable technical solution for the genetic improvement of Huaxi cattle. The optimized electroporation parameters and dual-vector strategy enhanced editing efficiency， with no detectable off-target effects， laying an important foundation for the further development of biotechnological breeding in beef cattle.

Objective This study aimed to explore the codon usage bias patterns and influencing factors of tyrosinase （TYR） gene in Wumeng Black-bone chickens during the evolution process. The relationship between phylogenetic evolution and codon usage bias among different species were analyzed， so as to provide reference for efficient heterologous genetic transformation of TYR gene in Black-bone chickens. Method Using the CDS of TYR gene in Wumeng Black-bone chickens and 22 other species as materials， and CodonW 1.4.4， CUSP， CHIPS and EMBOSS software were used to analyze the codon usage bias patterns of TYR gene. The possible factors causing codon preference were analyzed by Neutrality-plot and PR2-plot. The codon usage bias clustering heat map of TYR gene were constructed based on relative synonymous codon usage （RSCU） value， and phylogenetic relationship of TYR gene CDS among different species were analyzed by ClustalX 1.83 and Mega 7.0 software. The codon usage frequency of TYR gene in Wumeng Black-bone chickens was compared with that of genomic codons in model organisms such as Mus musculus， Danio rerio and Escherichia coli. Result There were 27 preferred codons （RSCU>1.0） of TYR gene in Wumeng Black-bone chickens， and ATC， CTG， AGA， TCT， TCA， and ACT were dominant codons （RSCU>1.6）. The GC_3s， GC， ENC and CBI values of TYR gene were 50.38%， 47.30%， 54.57， and 0.025， respectively， indicating weak preference. The content of GC_3s and GC in Cyprinidae were all higher than 55%， which indicated that the third base prefered to use G/C. Neutrality-plot and PR2-plot analysis results showed that natural selection pressure was the main factor in the formation of codon usage preference of TYR gene in different species. The clustering heat map and phylogenetic evolution results based on RSCU value and CDS were not completely consistent， but both clustering results suggested that the CDS codon usage preferences of TYR gene in Galliformes， such as Wumeng Black-bone chickens， Gallusgallus， and Coturnix japonica， were similar. Compared with Mus musculus and Escherichia coli， Danio rerio was more suitable as the heterologous expression system for TYR gene in Wumeng Black-bone chickens. Conclusion There were differences in codon usage bias patterns of TYR gene in different species， and natural selection was the main influencing factor in the formation of these preferences. Danio rerio might be the genetic transformation receptor for functional analysis of TYR gene in Wumeng Black-bone chickens.

Objective Early onset muscle weakness syndrome （MW） is a recently discovered genetic defect in Holstein cattle. The sick calves exhibit symptoms such as lying down motionless after birth and atrophy of the hind limb muscles. Its genetic mechanism is related to a single-base mutation in the gene CACNA1S， which encodes the α1S subunit of the L-type calcium channel protein. This study aimed to establish a molecular detection method for this genetic defect and explore its spread within the Chinese Holstein cattle population. Method Based on the specific DNA sequence of MW pathogenic site， the primers were designed for amplification refractory mutation system PCR （ARMS-PCR）. A genetic defect gene screening was conducted on 317 samples of Holstein bull frozen semen and hair follicles， and the results of ARMS-PCR were verified through Sanger sequencing. Pedigree data was used to trace the source of the MW mutation. Result ARMS-PCR detected 21 carriers of MW mutation with a carrier rate of 6.62% （21/317）， and no pure heterozygotes for the defective gene were found， suggesting that the mutation was recessive and purely lethal. Sanger sequencing showed the consistent genotypes with those obtained by ARMS-PCR， demonstrating the high accuracy of this technique. Furthermore， pedigree analysis traced the origin of MW carriers to the Holstein bull Southwind Bell of Bar-Lee， born in 1984. Conclusion The MW genetic defect had already spread within Chinese Holstein cattle populations and was present at a relatively high frequency. It was recommended that ranchers should carry out genetic testing and risk assessment of MW genetic defects as early as possible， and take scientific selection and mating measures to effectively reduce the economic losses caused by genetic defects.

Objective This study was aimed to investigate the correlation between phosphotriesterase related gene （PTER） and calbindin 2 （CALB2） gene polymorphisms and growth traits in American Yorkshire pigs， aiming provide new marker resources for molecular breeding of growth traits in this breed. Methods A total of 385 American Yorkshire pigs reaching 100 kg body weight were selected. Backfat thickness was measured， and ear tissue samples were collected for DNA extraction. Primers were designed based on the reference porcine genome sequence （Sscrofa 11.1）. The single nucleotide polymorphism （SNP） of PTER and CALB2 genes was identified through direct sequencing of PCR products. Genetic diversity analysis of SNP was performed using POPGENE software， and correlations with growth traits in American Yorkshire pigs were analyzed using SPSS 23.0 software. Result Polymorphism detection results revealed that there were 3 genotypes （GG， GC， and CC） at g.45384562 G>C of PTER gene in American Yorkshire pigs， the dominant genotype was GG （0.5403）， with a dominant allele was G（0.7468）. There were 3 genotypes （GG， GA， and AA） at g.14391325 G>Aof CALB2 gene， with GA as the dominant allele （0.4545） and A as the dominant allele frequency （0.5545）. Population genetic analysis results indicated that the polymorphic information content （PIC） of 2 SNPs of PTER and CALB2 genes was 0.31 and 0.37， respectively， representing moderate polymorphism （0.25<PIC<0.5）. Phenotype association analysis results revealed that g.45384562 G>C of PTER gene had no significant effect on the corrected days to 100 kg in American Yorkshire pigs （P>0.05）. However， the corrected live backfat thickness at 100 kg of GG genotype was significantly higher than that of GC and CC genotypes （P<0.05）. g.14391325 G>A of CALB2 gene was extremely significantly associated with both the corrected days to 100 kg and corrected live backfat thickness at 100 kg in American Yorkshire pigs （P<0.01）. Combined effect analysis indicated that CCAA genotype （combining PTER gene CC genotype and CALB2 gene AA genotype） represented an optimal genotype combination associated with shorter corrected days to 100 kg and reduced corrected live backfat thickness at 100 kg， with corresponding values for both traits being lower than those in other individuals. Conclusion This study identified 2 SNPs of PTER and CALB2 genes and confirmed their significant association with growth traits in American Yorkshire pigs. The results suggested that 2 SNPs （particularly the CCAA combined genotype） hold potential for genetic improvement in American Yorkshire pigs.

Objective L-alanine （L-Ala） is a multifunctional additive that functions both as a sweetener and a pH buffer. This study aimed to investigate the effect of L-Ala on the effect of boar semen storage at room temperature and provide a theoretical basis for optimizing the formulation of semen diluents. Method Semen was collected from three healthy adult Duroc boars aged 1 to 2 years. Various concentrations （0， 20， 40， 80， 160， and 320 μg/mL） of L-Ala were added to Modena diluent. Sperm motility and motion parameters （straight-line （rectilinear） velocity （VSL）， curvilinear velocity （VCL）， average path velocity （VAP）， and amplitude of lateral head （ALH）） were assessed at 17 ℃ on days 1， 3， 5， and 7 of storage. Samples showing significant changes were further analyzed for acrosome integrity， plasma membrane integrity， and oxidative stress indices. Result The effects of L-Ala on boar semen storage at room temperature were concentration- and time-dependent. During the first five days of storage， sperm motility parameters showed no significant differences in different concentration groups （0-320 μg/mL） （P>0.05）. By days 7， 320 μg/mL L-Ala group exhibited the best performance， among which sperm motility increased by 9.73% （P<0.05）， motility parameters （VSL， VAP， and ALH） increased by 11.33%， 13.08%， and 12.17% （P<0.05）， and acrosome integrity rate and plasma membrane integrity rate increased by 43.71% and 26.54% （P<0.05）， respectively， the total antioxidant capacity （T-AOC） increased by 1.14-fold （P<0.05）， while malondialdehyde （MDA） content decreased by 54.91% （P<0.05）. Conclusion Under the conditions of this experiment， L-Ala could effectively prolong the storage time of boar semen at room temperature， with 320 μg/mL being the optimal concentration. It significantly maintained sperm quality in the late storage period （7 days）， the mechanism of action might be related to enhancing antioxidant capacity and reducing lipid peroxidation damage.

Objective This study aimed to screen single nucleotide polymorphism （SNP） of adipose differentiation-related protein （ADRP） and fatty acid synthase （FASN） genes in Xuanhe pigs， and reveal the association between SNP and meat quality traits in Xuanhe pigs. Method The meat quality traits in 120 Xuanhe pigs， including meat color， marbling， pH₁， drip loss， and intramuscular fat （IMF） content， were systematically measured. Furthermore， PCR amplification and Sanger sequencing were performed on ADRP and FASN genes， and the association between different genotypes at each SNP and meat quality traits in Xuanhe pigs was analyzed using a least squares model. Result Three SNPs were identified in exon 8 of ADRP gene， among which rs325276230 was synonymous mutation， and rs321564137 and rs81217193 were missense mutation， with dominant genotypes AA， AA， and GG， and dominant alleles A， A， and G， respectively. Three SNPs were in Hardy-Weinberg equilibrium （P>0.05）. Six SNPs were detected in FASN gene， including 4 synonymous mutation sites （rs337531100， rs324640280， rs81478148， and rs319161464） and 2 missense mutation sites （rs337267671 and rs342967881）. The dominant genotypes were TC， CC， TT， GG， GG， and CT， corresponding to dominant alleles C， C， T， G， G， and T， respectively. rs81478148 was deviated from Hardy-Weinberg equilibrium （P<0.01）. The association analysis results demonstrated that SNP of ADRP and FASN genes were significantly associated with meat traits in Xuanhe pigs. The IMF content， marbling， and pH₁ of AA genotype at rs325276230 of ADRP gene were extremely significantly or significantly higher than those of GG genotype （P<0.01 or P<0.05）. The IMF content of AA genotype at rs321564137 was significantly higher than that of AG genotype， and the meat color of AA genotype was significantly lower than of AG genotype （P<0.05）. The IMF content of GG genotype at rs81217193 was significantly higher than that of GA and AA genotypes （P<0.05）. The IMF content of CC genotype at rs337531100 of FASN gene was significantly higher than that of TT and TC genotypes （P<0.05）. The meat color of TT genotype at rs324640280 was significantly higher than of CT genotype （P<0.05）. The meat color， pH₁， and drip loss of CT genotype at rs81478148 were extremely significantly or significantly higher than those of TT genotype （P<0.01 or P<0.05）. pH₁ of AA genotype at rs319161464 was significantly lower than that of GG and GA genotypes （P<0.05）. The marbling of GG genotype at rs337267671 was significantly higher than that of GA and AA genotypes （P<0.05）. The IMF content and marbling of TT genotype at rs342967881 were significantly higher than those of CC genotype （P<0.05）. Conclusion SNP of ADRP and FASN genes were significantly associated with meat quality traits in Xuanhe pigs， among which rs325276230， rs321564137， and rs81217193 of ADRP gene， and rs337531100 and rs342967881 of FASN gene could be used as potential molecular markers to improve the IMF content of Xuanhe pigs.

Objective In order to provide a basis for further optimizing the estrus synchronization strategy of Guanling cattle and improving the reproductive efficiency of large-scale cattle farms，this experiment was designed to investigate the effects of treatment methods， treatment months， fat conditions and reproductive status on the estrus synchronization of Guanling cattle. Method 150 healthy female Guanling cattle over 2 years old were selected and randomly divided into 5 groups， 30 cattle in each group. The following experiments were carried out， and artificial insemination was carried out after estrus identification. Experiment 1： 30 multiparous Guanling cattle with moderate to upper fatness were selected and treated with CIDR+ PGF_2α （group 1） and twice PGF_2α injection （group 2） in July to compare the effect of two treatment methods on estrus synchronization. The following experiments were carried out by twice PGF_2α injection. Experiment 2： 30 multiparous Guanling cattle with moderate to upper fatness were treated in March （group 3）. Experiment 3： 30 multiparous Guanling cattle with lean fatness were treated in July （group 4）. Experiment 4： In July， 30 reserve Guanling cattle with moderate to upper fatness were treated （group 5）. The estrus synchronization rates and non-return rates obtained from groups 3-5 were compared with the data of group 2 to explore the effect of Guanling cattle in different treatment months， fat conditions and reproductive status on estrus synchronization effects. Result There were no significant difference in estrus rate （93.33% and 90.00%） and non-return rate （89.29% and 88.89%） of Guanling cattle between groups 1 and 2 （P>0.05）. The estrus rate （66.67% and 36.67%） and non-return rate （75.00% and 54.55%） of Guanling cattle in groups 3 and 4 were significantly or extremely significantly lower than that in group 2 （P<0.05 or P<0.01）. There were no significant difference in estrus rate （83.33%） and non-return rate （88.00%） of Guanling cattle between groups 5 and 2 （P>0.05）. Conclusion The estrus synchronization treatment method and reproductive status had no significant effect on the estrus synchronization effect of Guanling cattle. The treatment month and fat condition could affect the estrus synchronization effect of Guanling cattle. It was appropriate to select the healthy cows with moderate to upper fatness， and the estrus synchronization treatment should be carried out in the warm season with abundant forage to obtain a relatively better synchronization effect.

Objective This study was conducted to identify the pathogens responsible for bovine mortality， aiming to provide a scientific reference for the prevention and control of diseases caused by co-infections with multiple pathogens in dairy cow. Method In this study， pathogenic bacteria were isolated and cultured from milk and blood samples of dead dairy cow using the plate streaking method. The isolates were subjected to species identification through Gram staining， 16S rRNA sequencing analysis， and virulence gene characterization. Antimicrobial susceptibility profiles were determined by the disk diffusion method， while pathogenicity was evaluated via animal challenge experiments. Result The bacterial isolate in the milk sample presented moist pink colonies of Gram-negative short bacilli on the McConkey medium. The results of 16S rRNA sequence analysis showed that the sequence similarity of the isolated bacteria to the representative strain of Klebsiella in GenBank was >99%， and it was clustered in the same branch as the standard strain of Klebsiella pneumoniae （K. pneumoniae） ATCC 13883. It was identified as K. pneumoniae and named BJ-B004. The results of virulence gene testing indicated that the isolate contained seven virulence genes， including fimH， uge， wabG， ureA， entB， mrkD and ycf. The results of the drug sensitivity test showed that it was resistant to streptomycin， penicillin， amoxicillin， ampicillin， and cotrimoxazole， but was sensitive to antimicrobials such as enrofloxacin， doxycycline， florfenicol， tetracycline， spectinomycin， and cefoperazone. A small Gram-negative bacillus with hemolytic properties and rounded ends was isolated from the blood sample of the dead dairy cow. It was mostly arranged singly or in pairs and showed two-level concentrated staining by Wright-Giemsa staining. The results of 16S rRNA sequence analysis showed that its sequence similarity with the Mannheimia strain was >98%， and it was clustered in the same branch as the Mannheimia strain ZY190616 of bovine origin. It was identified as Mannheimia hemolyticus （M. hemolyticus）and named BJ-B007. The results of the drug sensitivity test indicated that it was not resistant to any antibacterial drugs except gentamicin. BALB/c mice were respectively infected with K. pneumoniae， M. hemolyticus and the mixed bacterial solution at dose of 3×10⁷ CFU， with a PBS blank control group （n=5 per group）， and the results showed that all the mice in the challenge group presented symptoms of the disease. All died within 48 h in mixed infection group， 4 died in M. hemolyticus group and 1 died in K.pneumoniae group. Pathological examination revealed that M. hemolyticus caused severe congestion， hemorrhage and necrotic lesions in multiple organs such as the liver， spleen， lung and kidney. Conclusion This study successfully isolated a multidrug-resistant K. pneumoniae strain and a highly pathogenic M. haemolytica strain from pathological samples of a dead dairy cow. The mortality was determined to be primarily caused by K. pneumoniae-induced mastitis， followed by secondary bacteremia due to opportunistic infection with M. haemolytica. The results provided a scientific foundation for the integrated diagnostic strategies and comprehensive prevention protocols targeting bovine mastitis-respiratory syndrome co-infections.

Objective The purpose of this study was to construct infectious clone of Infectious bursal disease virus （IBDV） BJQ902 strain using reverse genetics technology， and provide important tools for vaccine development and the research of viral replication and pathogenic mechanisms. Method Based on the whole genome sequence of BJQ902 strain， PCR was used to amplify A and B fragments of IBDV BJQ902 strain’s genome. The RiboJ and HDVrz sequences were introduced at both ends of A and B fragments， respectively. Homologous recombination technology was employed to insert A and B fragments with ribozyme structures into vector pCAGGS， constructing the recombinant plasmids pCAGGS-BJQ-RAH and pCAGGS-BJQ-RBH. These vectors were co-transfected into DF-1 cells， and rBJQ902 strain was rescued after three consecutive passages in blind transmission. DF-1 cells were infected with both BJQ902 and rBJQ902 strains， and rBJQ902 strain was identified using PCR， Western blotting and indirect immunofluorescence assay （IFA）. Result The BJQ902 strain genome A and B fragments were successfully obtained， with sizes of 3 260 and 2 827 bp， respectively. The self-splicing ribozymes RiboJ and HDVrz sequences were ligated to the ends of fragment A and B， with sizes of 3 436 and 3 033 bp， respectively. The ribozyme-containing fragments A and B were then ligated to the pCAGGS vector， resulting in the successful acquisition of the recombinant plasmids pCAGGS-BJQ-RAH and pCAGGS-BJQ-RBH. rBJQ902 strain was obtained by blind passage for three consecutive generations in DF-1 cells. Compared with the parent strain BJQ902， rBJQ902 strain could also amplify the VP2 fragment after infecting DF-1 cells for 48 h. The lesions caused by rBJQ902 strain were consistent with those of the parent strain. Both Western blotting and IFA could detect the expression of VP2 protein in rBJQ902 strain， demonstrating that this strain exhibited similar biological characteristics to the parent virus during cell culture. Conclusion This study successfully obtained the infectious clone strain rBJQ902 of BJQ902 strain， providing technical support for in-depth research on the biological characteristics and pathogenic mechanism of IBDV and laying a foundation for subsequent virological research.

Objective This experiment aimed to investigate the impact of Porcine deltacoronavirus （PDCoV） infection on the innate immunity and the interference mechanism of immune signal transduction， with a focus on the roles of signal transducer and activator of transcription 1 （STAT1） and tripartite-motif protein 21 （TRIM21） in this process. Method Morphological changes in IPEC-J2 cells infected with PDCoV at a multiplicity of infection （MOI） of 1 were observed via an inverted microscope at 1， 12， 24 and 48 hours post-infection.Meanwhile， Real-time quantitative PCR and Western blotting were used to detect the expression of PDCoV-N gene and protein， respectively， for screening the optimal conditions of the PDCoV-infected IPEC-J2 cell model. At the peak of PDCoV infection， the mRNA and protein expression of STAT1， TRIM21， and interferon-stimulated genes （ISGs） were detected by Real-time quantitative PCR and Western blotting，respectively. The effects of overexpression or inhibition of STAT1 on the replication of PDCoV and the activity of the interferon-stimulated response element （ISRE） promoter were detected using the dual-luciferase reporter gene system. Additionally， eukaryotic expression vectors of TRIM21 and STAT1 were co-transfected into IPEC-J2 cells. Laser confocal microscopy， co-immunoprecipitation （Co-IP）， and Western blotting were used to verify the interaction between TRIM21 and STAT1 as well as the regulatory effect of TRIM21 on STAT1. Result PDCoV reached replication peak at 24 hour post-infection in IPEC-J2 cells. At the same time， the protein expression of STAT1 and TRIM21， as well as the transcription level of ISGs in experiment group， were extremely significantly higher than those in control group （P<0.01）. Compared with control group， the activity of ISRE promoter was extremely significantly enhanced after overexpression of STAT1， and the expression of PDCoV-N gene and its protein were extremely significantly reduced （P<0.01） ， while inhibition of STAT1 by fludarabine extremely significantly promoted viral replication （P<0.01）. Laser confocal microscopy and Co-IP confirmed the co-localization and interaction between STAT1 and TRIM21. Moreover， compared with control group， the expression of STAT1 protein was extremely significantly reduced after overexpression of TRIM21 （P<0.01）. Conclusion This study illuminated the intricate interplay between PDCoV and the innate immune response of host IPEC-J2 cells. As a key antiviral factor， STAT1 enhanced innate immunity via the ISRE pathway to inhibit viral replication. In contrast， TRIM21 participated in viral immune evasion by interacting with STAT1 and downregulating its expression. These findings provided novel insights into the molecular pathogenesis of PDCoV， and laid a foundation for formulating PDCoV prevention and control strategies.

Objective This study was conducted to understand the epidemic situation of bovine diarrhea-related viruses in Shanghai， and to provide a scientific basis for the prevention and control of bovine diarrhoeal disease. Method RT-PCR method was applied to detect Bovine rotavirus （BRV）， Bovine enterovirus （BEV）， Bovine coronavirus （BCoV） and Bovine viral diarrhea virus （BVDV） from 115 anal swab samples of diarrheal cattle in Shanghai area， and the gene sequences of some positive pathogens were sequenced and analyzed for genetic evolution. Result BRV， BEV and BCoV were detected in 115 anal swab samples from diarrheal cattle， with detection rate of 1.74% （2/115） for each， and the infection types of the detected viruses were all single infections. BVDV was not detected. The genetic evolution analysis results based on BRV VP7 and VP4 genes showed that the VP7 genes of the two detected BRV strains had the highest similarity with the Canadian strain FMV1078090， which was 94.1%， and were closely related to the American Human rotavirus strain 3000015004， both belonged to type G6. The VP4 gene had the highest similarity with Chinese strain HB-3， which was 98.2%， and both belonged to type P［11］. Therefore， the BRV genotypes of the two strains were G6P［11］. The genetic evolution analysis results based on the BEV 5'-UTR gene showed that among the two detected BEV strains， one had the highest similarity to the German strain D8-01， which was 90.6%， and both belonged to type E. The other had the highest similarity to the American strain Wye 8875， which was 93.5%， and both belonged to type F. Moreover， both BEV strains were distantly related to the prevalent strains in China. The results of genetic evolution analysis based on the BCoV N gene showed that the two detected BCoV strains clustered into a large branch with most Chinese and American strains， both belonged to the GⅡb gene group and had a close genetic relationship， while had a distant genetic relationship with the American strain Mebus and the vaccine strain BC94. Conclusion BRV， BEV and BCoV were the main viral pathogens associated with bovine diarrhea in Shanghai， among which the prevalent genotype of BRV was G6P［11］， the epidemiological genotypes of BEV were E and F， and the prevalent genotype of BCoV was GⅡb. This study provided a scientific basis for the formulation of prevention and control strategies for bovine diarrheal disease in Shanghai.

Objective This study aimed to establish a rapid serological method for detecting antibodies against Short beak and dwarfism syndrome virus （SBDSV） in ducks， providing technical support for the development of SBDSV antibody detection and diagnostic kits. Method BALB/c mice were immunized with purified and inactivated SBDSV M15 strain. Splenocytes from the immunized mice were fused with SP2/0 myeloma cells. Hybridoma cell lines that could secrete SBDSV monoclonal antibody were screened by indirect ELISA. Ascites was prepared from the mice inoculated with screened hybridoma cells. The titers of monoclonal antibodies were determined by indirect ELISA. The characteristics of the monoclonal antibodies were identified by indirect immunofluorescence assay （IFA） and latex particle agglutination （LPA） assay. The neutralizing activity of monoclonal antibodies was determined by neutralization test. Monoclonal antibodies subclasses were tested by commercialized kit. The reaction system was optimized by using the checkerboard titration method， and a competitive ELISA detection method based on SBDSV monoclonal antibody was established. The cut-off value of this method was determined by detecting negative sera. The specificity， sensitivity and repeatability of the established competitive ELISA detection method were performed. The clinical sera were detected by this method， which results were compared with the results of latex particle agglutination inhibition （LPAI） assay. Result Three hybridoma cells stably secreting SBDSV monoclonal antibody were obtained by indirect ELISA， which were named D5-3， G11-23 and H2-27， respectively. The results of indirect ELISA showed that the antibody titers in the supernatant of three hybridoma cells D5-3， G11-23 and H2-27 were 1∶2⁶， 1∶2⁵ and 1∶2⁶， respectively， and the antibody titers in the ascites were 1∶10⁵， 1∶10⁴ and 1∶10⁶， respectively. The IFA results showed that the monoclonal antibodies D5-3， G11-23 and H2-27 all had good binding activity and could be used for IFA detection. The results of LPA assay showed that only monoclonal antibody H2-27 had the characteristic of sensitizing latex. The results of neutralization test showed that all three monoclonal antibodies had the ability to neutralize SBDSV， and monoclonal antibody H2-27 had the strongest neutralizing activity. The identification results of monoclonal antibody subclasses showed that monoclonal antibody D5-3 and G11-23 belonged to IgG1 subclass， while monoclonal antibody H2-27 belonged to IgM subclass. A competitive ELISA method for detecting SBDSV antibody was established by using purified and inactivated SBDSV （M15 strain） as coating antigen and monoclonal antibody H2-27 as competitive antibody. When inhibition rate （PI）≥23.70%， serum sample was determined to be positive for SBDSV antibody， when PI≤15.91%， it was determined to be negative. This method demonstrated good specificity to SBDSV positive sera and had no cross-reaction with the positive serum of Muscovy duck parvovirus （MDPV）， Muscovy duck reovirus （MDRV）， Duck paramyxovirus （DPMV）， Duck hepatitis virus-1 （DHV-1） and Duck Tembusu virus （DTMUV）. The test result of the hyper immune serum （LPAI titer 1∶2⁸） which was diluted at 1∶25 600 was remained positive. The coefficients of variation for inter-assay and intra-assay were both less than 6%， which indicated good repeatability of this method. The consistency between the established competitive ELISA and LPAI assay was good， which reached 89.10% （188/211）. Conclusion Three monoclonal antibodies against SBDSV were successfully prepared in this study. The competitive ELISA method based on monoclonal antibody H2-27 had strong specificity， high sensitivity and good repeatability， could detect SBDSV antibodies in high throughput， providing technical support for monitoring the prevalence of SBDSV in China and for evaluating maternal antibody levels in ducklings.

Objective This study aimed to investigate the evolution characteristics of complete genome of Duck astrovirus type 1 （DAstV1） in Xinyang region，and provide a genomic reference for future studies on its epidemiology， genetic evolution，and pathogenicity. Method Clinical samples from diseased ducks submitted by a commercial farm were screened by PCR/RT-PCR for twelve common avian viruses， including Fowl adenovirus， Avian astrovirus and Duck hepatitis A virus. Liver tissues were collected， sterilized， and inoculated into 10-day-old SPF duck embryos via the yolk-sac route， and passaged four times. Allantoic fluid from each passage was tested for DAstV by RT-PCR. The isolated virus was subjected to whole genome sequencing， and the nucleotide sequences of the ORF1a， ORF1b and ORF2 genes， as well as the amino acid variation sites of their encoded proteins， were analyzed and compared. Result Avian astrovirus was detected in the clinical samples. DAstV1 was consistently positive in allantoic fluids from the 1st to 4th passage duck embryos. The isolate was designated HN24XY06. All fourth-passage embryos died following inoculation， exhibiting poor development and subcutaneous hemorrhage. The genome of HN24XY06 was 7 755 nt in length and contained three open reading frames： ORF1a， ORF1b， and ORF2. Sequence alignment and phylogenetic analysis showed that HN24XY06 belonged to DAstV1 strain and was closely related to the Shandong isolates DAstV-SDZZ and DAstV-SDWF. Sequence analysis of the amino acid sites of the ORF1a， ORF1b and ORF2 proteins showed that these proteins had mutations to varying degrees， mainly in the amino acid sequence encoded by ORF1a. Conclusion A strain of DAstV1 was successfully isolated in this study， thereby enriching the molecular epidemiological data of DAstV1 in Xinyang region and providing a solid foundation for further investigations into its pathogenic mechanisms.

Objective This study aimed to investigate the structure and function of the chicken DDX21 RNA helicase and to explore its potential role in the innate immune response induced by H9N2 subtype Avian influenza virus （AIV）. Method The chicken DDX21 gene was amplified and analyzed using bioinformatics tools. The recombinant chicken DDX21 protein was expressed in an Escherichia coli expression system through IPTG induction. Polyclonal antibody against chicken DDX21 were generated by immunizing rabbits with the purified protein. Antibody titers were quantified via indirect ELISA， and the reactivity of the polyclonal antibody to the endogenous chicken DDX21 protein was detected by Western blotting. Result The chicken DDX21 gene coding sequence （CDS） was 2 145 bp， encoding 714 amino acids. The nucleotide sequence of the chicken DDX21 gene cloned into the pMD18-T vector was verified by Sanger sequencing， and was consistent with the reference sequence available in the GenBank database （accession No.： XM_040675361.2）. Amino acid sequence alignment revealed high homology between chicken DDX21 and other avian species， with similarity ranging from 68.8% to 89.7%. Phylogenetic analysis indicated that the amino acid sequence of chicken DDX21 clustered within the avian clade， exhibiting close relationships with those of avian species. The protein contained conserved DEXDc， HELICc and GUCT domains， and its tertiary structure shared a similarity of 82.71% with that of human DDX21. The molecular weight of the recombinant protein was determined to be 101 ku， aligning with expectation. The polyclonal antibody titer reached 1∶320 000， and the polyclonal antibody successfully recognized endogenous DDX21 protein. The expression level of chicken DDX21 protein was upregulated in multiple tissues of chicken infected with H9N2 subtype AIV， revealing the involvement of chicken DDX21 in the response to viral infection. Conclusion This study successfully characterized chicken DDX21 gene， and the developed polyclonal antibody recognized the endogenous chicken DDX21 protein. These findings provided a foundation for further investigation into the mechanisms of chicken DDX21 in regulating the type Ⅰ interferon signaling pathways.

Objective This study aimed to isolate and screen β-lactam antibiotic-degrading bacteria，analyze their degradation activity and whole genome， and provide new ideas for repairing β-lactam antibiotic pollution in the environment. Method In this study， selective media containing penicillin potassium， amoxicillin， and ceftriaxone sodium were used to screen bacteria from seawater in Zhanjiang city in Guangdong province. The degradation rates were determined through rapid biodegradation tests. Molecular biological identification was performed on strains with degradation activity， and time-degradation curves were drawn. Degrading bacteria that degraded more than 90% of antibiotics within 120 h were selected to analyze the degradation effect at higher initial antibiotic concentrations， and the strain with the best degradation effect was screened for whole genome sequencing analysis. Result 18 strains of β-lactam antibiotic-resistant bacteria were screened from seawater in Zhanjiang， among which 5 strains could rapidly degrade β-lactam antibiotics. By drawing time-degradation curves， it was found that strains DA02 and DA03 could degrade 99.12% and 91.21% of ceftriaxone sodium with an initial concentration of 100 mg/L within 120 h， and DA02 could rapidly degrade ceftriaxone sodium with an initial concentration of 300 mg/L. The 16S rRNA gene sequencing analysis results showed that strain DA02 was Stenotrophomonas maltophilia. Whole genome sequencing identified strain DA02 with a genome size of 4 786 356 bp， a GC content of 66.58%， and a total of 4 253 predicted coding genes， 73 tRNAs， and 7 rRNAs. Multiple genes encoding β-lactamases were found， including L1_BLA， LRA-18， NmcR， PSV-1 and SRT-1. The bacterium also contained various antibiotic resistance genes， such as AAC（6'）， APH（3'）， cmlv and arr-2. It also possessed abundant carbohydrate metabolic pathways， with 91， 12 and 13 genes involved in carbon metabolism， sulfur metabolism， and nitrogen metabolism， respectively. Conclusion Highly efficient β-lactam antibiotic-degrading bacteria existed in seawater. Whole genome analysis revealed the possible degradation mechanism and bioremediation potential of ceftriaxone sodium-degrading bacterium DA02， providing a potential effective method for repairing β-lactam antibiotic pollution in the environment.

Objective Through the use of network pharmacology， molecular docking， and cell experiments， this study aimed to explore the therapeutic mechanism of Maxing Shigan decoction against Mycoplasma bovis infection， providing scientific basis for the modernization of traditional Chinese medicine and new strategies for the treatment of Mycoplasma bovis infection. Method Relying on bioinformatics platforms such as TCMSP， PubChem and SwissTargetPrediction， the active ingredients of the constituent herbs in Maxing Shigan decoction and their related targets were screened. The targets of Mycoplasma bovis related diseases were obtained through GeneCards and OMIM databases. The STRING database was used to construct protein-protein interaction network， and Cytoscape 3.9.1 software was used for network topology analysis. Meanwhile， the DAVID platform was used for GO function and KEGG pathway enrichment analysis. Molecular docking and visualization were completed using PyMOL 2.4.0 and AutoDockTool 1.5.6 software. MDBK cells were divided into blank control group， model group， and low， medium， and high dose groups （2.5， 5 and 10 mg/mL） of Maxing Shigan decoction. Except for blank control group， all other groups were infected with Mycoplasma bovis PG45 with a multiplicity of infection of 1 000 for 4 h. After cleaning， they were replaced with drug containing medium or complete medium， and continued to be cultured for 24 h. Real-time quantitative PCR was used to detect the Ct value and the mRNA expression of relevant inflammatory factors in each group of cells. Result Network pharmacology analysis revealed that Maxing Shigan decoction contained 134 active ingredients， and there were 93 targets related to Mycoplasma bovis-related diseases. A total of 20 intersection targets were identified between them， among which interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， matrix metallo proteinase 9（MMP9）， caspase 3 （CASP3） and prostaglandin peroxide synthase 2 （PTGS2） were the key core targets. GO function enrichment analysis mainly involved positive regulation of transcription by RNA polymerase Ⅱ， extracellular space， cytoplasm， nucleus， cytokine activity， etc. KEGG pathway enrichment analysis mainly covered signaling pathways related to IL-17， TNF， mitogen-activated protein kinase （MAPK），etc. The molecular docking results showed that the binding energy of the active ingredients of Maxing Shigan decoction was less than -17.8 kJ/mol， and had good binding ability. Among them， quercetin had the smallest binding energy to CASP3， which was -28.87 kJ/mol. Cell experiments showed that compared with model group， the Ct values were significantly increased （P<0.05）， the expression of IL-2 and IL-10 genes were significantly upregulated （P<0.05）， while those of IL-6， IL-8， TNF-α， IL-17A，CASP3， MMP9 and PTGS2 genes were significantly downregulated （P<0.05）， in each dose group of Maxing Shigan decoction. Conclusion Through network pharmacology prediction and cell experimental validation， this study revealed that Maxing Shigan decoction exered anti-Mycoplasma bovis infection effects by acting on targets such as IL-6， TNF and MMP9， regulating the secretion of inflammatory factors， and inhibiting inflammatory responses.

Objective This study aimed to screen microbial resources with high-temperature tolerance and high protease production from meat and bone residue， optimize the enzyme production process， and provide a theoretical basis for industrial development and application. Method Using protein-rich meat and bone residue as the source for strain isolation， thermotolerant and protease-producing strains were screened based on their high-temperature survival ability and casein-degrading activity. Species identification was performed through morphological observation， physiological and biochemical characteristics， and 16S rRNA sequence analysis. Using protease activity as the indicator， culture conditions were optimized by single-factor tests and response surface methodology （RSM）. Ultraviolet （UV） mutagenesis and high-temperature acclimation were applied to enhance the enzyme activity and thermotolerance of the strain. Result A strain identified as Aeribacillus pallidus 60 was isolated from fermented meat and bone residue. The optimal enzyme production conditions were determined as follows： 0.89% NaCl， 1.37% Ca²⁺， 0.5% Mg²⁺， pH 8.87 and incubation time 86.29 h. Under these conditions， protease activity increased by 59.51% compared to the non-optimized strain. After three rounds of UV mutagenesis， protease activity was significantly increased by 24.65% （P<0.01） on the basis of the optimized conditions. Subsequent high-temperature acclimation further enhanced the strain’s heat tolerance to 70 °C， and protease activity was significantly increased by an additional 24.16% （P<0.01）. Conclusion Aeribacillus pallidus 60 exhibited excellent high-temperature tolerance and protease-producing capacity. Culture condition optimization， coupled with UV mutagenesis and thermal acclimation， significantly enhanced its enzyme activity. Results provided a theoretical foundation and microbial resource for industrial applications.

Objective This experiment aimed to explore the protective effect of catechin on mice with mastitis， and to study the regulatory role of the glutathione peroxidase 4 （GPX4）/solute carrier family 7 member 11 （SLC7A11） signaling pathway in ferroptosis. Method Twenty female mice that had given birth within 7-10 days were selected and randomly divided into 5 groups： Blank control group， LPS model group， catechin low-dose group （40 mg/kg）， catechin high-dose group （80 mg/kg）， and dexamethasone positive control group. The mice in low- and high-dose catechin groups were intragastrically administered with catechin at the corresponding concentration， while the mice in other groups were given the same dose of normal saline （0.3 mL per mouse）， for 7 days. After the intragastric administration was completed， the mice in positive control group were intraperitoneally injected with 100 μL of 2 mg/mL dexamethasone. Two hours later， except for blank control group， the mice in other groups were injected with 50 μL of 0.2 mg/mL LPS on each side of the fourth pair of nipples through mammary duct to establish the mastitis model. The weight changes of the mice in each group after the modeling were compared. The histological changes of the mammary glands in each group of mice were observed through HE staining. The content of malondialdehyde （MDA） and the activity of superoxide dismutase （SOD） in mammary gland were detected by biochemical kits. The expression levels of GPX4， SLC7A11 and ferritin heavy chain （FTH） proteins in mammary gland were detected by Western blotting. Immunofluorescence was used to detect the fluorescence intensities of FTH and GPX4 proteins. Result Compared with blank control group， the body weight of mice in LPS group showed a downward trend， while the body weight of mice in catechin treatment groups showed an upward trend. HE staining results showed that the mammary gland acini of mice in blank control group were intact and no inflammatory cell infiltration was observed. In LPS group， there was a significant infiltration of inflammatory cells and atrophy of the mammary gland lobules in the mice.These pathological damages were substantially alleviated in both the high- and low-dose catechin groups， as well as in dexamethasone positive control group. Compared with blank control group， the content of MDA in mammary gland of mice in LPS group significantly increased， and the activity of SOD significantly decreased （P<0.05）. Compared with LPS group， the content of MDA in mammary gland of mice in high- and low-dose catechin groups significantly decreased， and the activity of SOD significantly increased （P<0.05）. Western blotting results showed that compared with blank control group， the expression levels of GPX4， SLC7A11， and FTH proteins in mammary gland of mice in LPS group were significantly decreased （P<0.05）.Compared with LPS group， the expression levels of GPX4， SLC7A11， and FTH proteins in mammary gland of mice in high- and low-dose catechin groups were significantly increased （P<0.05）. The results of immunofluorescence detection showed that， compared with blank control group， the relative fluorescence intensities of GPX4 and FTH proteins in mammary gland of mice in LPS group significantly decreased （P<0.05）. Compared with LPS group， the relative fluorescence intensities of GPX4 and FTH proteins in mammary gland of mice in high-dose catechin group significantly increased （P<0.05）. Conclusion Catechins could significantly alleviate the damage caused by LPS-induced mastitis in mice， and the protective effect was closely related to the inhibition of ferroptosis. Catechins enhanced the antioxidant capacity of the body by upregulating the expression of SLC7A11， promoting the activity of GPX4， and ultimately reducing the accumulation of MDA， thereby exerting an inhibitory effect on ferroptosis.

Objective The purpose of this experiment was to investigate the effects of rutin on the barrier function and inflammatory injury of sheep rumen epithelial cells induced by lipopolysaccharide （LPS）. Method Sheep rumen epithelial cells were cultured in vitro. Different concentrations of LPS （0， 1， 5， 10， 50， and 100 μg/mL） were used to stimulate and establish a model of rumen epithelial cell injury. The optimal concentration and duration of LPS action were determined. The rumen epithelial cells were pre-treated with different concentrations of rutin （0， 25， 50， 100， and 150 μg/mL）， and the cell viability was detected by MTT assay. The relative proliferation rate of the cells was calculated to determine the appropriate concentration and duration of action of rutin. Based on the selected concentrations and durations of LPS and rutin， the cells were grouped and treated accordingly. They were respectively designated as control group （CON）， LPS group， rutin group （R）， and rutin intervention group （R+LPS）. The contents of interleukin-1β （IL-1β）， tumor necrosis factor-α （TNF-α）， and IL-10 in the cell supernatant were determined by ELISA kit. The relative mRNA expression levels of IL-1β， IL-4， IL-6， IL-8， IL-10， IL-12， TNF-α， IFN-γ， CXCL8， CXCL9， Occludin， ZO-1， Claudin-1， and Claudin-4 were detected by Real-time quantitative PCR. Result Through experimental screening， it was ultimately determined that 1 μg/mL LPS， when applied for 9 hours， was the optimal condition for establishing the rumen epithelial cell injury model. A treatment of 100 µg/mL rutin for 8 hours was identified as the optimal condition for the pre-protective effect of rutin on rumen epithelial cells in vitro. Compared with CON group， the contents of IL-1β and TNF-α in the cell supernatant of LPS group were significantly increased （P<0.05）， while IL-10 content was significantly decreased （P<0.05）. Compared with LPS group， the contents of IL-1β and TNF-α in the cell supernatant of R+LPS group were significantly decreased （P<0.05）， while IL-10 content was significantly increased （P<0.05）. The Real-time quantitative PCR results showed that compared with CON group， the mRNA expression levels of IL-1β， IL-6， IL-12， TNF-α， IFN-γ， CXCL8， and CXCL9 in the rumen epithelial cells of LPS group were significantly increased （P<0.05）， the mRNA expression levels of IL-4， IL-10， Claudin-4， and ZO-1 were significantly decreased （P<0.05）. Compared with LPS group， the mRNA expression levels of IL-1β， IL-6， IL-12， TNF-α， INF-γ， CXCL8， and CXCL9 in the rumen epithelial cells of R+LPS group were significantly decreased （P<0.05）， the mRNA expression levels of IL-4， IL-10， ZO-1， and Claudin-4 were significantly increased （P<0.05）. Conclusion Under this experimental conditions， 100 µg/mL rutin， when applied for 8 hours， exhibited a significant protective effect on the barrier function and inflammatory injury of the rumen epithelial cells induced by LPS.

Objective This experiment combined liquid chromatography-mass spectrometry （LC-MS） technology with network pharmacology methods to explore the key active components， potential target sites and regulatory mechanisms of the anti-tumor effects of Scutellariae barbatae D.Don， providing theoretical support for clarifying the pharmacological basis of its efficacy. Method LC-MS technique was employed to conduct mass spectrometric analysis of the extract of Scutellariae barbatae D.Don in both positive and negative ion modes， thereby identifying its main chemical components. Based on the network pharmacology approach， a comprehensive exploration was conducted using multiple databases and bioinformatics tools to identify the key active components of Scutellariae barbatae D.Don in anti-tumor effects and the underlying mechanisms of action. Through the TCMSP platform， candidate compounds that meet the criteria of oral bioavailability （OB） ≥30% and drug-likeness （DL）≥0.18 were selected， and the potential targets were predicted using SwissTargetPrediction.Tumor-related genes were retrieved from GeneCards and OMIM databases， and intersection analysis was conducted to identify key therapeutic targets. Further， the protein-protein interaction network （PPI） was constructed using the STRING software and topological analysis was conducted to screen core targets. GO functional annotation and KEGG pathway enrichment analysis was performed using Metascape to elucidate the biological functions and signaling pathways involved. The “component-target-disease” and “target-pathway” network diagrams were constructed using Cytoscape software to visually present the potential molecular mechanisms of the anticancer effect of Scutellariae barbatae D.Don. Molecular docking was employed to validate the binding affinity between core active components and key targets. Result LC-MS analysis identified 14 major components in Scutellariae barbatae D.Don under negative ion mode， primarily flavonoids including luteolin， baicalein， and wogonin. Among these， compounds such as moslosooflavone， baicalein， and wogonin were associated with a higher number of targets. A total of 29 potential active ingredients and 330 action targets were identified. The intersection with tumor targets resulted in 95 key targets （such as AKT1， VEGFA， EGFR， and ESR1）. PPI network indicated that AKT1， VEGFA， and EGFR were the key targets. GO functional enrichment analysis revealed that the core targets mainly involved cell proliferation， apoptosis， angiogenesis， etc. KEGG pathways were significantly enriched in tumor-related signaling pathways such as PI3K-Akt， FoxO， and p53. Molecular docking results indicated that the core active components baicalein and hankwintin had good binding activities with the key targets AKT1， EGFR， VEGFA， and ESR1， further verifying the reliability of the network pharmacology predictions. Conclusion Scutellariae barbatae D.Don mainly exerted its effects through flavonoid active components such as baicalin and wogonin， which act in synergy on key targets such as AKT1， VEGFA， and EGFR，which significantly regulated signaling pathways such as PI3K-Akt， p53， and FoxO， thereby inhibiting tumor cell proliferation， inducing cell apoptosis， and blocking angiogenesis. The results of this experiment comprehensively elucidated the multi-pathway and multi-component collaborative anti-tumor molecular mechanism of Scutellariae barbatae D.Don， providing an important theoretical basis for its clinical application and new drug development.