In recent years， an increasing number of intelligent devices have been put into use in cattle and sheep production， providing a new way to accurately and efficiently obtain individual and environmental phenotypic data. However， how to scientifically process and analyze large volumes of data poses new challenges for managers. Machine learning can independently learn high-dimensional data from various sensors through algorithms and models， extract key information from it， conduct regression prediction or build different disease early warning models， assist managers in timely intervention， and thereby improve management efficiency. This paper mainly reviews the basic process of machine learning， commonly used machine learning algorithms in pastures， and their applications in weight prediction， heat stress early warning， mastitis early warning， estrus monitoring， etc. It also summarizes and analyzes the existing problems， with the aim of providing theoretical references for the development of smart animal husbandry in China.

Ovarian granulosa cells （GCs） play a critical role in follicular development and the maintenance of ovarian function. Dysfunction of GCs is closely associated with reproductive disorders in animals. As a class of endogenous non-coding small RNAs， miRNAs regulate the expression of target genes at the post-transcriptional level and participate in various biological processes of GCs. This article systematically elucidates the biological relationship between GCs and follicular development， with a focus on the molecular mechanisms by which miRNAs influence follicular development through the specific regulation of GC metabolism， including glucose metabolism， lipid metabolism， and biological oxidation. By integrating relevant research advances， this review aims to reveal the functions of miRNAs in the metabolic regulation of GCs and provide a theoretical basis for improving animal reproduction.

In modern intensive poultry production， skeletal disorders have become one of the major constraints affecting poultry health and production efficiency. Conventional nutritional research has primarily focused on the direct regulatory roles of macronutrients such as calcium， phosphorus， and vitamin D in bone development. In recent years， advances in gut function studies have introduced the concept of the "gut-bone axis"， which provides a new perspective for understanding the complex interactions among intestinal nutrient absorption， microbiota and their metabolites， and gut hormones in regulating bone growth and metabolism. Although numerous reviews on the "gut-bone axis" have been published in mammals， systematic reviews focusing on poultry， particularly laying hens， remain scarce， and the role of intestinal hormones in avian bone metabolism has not been thoroughly summarized. This review aims to elucidate the mechanisms by which intestinal health and its key components， including digestive and absorptive functions， microbial communities， and gut hormones， modulate skeletal homeostasis in chickens. The findings are expected to provide a theoretical basis for nutritional strategies aimed at improving bone health in poultry and to offer novel insights into future research on the gut-bone axis.

The quest for safe and effective feed additives to promote the healthy growth of livestock and poultry has long been a research hotspot both domestically and internationally. Functional oligosaccharides， which possess multiple physiological functions such as modulating intestinal microbiota， enhancing immune function， and improving intestinal barrier integrity， have demonstrated promising application prospects in livestock and poultry farming. This review systematically reviews the main categories of functional oligosaccharides and their mechanisms for regulating intestinal health in livestock and poultry， provides an in-depth analysis of the critical challenges encountered during their use， and outlines future research directions. The study aims to furnish theoretical foundations and practical guidance for the scientific application of functional oligosaccharides in livestock and poultry production， thereby fostering the healthy and sustainable development of the industry.

With the rapid growth of the exotic pet market， the risk of zoonotic diseases originating from exotic pets has become increasingly prominent， posing a potential threat to public health security. Therefore， accurately distinguishing the risks of zoonoses originating from exotic pets and implementing practical scientific measures are the key components in strengthening the frontline of our public health safety. In order to scientifically evaluate and prevent such risks， this study， based on the recent trends of China's exotic pet industry， systematically reviewed the types of zoonotic diseases originating from exotic pets with high prevalence and high hazard. It summarized their epidemiological characteristics and the comprehensive prevention and control system. Meanwhile， this study analyzed the risk factors for zoonotic diseases in the trading and breeding of exotic pets and proposed targeted prevention and control strategies. This study aimed to provide a scientific basis and theoretical support for suppressing the prevalence of exotic pet-related zoonotic diseases. It also provides professional references for industry practitioners to formulate prevention strategies and reduce the risk of epidemic spread.

Infectious bronchitis virus （IBV） is one of the important pathogens that seriously threatens the global poultry industry. Among them， nephropathogenic infectious bronchitis virus （NIBV） infection can cause severe kidney damage in poultry， leading to high mortality and huge economic losses. Studies have shown that excessive inflammatory responses mediated by the activation of the NOD-like receptor protein 3 （NLRP3） inflammasome are one of the important mechanisms of NIBV-induced kidney damage in chickens， and the NLRP3 inflammasome may be an important target for preventing and controlling NIBV infection and its related kidney damage. Traditional Chinese medicine （TCM）， with its advantages of multiple components， multiple targets， low residues， and low likelihood of developing resistance， plays an important role in preventing and treating NIBV. Recent studies have found that the natural active components of TCM are closely related to alleviating NIBV-induced kidney damage in chickens by regulating the activation of the NLRP3 inflammasome. This paper systematically reviews the activation and regulation of the NLRP3 inflammasome and its underlying mechanisms in NIBV-induced kidney damage in chickens， and analyzes the research progress of natural active components from TCM in regulating the NLRP3 inflammasome via related signaling pathways to alleviate such kidney injury. This work aims to provide a theoretical basis for developing novel TCM derived feed additives or therapeutic agents for prevention and treatment of NIBV infection in chickens.

Traditional veterinary drug formulations commonly face issues such as low bioavailability， high dosing frequency， poor targeting， and high risk of tissue residue. With breakthroughs in novel delivery systems and formulation technologies， new veterinary drug formulations show significant potential in improving therapeutic efficacy， enhancing medication adherence， and achieving sustained and intelligent release. This paper systematically reviews the current research status of veterinary drug formulations and the development of new formulations， focusing on key innovations such as microencapsulation technology， self-emulsifying systems， and 3D-printed personalized formulations. It evaluates advancements in enhancing pharmacokinetic performance， improving targeting capabilities， and expanding clinical application scenarios. Additionally， it analyzes the challenges faced during the translation of new formulations， including technical bottlenecks， carrier biocompatibility， scalability of manufacturing processes， and cross-species applicability. The aim is to provide a reference for the innovative development and industrial application of new veterinary drug formulations. Furthermore， the development of veterinary drug formulations will increasingly rely on interdisciplinary collaboration and digital strategies， driving the veterinary drug industry towards more efficient， precise， safe， and environmentally friendly directions. This progress lays a theoretical foundation for significantly improving animal health and ensuring public health safety.

Porcine reproductive and respiratory syndrome （PRRS） is a viral infectious disease of significant economic importance in pigs， posing a serious threat to the global pig farming industry. Inducing programmed cell death （PCD） in host cells is considered a key cellular mechanism in the pathogenesis of PRRS. In this review， we summarized the signal regulatory mechanism of PRRSV-induced PCD， clarified its role in viral replication and immune regulation， and provided a theoretical basis for the design of new vaccines and antiviral strategies.

To promote the development of molecular breeding systems for pigs in China， particularly for the genetic improvement and conservation of local breeds， this study aimed to construct a liquid-phase genotyping chip with both functional representativeness and population discrimination power， providing an efficient genomic tool for precision breeding. Publicly available pig genomic sequencing data and data reported in published studies were used in this study， including whole-genome resequencing data from 1 176 individuals， long-read sequencing data from 80 individuals， and graph-pangenome genotyping results derived from 27 high-quality pig genomes. Candidate marker loci were identified through population differentiation analysis， quantitative trait locus （QTL） annotation， and linkage disequilibrium （LD） filtering. A porcine 50K liquid-phase chip was constructed through commercial probe design and quality control procedures. Chip performance was evaluated using target region coverage and sequencing depth. The final pig 50K liquid-phase chip comprised 50 023 high-quality loci， including 45 113 single nucleotide polymorphisms （SNPs） and 4 910 structural variations （SVs）， and incorporated a full-length mitochondrial genome capture design. These loci were evenly distributed across the genome， enabling joint detection of SNPs and SVs. Performance evaluation demonstrated that the chip exhibited high target-region coverage， sequencing depth， and detection consistency across both Chinese local and commercial pig breeds. By integrating SNPs and SVs markers， the pig 50K liquid-phase chip developed in this study showed a well-balanced locus distribution and stable detection performance， providing a reliable genotyping tool for pig population genetic analyses， molecular breeding， and related studies.

This study aims to dissect the microbiological basis of feed conversion ratio （FCR） differences in Duroc pigs fed high-fiber diets， using multi-omics to reveal the interplay between gut microbiota functional profiles and host responses. Duroc barrows fed a diet containing 30% soybean hulls （crude fiber： 13.11%） were selected as experimental subjects. According to the FCR phenotype， they were divided into high feed conversion rate group （HFCR） and low feed conversion rate group （LFCR）， with 3 pigs in each group. Rectal contents and mucosal tissues were collected and subjected to integrated metagenomic， transcriptomic， and metabolomic analyses to assess microbial community composition， functional gene expression， and metabolite profiles. Multi-omics integration analysis identified key microbes and potential pathways influencing FCR. The LFCR group demonstrated significant enrichment in fiber-degrading taxa， including Prevotella， Oscillibacter， and Ruminococcus. The microbial CAZyme profile of the LFCR group exhibited increased abundances of glycoside hydrolases， including GH5 and GH13_28， indicating enhanced polysaccharide degradation capacity. Host transcriptomics showed upregulated expression of smooth muscle contraction genes （MYH11， MYL9） in LFCR， in contrast to elevated chemokine genes （CCL19， CXCL2） in HFCR. Metabolomics analysis revealed significantly reduced cellobiose levels in the LFCR group. Multi-omics correlation analysis further indicated significant positive associations between energy metabolism genes （ND5， ND6） and Prevotella sp. CAG：485， P. P5-92， P. P2-180， and Clostridium sp. CAG：413， suggesting a potential synergistic process linking efficient fiber degradation to enhanced host energy metabolism in low-FCR individuals. Under high-fiber diet conditions， low-FCR pigs exhibit enhanced polysaccharide degradation and energy-linked host gene expression， whereas high-FCR counterparts exhibit upregulation of chemokine signaling-related genes. These insights implicate gut microbial functionalities and host-microbe interplay in FCR disparities， offering targets for microbiota-targeted enhancements in fibrous feed efficiency.

This study aimed to construct a single-cell atlas of the chicken retina and to explore its regulatory role in key growth traits， identifying potential critical cell types and gene regulatory mechanisms. We utilized phenotypic data from 1 047 B-line White-feathered broilers from the 10th and 11th generations， including weight gain （WG）， feed intake （FI）， body weight at 28 days （BW28）， body weight at 40 days （BW40）， feed conversion ratio （FCR）， and residual feed intake （RFI）. All these traits were measured during the 28-40 day growth period. Genome-wide association studies （GWAS） were performed using a mixed linear model， and heritability and correlations among traits were estimated. Then， we obtained 18-day-old chicken embryonic retina single-cell RNA-seq data from a public database， and constructed a single-cell atlas of the chicken retina using UMAP dimensionality reduction and clustering analysis. The study further performed a joint analysis by integrating the single-cell transcriptome with the broiler GWAS results through single-cell RNA sequencing-assisted genome-wide association studies （scGWAS）. To validate the gene modules identified by scGWAS， 60 White-feathered broilers with no significant difference in body weight （P>0.05） but a highly significant difference in feed intake （P<0.001） were selected and divided into a high feed intake group （HFI， n=30） and a low feed intake group （LFI， n=30）. Transcriptome sequencing and differential genes expression analysis were then performed on their retinal tissues. The results showed that the phenotypic correlations among the traits were highly significant （P<0.001）. All traits exhibited low to moderate heritability， with BW28 having the highest heritability （0.25） and RFI the lowest （0.07）. After quality control and filtering， 30 005 high-quality cells were retained， and a chicken retinal single-cell atlas comprising 8 cell types was constructed. scGWAS analysis revealed that genetic variations underlying FI and RFI were significantly associated with the retina， and these genetic signals were specifically enriched in distinct retinal cell types. Additionally， we uncovered 5 gene modules that were simultaneously enriched for both FI genetic signals and retinal transcriptome signals. Finally， the study revealed that APP was significantly highly expressed in the retinal tissue of the high-feed-intake group （P<0.01）， whereas NTM was significantly upregulated in the retinal tissue of the low-feed-intake group （P<0.01）. Collectively， this study is to integrate single-cell transcriptomics with GWAS analysis， revealing the significant role of the retina in regulating feed intake in chickens. We further focused on Müller glial cells， photoreceptor cones， and retinal ganglion cells， as their genes expression was found to be significantly correlated with genetic signals for feed intake. Furthermore， the genes APP and NTM were identified as potential key regulators of feed intake in broilers.

The study aimed to construct a melanin deposition obstruction model by exogenous addition of arbutin， and study the effects of lncRNA-7896 on melanin deposition-related genes， tyrosinase activity and melanin content in Xichuan Black-bone chickens. Eighty breeding eggs of Xichuan Black-bone chicken were selected and randomly divided into 4 groups. The control group （CON） was injected with 4 μL of PBS solution， the arbutin treatment group （ARB） was injected with 4 μL of 2 μmol·L^-1 arbutin， the empty vector control group （A-NO） was injected with 4 μL of 2 μmol·L^-1 arbutin and the empty vector of transposon， and the lncRNA-7896 overexpression group （A-LNC） was injected with 4 μL 2 μmol·L^-1 arbutin and transposon lncRNA-7896 vector （transposon vector： transposon coenzyme vector was 2∶1）. The test period was 28 d， starting from the hatching of the breeding eggs to the 7th embryo age， and the test period was 21 d. After the chicks hatched and were raised for one week， samples of heart， liver， pectoral muscle and leg muscle of Xichuan Black-bone chickens were collected. The collected tissue samples were extracted with RNA and reverse transcribed into cDNA. The expression levels of lncRNA-7896， MITF， TYR， and TYRP-1 were detected by real-time fluorescence quantitative PCR （qRT-PCR）. Meanwhile， the tyrosinase activity in pectoral muscles and leg muscles was detected by ELISA kits. The melanin content in the pectoral and leg muscles was detected by the colorimetric reagent kit. The melanin content in the pectoral and leg muscles was visualized by the Masson-Fontana staining reagent kit. Finally， the data were statistically analyzed by one-way analysis of variance using SPSS 23.0 software. The results showed that， compared with the control group， the addition of 4 μL 2 μmol·L^-1 arbutin significantly downregulated the expression levels of MITF， TYR and TYRP-1 genes （P<0.05）； Meanwhile， the activities of tyrosinase and melanin contents in pectoral muscle and leg muscle were also markedly decreased after 4 μL 2 μmol·L^-1 arbutin treatment （P<0.05）. Notably， exogenous introduction of lncRNA-7896 in Xichuan Black-bone chickens with inhibited melanin deposition could distinctly upregulate the expression of MITF， TYR and TYRP-1 genes， and effectively increase tyrosinase activity as well as melanin content in pectoral and leg muscles （P<0.05）. In summary， arbutin can suppress melanin deposition in Xichuan Black-bone chickens， whereas lncRNA-7896 can significantly restore the expression of melanogenic genes and increases both tyrosinase activity and melanin content in the pectoral and leg muscles after the deposition has been blocked.

This study aimed to establish a PGCs-based system for site-specific insertion of the EGFP gene into the chicken Z chromosome， improve the efficiency of positive PGCs acquisition and the stability of germline transmission， and provide technical support for early sex identification of chicks and precision breeding in poultry. White Leghorn chicken PGCs and 2-2.5-day-old yellow-feather broiler recipient embryos were used as experimental materials. A PGK-EGFP donor plasmid carrying homology arms and a CRISPR/Cas9 editing plasmid were constructed， with a donor plasmid lacking homology arms used as the control. The EGFP gene was introduced into PGCs using optimized electroporation. After fluorescence-activated cell sorting of positive PGCs， the cells were microinjected into recipient embryos to generate G0 germline chimeras， followed by crossbreeding to obtain G1 chickens. PCR， Sanger sequencing， and fluorescence detection were used to examine the site-specific insertion and expression of the exogenous gene at both the DNA and phenotypic levels. The detected indicators included PGCs transfection efficiency， the proportion of positive PGCs， the proportion of germline chimeras， and the positive rate of G1 heterozygotes. In the experimental group， the proportion of EGFP-positive PGCs reached 81.51% at 48 h post-electroporation， maintaining 28.26% after 7 days， which was significantly higher than the 0.09% observed in the control group. Among 88 microinjected embryos， 25 hatched successfully， including 12 roosters. After sexual maturity， 11 out of 12 roosters were confirmed as germline chimeras， yielding a colonization efficiency of 91.60%. Mating of germline chimeras with wild-type hens yielded 286 G1 offspring， of which 36 were positive heterozygotes， corresponding to a transmission rate of 12.60%. Sanger sequencing verified precise integration of the exogenous fragment into the target Z chromosome locus， and EGFP expression was detectable in the beaks and toenails of positive G1 chickens. This study established a highly efficient PGCs-mediated transgenesis platform enabling site-specific integration of the EGFP reporter gene into the chicken Z chromosome. The integrated transgene was intact and stably expressed， with both transfection efficiency and germline transmission rates reaching internationally advanced levels. This study results provides reliable animal models and technical support for the study of avian gene function， precision breeding， and innovation of germplasm resources.

This study aimed to investigate the impact of Salmonella Enteritidis infection on the serum metabolomic profile of "Guangming 2" White-feathered broilers using metabolomics， and to screen the key metabolites and core pathways associated with the infection. A total of 64 6-day-old "Guangming 2" White-feathered broilers， reared in isolators， were randomly assigned to an infected group （SE） and a control group （CT）. The broilers in infected group were orally gavaged with Salmonella Enteritidis suspension for 3 d， while the broilers in control group received an equal volume of PBS. Serum was collected 24 h post-challenge and analyzed using liquid chromatography-mass spectrometry （LC-MS） for non-targeted metabolomics. Multivariate statistical methods were employed to screen differential metabolites， followed by KEGG pathway enrichment analysis. Furthermore， weighted gene co-expression network analysis （WGCNA） was applied to correlate metabolite expression patterns with immune indices （IgA， IL-6， IL-8， IFN-γ） and intestinal morphological phenotypes to identify hub metabolites. Serum levels of the pro-inflammatory factor IL-6 and IgA were significantly elevated in the infected group， accompanied by pathological changes such as reduced intestinal mucosal thickness and decreased glandular depth， indicating successful model establishment. A total of 1 897 serum metabolites were identified， with 111 showing significant differences. Pathway enrichment analysis revealed these differential metabolites were significantly enriched in pathways including pyrimidine metabolism， lysine degradation， and D-amino acid metabolism. WGCNA constructed 5 co-expression modules， among which the "brown" module and others showed significant correlations with infection status and multiple pathological phenotypes. From these， 186 hub metabolites were screened， 49 of which were also differential metabolites. The metabolites Veranisatin A and D-Myo-Inositol （1，2，3） Trisphosphate were highly correlated with infection， multiple immune factors， and intestinal damage indicators. Hub metabolites were significantly enriched in 11 pathways， including glycerophospholipid metabolism， D-amino acid metabolism， and tryptophan metabolism， ect. Early-stage Salmonella infection extensively reprograms the serum metabolome of Guangming-2 broilers， markedly altering nucleotide， amino acid and lipid metabolism. The study identified a set of differential and hub metabolites closely associated with infection， among which Veranisatin A and D-Myo-Inositol （1，2，3） Trisphosphate may serve as key nodes connecting immune response and metabolic regulation. These findings provide a new basis for elucidating the mechanisms of Salmonella infection in broilers from a metabolic perspective and for exploring potential biomarkers.

To explore the molecular basis of the color difference between red and white cashmere goats， the genes and metabolites related to skin and hair follicles in Inner Mongolia red and white cashmere goats were analyzed by transcriptome and metabolome analysis. During the active phase of secondary follicles in cashmere goats （September）， 6 healthy adult female Erlangshan-type white cashmere goats and 6 adult female Urad Zhongqi local red cashmere goats （around 3 years old， weighing 25-30 kg） were selected. Skin follicle tissue samples from the lateral side of the body were collected for transcriptomic and metabolomic analyses. A total of 125 differentially expressed genes were screened from the transcriptome， comprising 64 up-regulated genes， 61 down-regulated genes， and 7 genes associated with pigment. GO functional enrichment analysis revealed that these differential genes were significantly enriched in 9 pigment-related categories， including pigmentation， melanosome organization， melanin biosynthetic process， positive regulation of melanin biosynthetic process， melanin metabolic process， tyrosine metabolic process， melanosome， melanosome membrane， and tyrosine binding. KEGG enrichment analysis indicated that the tyrosine metabolism pathway was the most significantly enriched. The metabolomics analysis detected a total of 188 differential metabolites under both positive and negative ion modes， with 37 down-regulated and 151 up-regulated. HMDB enrichment analysis demonstrated that these differential metabolites were significantly enriched in pathways such as nucleotide sugar metabolism， arginine and proline metabolism， glycine and serine metabolism， betaine metabolism， and estradiol metabolism. Furthermore， KEGG enrichment analysis showed that the differential metabolites were involved in multiple pathways， including glycine， serine， and threonine metabolism， ABC transporters， cysteine and methionine metabolism， etc. Combined analysis revealed that the majority of the differentially expressed genes and metabolites were significantly correlated and collectively enriched in 8 pathways. The results showed that TYRP1， TYR， PMEL， SOX10， SLC45A2， GPR143 and TRPM1 were the key genes that led to the difference of hair color phenotype between Erlangshan white cashmere goat and Wulatezhong local red cashmere goat. It is suggested that S-adenosylhomocysteine may serve as a potential source of cysteine required in the pheomelanin biosynthesis pathway.

This study aimed to systematically estimate genetic parameters of body weight and body size traits in Chinese Holstein cattle from birth to 12 months of age and to analyze the effect of early body weight on first-lactation 305-day milk yield， providing a theoretical basis for early selection. A total of 36 837 phenotypic records from 4 692 Holstein heifers born between 2013 and 2022 in two large-scale dairy farms in Shandong and Henan provinces were collected， covering 24 growth traits including body weight and body measurements from birth to 12 months. Additionally， first lactation 305-day milk yield data were tracked for 1 041 of these animals. Genetic parameters were estimated using DMU software with the AIREML-EM algorithm under an animal model. Pearson correlation analysis was conducted using R software to assess the phenotype relationship between body weight at different months and first-lactation 305-day milk yield. Heritability estimates for all traits ranged from 0.18 to 0.81， indicating moderate to high genetic control. The heritability of body weight at 2 months and heart girth at 3 months were the highest， at 0.54 and 0.81， respectively. High genetic correlations （ranging from 0.10 to 0.91） were observed between weight and body measurement traits across different months. Positive genetic correlations （0.02 to 0.60） were found between first lactation 305-day milk yield and birth weight， body weight at 2， 3， and 5 months， as well as body height at 12 months. In contrast， the remaining weight and body measurement traits showed negative genetic correlations with first lactation 305-day milk yield， ranging from -0.75 to -0.05. Body weight at 11 and 12 months showed highly significant positive correlations with first-lactation 305-day milk yield （P<0.01， r=0.125， 0.138）. Body weight and body size traits in Chinese Holstein cattle exhibit considerable potential for genetic improvement. Early body weight， especially in later growth stages， has a significant impact on first lactation 305-day milk yield performance. This study provides foundational support for the genetic evaluation and selection of growth traits in Chinese Holstein cattle.

This study aimed to analyze the effect of somatic cell count （SCC） on milk yield， essential amino acid content， and convential milk components using a large-scale Chinese dairy cow dataset. This analysis sought to characterize SCC at the population level and deepen understanding of milk quality and mastitis in dairy cows. The study utilized 26 782 milk samples from 5 693 lactating Holstein cows in North China. The spectral data， routine milk composition， and somatic cell count （SCC） information from milk samples were collected. The laboratory to estimate the content of essential amino acids （isoleucine， leucine， phenylalanine， valine， lysine， and total essential amino acids） was estimated using a predictive model established in the laboratory. Outliers were removed based on Mahalanobis distance， individual cow information， and milk composition data. The analysis of variance was performed on milk yield and milk quality traits （conventional components and essential amino acid content）， with parity， lactation stage， calving season， and somatic cell score （SCS） as fixed effects； cow ID， herd-test date， and residuals as random effects. The overall mean SCC for the dairy cows was 172.95×10³ cells·mL^-1， indicating a low incidence of mastitis within this herd. SCS showed negative correlations with milk yield， lactose percentage， and urea nitrogen， while exhibiting positive correlations with milk fat percentage， milk protein percentage， total solids， and all essential amino acids contents. As SCS increased， milk yield and lactose percentage decreased significantly （P<0.001）， while milk fat percentage， milk protein percentage， total solids， urea nitrogen， and essential amino acid content increased significantly （P<0.001）. The effect of increasing SCS on milk fat percentage was greater than its effects on other milk quality traits， while differences in milk protein percentage among SCS groups were minimal. SCS had a greater impact on essential amino acid content than on milk protein percentage. In conclusion， elevated SCS levels in milk （indicating mastitis risk） significantly impact dairy cows' milk yield， essential amino acid content in milk， and milk composition. To enhance milk yield and ensure consistent milk quality， dairy farms should monitor SCS indicators and strengthen mastitis prevention and management.

The study aimed to investigate the transcriptional regulatory mechanisms of breast muscle development in Gang geese. The breast muscle tissues were collected from healthy male Gang geese at 0， 6， 10， and 30 weeks of age（w）， which were hatched in the same batch （n=10 per age group； total N=40）. Gang geese were divided into 4 groups according to age， with 3 biological replicates per group for transcriptomic analysis. Transcriptome sequencing was performed to screen for key genes and pathways associated with breast muscle development. Through KEGG enrichment analysis， candidate genes regulating breast muscle development in Gang geese were identified. The results showed that body weight， breast muscle weight， and breast muscle index of Gang geese increased consistently from 0 to 30 weeks of age， and 10 weeks of age was identified as a turning point for breast muscle development. Based on this turning point， transcriptomic analysis identified 1 878， 2 258， and 1 079 differentially expressed genes （DEGs） in the 0 w vs 10 w， 6 w vs 10 w， and 10 w vs 30 w comparisons， respectively. KEGG enrichment analysis was performed on the DEGs， and 7 common signaling pathways， including ECM-receptor interaction， focal adhesion， regulation of actin cytoskeleton， apelin signaling pathway， adipocytokine signaling pathway， FoxO signaling pathway， and cell adhesion molecules （CAMs）， were enriched （P<0.05）. Further PPI analysis identified key candidate genes， including COL1A2， COL2A1， THBS4， and MYLK4. The candidate genes were finally mapped to the ECM-receptor interaction， focal adhesion， regulation of actin cytoskeleton， and Apelin signaling pathways according to the KEGG database. Based on the results， COL1A2， COL2A1， THBS4， and MYLK4 were preliminarily identified as candidate genes regulating the turning point of breast muscle development in Gang geese. These candidate genes jointly regulate breast muscle development in Gang geese through crosstalk among the ECM-receptor interaction， focal adhesion， regulation of actin cytoskeleton， and Apelin signaling pathways.

This study aimed to systematically screen independent INDEL loci significantly associated with body weight and body size traits in Lion-head geese through whole-genome resequencing and INDEL-GWAS strategy， and to explore candidate genes， providing theoretical support for analyzing their genetic regulatory mechanisms and molecular breeding. A total of 504 Lion-head geese （257 males and 247 females） at 120 days of age was phenotyped for body weight and 11 body measurement traits. Whole-genome resequencing was performed at an average depth of 10×. Raw data were quality-controlled using SOAPnuke， aligned to the Anser cygnoides reference genome （1.1 Gb） via BWA， and subjected to INDEL detection via the GATK. After strict filtering （MAF>0.01， biallelic）， 828 017 high-quality INDELs were obtained. GWAS was conducted using the GEMMA mixed linear model， and the significance threshold was set at the chromosome-wide level （0.05/number of chromosome variants）. To identify independent associated INDEL loci， the linkage disequilibrium （LD） SNPs （r²≥0.20） within a 1 Mb upstream/downstream region of significant INDEL were evaluated. Conditional GWAS analysis with the most strongly linked SNP in the region as the covariate was employed to verify the independence of INDELs. A total of 828 017 high-quality INDELs （388 574 insertions， 439 443 deletions） were identified across the genome-wide， with an average density of 0.75 INDELs·kb^-1. The 89.8% of relatively independent INDELs exhibited moderate to high LD （r²≥0.20） with neighboring SNPs. Initial GWAS screening identified 28 chromosome-wide significant INDEL loci. Conditional GWAS analysis excluded 6 INDELs due to strong linkage （r²≥0.20） with neighboring significant SNPs， ultimately confirming 22 independently associated INDEL loci. These loci implicated 43 candidate genes， including known regulators such as ASH1L （bone metabolism）， Cysltr1 （cartilage development）， TPST2 and COL5A1 （skeletal development）， and Vgll4 （skeletal muscle metabolism）. Among these， 34 genes （e.g.， AARS1， DENND1A） were reported for the first time to be associated with growth and development traits. This study establishes the INDEL-GWAS analytical framework for Lion-head geese， successfully identifying 22 independently associated INDEL loci and 43 candidate genes. The INDEL-GWAS effectively complements SNP analysis by revealing novel genetic loci， providing new perspectives and targets for dissecting molecular mechanisms and enabling precision breeding in Lion-head geese.

The purpose of this study was to investigate the effect of hypoxic environment on the synthesis of extracellular matrix （ECM） synthesis-related proteins in yak lungs. In this study， the healthy lungs of adult yak and cattle （5 heads each） were used as the research object. The distribution of collagen fibers in the lungs was detected by Masson trichrome and Sirius red staining. The distribution of ECM-related and fibrosis-related proteins in the lungs was detected by immunohistochemistry. The expression of α-SMA， ECM-related and fibrosis-related proteins in the lungs was detected by Western blot. The results of Sirius red staining， Masson's trichrome staining， and immunohistochemical staining showed that the distribution of collagen fibers in the pulmonary artery smooth muscle layer and adventitia was more abundant in yaks than in yellow cattle. ECM- and fibrosis-related proteins were expressed to varying degrees mainly in the bronchial epithelium at all levels， adventitia， muscular layer， pulmonary artery endothelium， adventitia， muscular layer， and alveolar septa. Western blot results indicated that the protein expression levels of Collagen I， Elastin， and Collagen III in the lungs of adult yaks were significantly higher than those in adult yellow cattle （P<0.001）； while the expression levels of α-SMA， Laminin， Fibronectin， and Collagen IV in the lungs of adult yaks showed no significant differences compared with those in adult yellow cattle. After hypoxic treatment of yak lung fibroblasts （YLFs）， Western blot results demonstrated that the expression levels of PCNA and the Bax/Bcl-2 ratio first increased and then decreased， reaching a peak at 24 h， and were significantly higher in the hypoxia group than in the normoxia group； the expression of Caspase-3 gradually increased over time and was significantly higher in the hypoxia group than in the normoxia group at each time point. Regarding fibrosis-related proteins （Laminin， Collagen IV， and Fibronectin）， during acute hypoxia （≤24 h）， Collagen I showed no significant difference between hypoxia and normoxia， while Laminin and Fibronectin were significantly higher than normoxic levels； during chronic hypoxia （>24 h）， no significant differences were observed compared with the normoxia group. For extracellular matrix-related proteins （Collagen I， Elastin， and Collagen III）， their expression levels were significantly higher in the acute hypoxia group （≤24 h） than in the normoxia group； during chronic hypoxia， Elastin maintained the difference observed at 24 h， while the differences for Collagen I and Collagen III became even more pronounced. The results of this study show that hypoxic environment can promote the proliferation of YLFs and the synthesis of ECM in yak lungs， which provides some basic data for further exploring the mechanism of yak lungs adapting to hypoxia.

The aim of this study was to investigate the effect of activating transcription factor 3 （ATF3） on the proliferation and apoptosis of rabbit dermal papilla cells （DPCs）. In this study， 6-month-old long-haired male rabbit was selected to isolate and obtain primary DPCs， which were divided into empty control group and overexpression ATF3 treatment group， with 3 replicates in each group. The effects of ATF3 on the proliferation， apoptosis and related gene expression of DPCs were detected by immunofluorescence staining， real-time fluorescence quantitative polymerase chain reaction （qRT-PCR）， Western blot and CCK-8. The following results were obtained： 1） Rabbit DPCs were successfully isolated and identified， and ATF3 overexpression vector was constructed. 2） Overexpression of ATF3 significantly promoted the expression of apoptotic genes BAX and Caspase-9 （P<0.05）， and significantly inhibited the expression of anti-apoptotic gene BCL-2 and proliferation marker PCNA （P<0.05）. 3） Overexpression of ATF3 significantly down-regulated the expression of Wnt2 and CCND1 （P<0.05）， and up-regulated the expression of SFRP2 and TGF-β1 （P<0.05）. 4） Overexpression of ATF3 significantly inhibited the proliferation and induced apoptosis of DPCs （P<0.01）. This study clarified that ATF3 inhibited the proliferation of rabbit DPCs and induced their apoptosis by regulating apoptosis and hair follicle development-related genes， revealed the negative regulatory role of ATF3 in rabbit hair follicle development， and provided an important theoretical basis for further understanding the molecular mechanism of rabbit hair follicle development.

Based on genome-wide association study （GWAS） technology， this research aimed to identify single nucleotide polymorphisms （SNPs） and candidate genes associated with semen cryotolerance traits in roosters， in order to provide reference information for molecular breeding aimed at improving semen cryotolerance of roosters. A total of 215 healthy 65-week-old White Leghorn breeding roosters were selected as the experimental population. Fresh semen and frozen-thawed semen samples were collected， and freeze-tolerance indexes including variations in sperm motility， sperm viability and sperm abnormality rate were determined and calculated， with 3 biological replicates. Heritability was estimated using DMUAI software. Genomic DNA was extracted from wing vein blood samples， and genotyping was performed using the chicken 55K SNP array. After quality control with Plink v1.9 software， GWAS was conducted using the BLINK model embedded in GAPIT， and genetic effect analysis was carried out for SNP-linked candidate genes. The variation values of sperm motility and sperm viability presented low heritability ranging from 0.070 to 0.154， while the variation of sperm abnormality rate showed moderate heritability of 0.397. A total of 7 significant SNP loci were identified by GWAS. Three significant SNPs （chr6：31460756， chr24：4865901， chr24：5049139） associated with sperm viability variation were annotated to 3 candidate genes， namely CADM1， APOC3 and APOA1. Four significant SNPs （chr6：4901210， chr6：4902911， chr7：15775500， chr7：26741761） related to sperm abnormality rate variation corresponded to 4 candidate genes including DYDC2， MTX2， ADCY5 and MYLK. This GWAS on semen cryotolerance in White Leghorn roosters identified 7 key candidate genes， including APOA1， MTX2， and APOC3. The results provide valuable information for marker-assisted selection breeding aimed at improving semen cryotolerance in roosters.

This study aimed to investigate the effects of low glucose-induced activation of Hippo signaling pathway on apoptosis and steroid hormone secretion of yak follicular granulosa cells （GCs）. Fresh ovaries were collected from healthy adult yaks. GCs were isolated from 3-5 mm follicles and cultured in low-glucose medium for 0， 12， 24， and 48 h. Western blotting was performed to assess the impact of low glucose on the expression of apoptosis-related proteins（including Bax， Bcl-2， and cleaved caspase-3）， and to determine the optimal time point at which low glucose induces GC apoptosis. Apoptosis was further confirmed by TUNEL assay. In parallel， Western blotting was used to evaluate the effects of low glucose on the expression of key steroidogenic enzymes （CYP11A1， CYP19A1， HSD3B1， and STAR） and core components in the Hippo signaling pathway （MST1， p-MST1， YAP， P-YAP， TAZ， and P-TAZ）. To functionally determine the role of YAP， the central effector of the Hippo pathway， GCs were treated with the YAP inhibitor verteporfin （VP） or the YAP activator PY-60. Subsequently， we examined the effects of YAP inhibition or activation on GC apoptosis， estradiol （E₂） and progesterone （P₄） secretion， as well as on the expression levels of Hippo pathway kinases， apoptosis-related proteins， and steroidogenic enzymes. The results showed that， after 24 hours of low-glucose treatment， the protein expression levels of Bax and cleaved caspase-3 in GCs were significantly upregulated （P<0.05）， while the Bcl-2 protein expression level was significantly downregulated at 48 hours （P<0.05）. The low-glucose environment significantly downregulated the expression levels of steroidogenic enzymes （CYP11A1， CYP19A1， HSD3B1， and STAR） in GCs （P<0.05） and significantly increased the ratios of P-MST1/MST1， P-YAP/YAP， and P-TAZ/TAZ （P<0.05）. Further studies found that VP treatment enhanced the low-glucose effects， increased the GC apoptosis rate， significantly reduced E₂ and P₄ concentrations （P<0.05）， elevated Bax and Cleaved-caspase 3 protein expression， downregulated Bcl-2 protein expression （P<0.05）， decreased steroidogenic enzyme protein expression levels （P<0.05）， and significantly increased the phosphorylation levels of MST1， YAP， and TAZ （P<0.05）. In contrast， PY-60 reversed the negative effects of low glucose on GCs. In summary， low glucose induces apoptosis of yak GCs by upregulating Bax and cleaved caspase-3 proteins expression and downregulating Bcl-2 protein expression. It also inhibits the secretion of E₂ and P₄ by downregulating the expression of steroidogenic enzymes （CYP11A1， CYP19A1， HSD3B1， and STAR）. Moreover， the Hippo signaling pathway is implicated in low glucose-induced functional impairments of granulosa cells in yaks. The study results provides a theoretical basis for further elucidating the regulatory mechanisms of follicular development and improving reproductive efficiency in yaks.

The aim of this study was to investigate the effects of bamboo leaf flavonoids （BLF） on liver injury， antioxidant function and related gene expression of diquat （DQ） stressed broilers.The experiment is divided into two parts. In the first part， 240 1-day-old yellow-feathered male broilers were randomly divided into 2 groups with 8 replicates per group and 15 broilers per replicate. Broilers in the blank group were fed a basal diet， and broilers in the BLF group were fed an experimental diet （1 000 mg·kg^-1 BLF was added to the basal diet）. The experiment lasted for 28 days. The growth weight and feed consumption of broilers were recorded to explore the effect of BLF on the early growth performance of yellow-feathered broilers. In the second part， 16 broilers close to the average body weight were randomly selected from the 28 d blank group and the BLF group， and divided into 4 groups， 8 birds in each group. The control group （CON） and stress group （DQ） were from the original blank group， while the BLF group and BLF-DQ group were from the original BLF group. On the 29th day， broilers in DQ and BLF-DQ groups were intraperitoneally injected with DQ （40 mg·mL^-1） at a dose of 1.0 mL·kg^-1 body weight （BW）， while the other two groups of broiler chickens were injected with the same dose of physiological saline. After 24 hours， samples were collected and indicators were measured to analyze the protective effect of BLF on DQ-induced liver injury in broilers. The results showed that： Compared to the blank group， the average daily gain （ADG） of the BLF group increased and the feed-to-gain ratio （F/G） decreased （P<0.05）. Compared with the CON group， the content of aspartate aminotransferase （AST） increased （P<0.05） in the serum of the DQ group， the level of interleukin-10 （IL-10） in the liver decreased （P<0.05）， and the contents of interleukin-6 （IL-6） and reactive oxygen species （ROS） increased （P<0.05）. The level of IL-10 in the liver increased （P<0.05）， the level of tumor necrosis factor-α （TNF-α） decreased （P<0.05）， the levels of total antioxidant capacity （T-AOC） and total superoxide dismutase （T-SOD） increased （P<0.05）， and the levels of malondialdehyde （MDA） and ROS decreased （P<0.05） in the DQ group. Compared with the DQ group， the levels of IL-6 and IL-1β in the liver of the BLF-DQ group decreased （P<0.05）， the contents of MDA and ROS decreased （P<0.05）， and the expression level of Trx-R2 gene in the liver increased （P<0.05）. Compared with the CON group， the content of IL-10 significantly increased （P<0.05）， the content of IL-1β significantly decreased （P<0.05）， and the level of ROS significantly decreased （P<0.05）.In conclusion， BLF can improve the early growth performance of broilers， and can upregulate the gene expression of SIRT1， PGC1-α， and Trx-R2， thereby enhancing the antioxidant capacity of the liver and alleviating the structural damage and inflammatory response caused by oxidative stress.

The purpose of this study was to systematically explore the dynamic effects of heat stress on oxidative damage and apoptosis of primary muscle cells in broilers and its molecular mechanism. Chicken primary muscle cells were randomly divided into control group （Normal Control， NC， 37 ℃） and heat stress group （ Heat Stress， HS， 43 ℃）. The heat stress group was treated for 6， 12， 18， 24， 30， 36， 42 and 48 h， respectively. The degree of cell damage was evaluated by detecting lactate dehydrogenase （ LDH ）， lactic acid （ LD ） and cell redox status in cell culture medium. TUNEL staining was used to evaluate the proportion of apoptosis. The expression levels of cell stress and apoptosis-related genes were detected by RT-PCR and Western blot. The results showed that： Compared with the NC group， the activity of lactate dehydrogenase （LDH）， the content of lactic acid （LD） and reactive oxygen species （ROS） increased significantly with the prolongation of heat stress time （P<0.05）. The content of malondialdehyde （MDA）， the activities of glutathione peroxidase （GSH-Px）， catalase （CAT）， total superoxide dismutase （T-SOD） and total antioxidant capacity （T-AOC） showed a trend of increasing first and then decreasing， and all reached the peak around 24 h， which was significantly higher than that of NC group （P<0.05）. Compared with the NC group， the proportion of apoptosis increased first and then decreased during 6~48 h of heat stress， and reached the highest value at 24 h （P<0.05）. The expression levels of HSP70 and TLR4 signaling pathways in cells were significantly up-regulated （P<0.05）. In summary， heat stress can significantly induce time-dependent oxidative stress and apoptosis in broiler primary muscle cells， which may be closely related to the activation of TLR4 signaling pathway. This study revealed the dynamic changes of heat stress-induced muscle cell injury， and provided a theoretical basis and in vitro model basis for further elucidating the molecular mechanism of heat stress-mediated muscle injury in broilers.

This study aimed to investigate the effects of slow-release urea on growth performance and rumen microbiota in yaks under low-altitude stall-feeding fattening conditions. Eighteen healthy male Leiwuqi yaks aged 1.5 years with similar body weight （143±25 kg） were randomly assigned to three groups， with six replicates per group and one yak per replicate. The animals were fed different protein diets： control group （CON， 12.5% CP）， low-protein group （LP， 9.5% CP）， and non-protein nitrogen group （NPN， 12.5% CP， replacing 3% soybean meal with 0.6% slow-release urea to provide an equal nitrogen level）. The pre-trial period lasted for 14 days， followed by a 35-day experimental period. The results showed that： 1） The average daily gain （ADG） of the LP group decreased by 13.7% compared with the CON group， while the feed-to-gain ratio increased by 14.67%； the ADG and feed-to-gain ratio of the NPN group were similar to those of the CON group. 2） Aspartate aminotransferase activity in the LP group showed an increasing trend compared with the CON group （P=0.082）， and the concentrations of triglycerides and glucose were higher than those in the CON and NPN groups. 3） Rumen microbial protein content in the CON and NPN groups was higher than in the LP group， and the ammonia nitrogen level was significantly higher in the CON and NPN groups than in the LP group （P<0.05）. In addition， the butyrate concentration in the NPN group was significantly lower than that of the CON group （P<0.05）. 4） Rumen microbial composition at the genus level differed among the three diets. PCoA analysis showed that the LP and NPN groups were significantly separated from the CON group （P<0.05）. Unique genera with a relative abundance >5% included norank_ f Puniceicoccaceae， norank_ f CAP-aah99b04， and p-1088-a5_gut_group in the CON group； Bergeyella， Acinetobacter， and norank_ f Carnobacteriaceae in the LP group； and unclassified_ f Erysipelatoclostridiaceae， Bifidobacterium， and norank_c Gracilibacteria in the NPN group. Among the shared genera， the phyla Spirochaetota and Fibrobacterota were significantly more abundant in the LP group than in the CON group （P<0.05）. At the genus level， Rikenellaceae_RC9_gut_group was significantly less abundant in both the LP and NPN groups compared with the CON group （P<0.05）. 5） PICRUSt functional pathway analysis showed that amino acid metabolism was significantly lower in the LP group than in the CON group （P<0.05）， whereas replication and repair and nucleotide metabolism were significantly higher in both the LP and NPN groups compared with the CON group （P<0.05）. In conclusion， under low-altitude stall-feeding conditions， reducing dietary protein levels decreased the growth performance of yaks， while appropriate supplementation with slow-release urea maintained performance close to the control level. Different protein diets significantly affected rumen fermentation parameters （e.g.， ammonia nitrogen， butyrate）， microbial community structure， and functional pathways. Overall， slow-release urea supplementation helps maintain growth performance and regulate rumen microecology in yaks.

Ligularia virgaurea is a widely distributed native functional forage on the Qinghai-Tibet Plateau. This study was conducted based on previous investigations into its regulatory effects on digestion and metabolism in Tibetan sheep. Based on this， This study systematically investigated the effects of Ligularia virgaurea supplementation on nitrogen metabolism， energy metabolism， and their coupling mechanism in grazing Tibetan sheep in alpine meadows. Thirty-two Tibetan sheep with similar body weight （（29±1.56） kg） and developmental status were randomly divided into four groups， with 8 sheep in each group. Four L.virgaurea supplementation levels were set： 0， 100， 200， and 300 mg·kg^-1 body weight （BW）， and the feeding trial lasted for 27 days. Measurements of nitrogen metabolism， energy metabolism and methane emission indices were conducted to evaluate the effects of dietary supplementation with Ligularia virgaurea on Tibetan sheep. The results showed that supplementation with Ligularia virgaurea significantly affected the relationship between general metabolism and energy metabolism of grazing Tibetan sheep （P<0.05）. The nitrogen requirement was highly significantly positively correlated with maintenance metabolizable energy （P<0.01）. At the supplemental dose of 200 mg·kg^-1 BW， Ligularia virgaurea exerted the most prominent regulatory effects on nitrogen metabolism， energy metabolism and their coupling effects in grazing Tibetan sheep， with the optimal performance in feed digestibility， nitrogen retention efficiency and energy utilization efficiency. Ligularia virgaurea significantly altered methane emissions from Tibetan sheep， and the 200 mg·kg^-1 BW group exhibited the greatest reduction in methane output. Supplementation of Ligularia virgaurea facilitated the coupling between maintenance metabolizable energy and nitrogen requirement of grazing Tibetan sheep. When maintenance metabolizable energy exceeded 7.585 MJ·d^-1， its growth rate was higher than that of nitrogen requirement. Additionally， Ligularia virgaurea promoted the coupling of energy cycle and nitrogen cycle in grazing Tibetan sheep. These findings provide a theoretical basis for the application of Ligularia virgaurea to improve nitrogen and energy metabolism as well as their coupling， and to inhibit enteric methane emissions in Tibetan sheep.

This study aimed to systematically elucidate the molecular mechanisms underlying host cell infection by Akabane virus （AKAV）. We performed integrated multi-omics analysis to delineate key response pathways and core regulatory molecules in African green monkey kidney cells （Vero cells） following AKAV infection. Vero cells were infected with goat-derived AKAV strain GZTS-1 at a multiplicity of infection （MOI） of 0.1， with uninfected cells serving as controls. Transcriptomic and proteomic profiling were conducted to identify differentially expressed genes （DEGs） and proteins （DEPs）. Bioinformatic analyses， including Gene Ontology （GO） functional annotation and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway enrichment， were performed to characterize the cellular response network. Nine DEGs were randomly selected and validated by qPCR. A total of 542 DEGs （337 upregulated， 205 downregulated） were identifiedby transcriptomics. The qPCR results correlated well with the sequencing data， confirming data reliability. Proteomics identified 579 DEPs （461 upregulated， 118 downregulated）. Integrated analysis revealed that both DEGs and DEPs were significantly enriched in critical immune and inflammatory pathways， including cytokine-cytokine receptor interaction， RIG-Ⅰ-like receptor signaling pathway and NF-κB signaling. Further screening pinpointed 22 key molecules with consistent differential expression at both transcriptional and translational levels， which are implicated in inflammatory responses， prostaglandin synthesis and immune signal transduction. In conclusion， Vero cells mount an antiviral response against AKAV primarily by activatingp the RIG-Ⅰ/NF-κB signaling axis and amplifying cytokine cascades. This study not only provides potential targets and theoretical foundations for AKAV vaccine development but also offers critical insights into its pathogenesis and host immune regulation strategies.

The significant lyophilization damage to pseudorabies virus （PRV） restricts the production of high-quality vaccines. To explore the lyophilization damage targets of PRV and provide a theoretical basis for the stable preservation of Pseudorabies Virus， this study took the Bartha-K61 strain as the research object， detected the viral titer， adsorption and internalization functions， morphological and structural integrity， and fusion activity of PRV before and after lyophilization， and comprehensively analyzed and discussed the lyophilization damage targets of Pseudorabies virus. Results showed that the viral titer loss was 2.0 lg TCID₅₀·mL^-1 after lyophilization. There was no significant change in the adsorbed nucleic acid copy number （P>0.05）， while the internalized nucleic acid copy number was extremely significantly reduced （P<0.01）， indicating that the adsorption function was not impaired but the internalization function was disrupted. The fusion activity decreased to 37.5%， showing an extremely significant difference （P<0.01）. Transmission electron microscopy （TEM） images revealed that although the viral envelope integrity was severely damaged， the nucleocapsid integrity remained intact. Consistent with this， the PMA-qPCR results also confirmed that the viral nucleocapsid was not destroyed. These findings illustrate that lyophilization damages the envelope and glycoproteins of PRV， thereby impairing its internalization and fusion capabilities.

Gosling gout caused by goose astrovirus （GAstV） is primarily characterized by the deposition of urate crystals in visceral organs and articular cavities. GAstV can be classified into two distinct genotypes， GAstV-1 and GAstV-2， and their coinfection is quite universal. The disease is transmitted horizontally via the fecal-oral route and vertically through contaminated hatching eggs， posing significant challenges for diagnosis and control. To establish a rapid ELISA for the simultaneous detection of specific antibodies against both GAstV-1 and GAstV-2 to screen and cull the seropositive breeding geese， the dominant antigenic epitopes within cap proteins of each GAstV genotype were analyzed via bioinformatics and identified by using positive serum. Immunogenic epitope peptides were fused in tandem and inserted into pET30a vector. A soluble recombinant protein containing the fused epitopes of both GAstV genotypes was successfully induced and served as the coating antigen. Reaction parameters were gradually optimized via a checkerboard titration method to establish a dual-genotype universal indirect ELISA for GAstV-specific antibody detection. Results showed that the optimal coating concentration was 60 ng per well， with the best coating condition being incubation at 37 ℃ for 1 hour. Optimal blocking was achieved using 5% skim milk at 4 ℃ overnight. The optimal dilution for positive sera was 1∶800， incubated at 37 ℃ for 1.5 hours. The optimal dilution for the HRP-conjugated rabbit anti-goose IgY secondary antibody was 1∶15 000， incubated at 37 ℃ for 1.5 hours. The optimal TMB substrate reaction condition was incubation at 37 ℃ in the dark for 5 minutes. The positive cut-off value （S/P） for this method was determined to be 0.37. The detection sensitivity for positive sera reached a dilution of 1∶3 200. Both intra-assay and inter-assay coefficients of variation （CV） were less than 10%. Furthermore， the assay showed no cross-reactivity with positive sera against other common goose viral pathogens. Testing results of clinical serum samples collected from goose farms and slaughters from Henan province showed that the concordance rate between fusion epitope-based ELISA and GAstV-1 monovalent ELISA was 86.2%， and 93.1% with a GAstV-2 counterpart. The established method provides significant technical support for the epidemiological surveillance of GAstV-1 and GAstV-2， and for the screening and culling of GAstV-seropositive breeding geese.

This study aimed to systematically evaluate the seropositivity and epidemiological characteristics of four vertically transmitted diseases—avian leukosis （AL）， reticuloendotheliosis （RE）， chicken infectious anemia （CA）， and avian reovirus disease （AR） in local breeder chickens of Sichuan Province. Using the ELISA method， serum samples collected from seven local chicken breeds across five regions—Northwestern， Northeastern， Southern， Central， and Southwestern of Sichuan—were tested for antibodies against the four pathogens. The results showed that the four pathogens exhibited varying degrees of infection in local breeder chickens in Sichuan Province. The total antibody positivity rate was highest for ARV （78.10%）， followed by CAV （55.90%） and REV （47.40%）， while the ALV-J antibody positive rate was the lowest （8.12%）. Among them， REV infection was the most widespread， with antibodies detected in all local breeder chicken flocks. Mixed infections with the four pathogens were severe， with dual infections being the most prevalent （36.80%）， while triple （20.77%） and quadruple （1.35%） infections were also observed. The severity and patterns of mixed infections varied across different geographical regions， with Southwestern Sichuan exhibiting a triple infection rate as high as 77.50%. Susceptibility to the four pathogens differed significantly among the breeds， and risk assessment further revealed the susceptibility risks of each breed. Meihua breeding chickens showed some resistance to CAV， ARV， and REV infections， while Jiuyuan Black chickens were susceptible to ALV-J and CAV. In summary， the four vertically transmitted pathogens exhibited high infection rates and extensive dissemination among the seven local chicken breeds in Sichuan Province， with severe mixed infections. This study will provide a theoretical basis for the development and conservation of local breeder chicken resources in Sichuan Province， thereby promoting the secure and sustainable development of local breeder chickens’ genetic resources.

The increasing prevalence of antibiotic resistance has driven research on bacteriophages. This study aims to isolate and screen Klebsiella pneumoniae bacteriophages， conduct research on their biological characteristics， and perform systematic genomic analysis， providing a theoretical basis for phage therapy against Klebsiella pneumoniae pneumonia. Klebsiella pneumoniae phages were isolated and purified from environmental sewage samples with Klebsiella pneumoniae KP-L9 （from a mallard duck） as the host bacterium. The biological characteristics of the phage， including its morphology， host range， multiplicity of infection （MOI）， one-step growth curve， and stability under various pH， temperature， organic solvent， and ultraviolet light conditions， were comprehensively evaluated. Furthermore， its whole genome was sequenced and analyzed. A phage PKP-2， capable of lysing multiple Klebsiella pneumoniae strains， was isolated from wastewater. Transmission electron microscopy revealed the presence of tailed bacteriophages， which can lyse four different strains of Klebsiella pneumoniae. The optimal MOI was 0.01. The latent period of PKP-2 was approximately 10 minutes， with a burst size of 132 PFU·cell^-1. It remained stable within a pH range of 3.0-12.0 and retained activity after a 30-minute incubation at 70 ℃. Moreover， it also demonstrated good tolerance to ultraviolet irradiation， though sensitivity to organic solvents varied. In vitro antibacterial assays confirmed its potent growth inhibition of host bacteria. The PKP-2 genome is a linear DNA approximately 46 909 bp in length， with a GC content of 50.747%， encoding a total of 65 genes， and no lysogenic， antibiotic resistance， or virulence-related genes were found. This study isolated the Klebsiella pneumoniae phage PKP-2， which shows strong lytic activity and stability against diverse host strains. The absence of virulence and resistance genes in its genome highlights its potential for clinical development in phage therapy against Klebsiellapneumoniae infections.

This study aimed to investigate the dynamic infection status and subtype distribution of Blastocystis in intensive pig farms. In this study， a total of 1 251 samples were collected from three large-scale pig farms in Zhumadian City， Xinyang City and Quzhou City， stratified by age group and divided into two batches. Detection was performed using a PCR method targeting the Blastocystis small subunit ribosomal RNA （SSU rRNA） gene. Positive samples were sequenced and subjected to sequence analysis. Subtypes of Blastocystis were identified using the NCBI database， and a phylogenetic tree was constructed using the neighbor-joining method to further validate the Blastocystis subtypes. The results showed that the overall infection rate of Blastocystis in the pig herds was 78.4% （981/1251）. The infection rates in Zhumadian， Xinyang， and Quzhou were 81.80%， 72.4%， and 87.5%， respectively， with statistically significant differences among the regions （χ²=21.273， P<0.01）. The infection rates across different age groups （in months） ranged from 67.8% to 85.4%， with the highest rate observed in the 4-month-old group （85.4%）. Subtype identification revealed four subtypes： ST1， ST3， ST5， and ST15. ST5 was the dominant subtype （75.56%）， and ST15 was detected in domestic pig herds for the first time in China. The results indicate that the three large-scale pig farms in this study had high infection rates of Blastocystis， with multiple zoonotic subtypes （ST1， ST3， ST5， and ST15） present， posing a significant public health risk.

This study aimed to establish an RT-qPCR detection method for rapid and accurate detection of H11 subtype avian influenza virus （AIV）. Based on the H11 subtype AIV HA gene sequences published by GISAID from 2020 to 2024 and the HA gene sequences of the same subtype detected through routine monitoring in the writer's laboratory， specific primers and probes were designed targeting conserved regions across lineages. Reaction conditions were optimized， standard curves were plotted， specificity， sensitivity， and repeatability were evaluated， and the method was applied to test 162 clinical samples preserved in the writer's laboratory. The results showed that the method demonstrated good specificity， with amplification curves observed only for the H11 subtype AIV， and no cross-reactivity with other AIV subtypes or common avian respiratory pathogens. The method exhibited good sensitivity， with the lowest detection limit of 1.08 copies·μL^-1. The method showed good repeatability， with both intra-assay and inter-assay coefficients of variation below 1%. The clinical sample test results showed that this method was consistent with virus isolation and identification results， with a 100% concordance rate. In conclusion， the detection method established in this study can provide technical support for the early monitoring， warning， prevention， and control of H11 subtype AIV.

The aim of this study was to investigate the role of exosomes derived from Echinococcus multilocularis and their RNA and proteins in inducing pathological angiogenesis in the host liver. The BALB/c mouse model of E. multilocularis infection was established by inoculating 500 protoscoleces via the hepatic portal vein. Liver tissues were collected 4 months post-infection for histopathological examination by HE staining， and the expression of the vascular endothelial marker CD31 and vascular endothelial growth factor A （VEGFA） was detected by immunohistochemistry （IHC）. Exosomes were isolated from the supernatant of in vitro-cultured protoscoleces via ultracentrifugation and subjected to different treatments including Triton X-100 alone， Triton X-100 combined with RNase， or proteinase K to obtain exosomes depleted of specific components. The treated exosomal samples were then co-cultured with human umbilical vein endothelial cells （HUVEC） to evaluate their effects on cell proliferation， cell cycle progression， migration， invasion， and tube formation. The results showed that significant pathological angiogenesis was observed in the liver tissues surrounding the alveolar hydatid cysts， with markedly upregulated expression of CD31 and VEGFA. Untreated E. multilocularis-derived exosomes significantly promoted HUVEC proliferation， cell cycle progression， migration， invasion， and tube formation. The pro-angiogenic activity of exosomes was almost completely abolished after treatment with Triton X-100 alone or Triton X-100 combined with RNase， whereas proteinase K treatment had a considerably weaker effect than Triton X-100 or Triton X-100 combined with RNase. In conclusion， exosome-derived RNA is a key factor in E. multilocularis-induced pathological angiogenesis in the host liver. This study provides a theoretical basis for elucidating the pathogenic mechanisms of E. multilocularis and developing novel therapeutic agents against pathological angiogenesis.

This study aimed to establish a quantitative real-time polymerase chain reaction （qPCR） assay with high specificity and sensitivity for the detection of Anaplasma marginale. Specific primers and probes were designed based on the msp1β gene sequence of Anaplasma marginale retrieved from GenBank. Reaction conditions and procedures were optimized to screen the optimal primer and probe concentrations as well as the annealing temperature. Subsequently， sensitivity， specificity and repeatability tests were performed， and clinical samples were further detected. The results showed that the established qPCR assay exhibited a good linear relationship， with a correlation coefficient （R²） of 0.999 2 and a slope of -3.284. The limit of detection （LOD） was 3.92 copies·μL^-1， which was 100 times more sensitive than the conventional PCR assay. This assay could only amplify the target gene of Anaplasma marginale， and no amplification was observed for 6 other blood pathogens， including Anaplasma bovis and Neospora caninum. Both the intra-assay and inter-assay coefficients of variation were less than 0.7%， showing good repeatability. When 374 clinical bovine blood samples were tested， the positive rate detected by the established qPCR assay was 5.41% higher than that detected by the conventional PCR assay.In conclusion， the qPCR method established in this study exhibits high sensitivity， strong specificity， and good reproducibility， providing reliable technical support for the early diagnosis， epidemiological investigation， prevention and control practices of Anaplasma marginale disease.

This study aimed to construct a HD11 cell line stably expressing chicken hexokinase II （HK-2） using a lentiviral vector system， thereby providing a cellular model to investigate the role of HK-2 in chicken hyperglycemia and viral infection. First， the chicken HK-2 gene sequence was amplified by PCR and cloned into the lentiviral vector pLV3-CMV-MCS-Puro. Subsequently， this construct was co-transfected with helper plasmids into 293T cells to package the lentivirus， while an EGFP-expressing lentivirus was packaged as a control. HD11 cells were infected with the viruses， and after 72 hours， a stable cell line was established through puromycin selection. The function of HK-2 was preliminarily validated by measuring glucose consumption and glucose 6-phosphate （glucose 6-phosphate， G6P） production. The established stable cell line was then infected with avian leukosis virus subgroup J （ALV-J） to assess the expression levels of glucose metabolism-related proteins and viral replication efficiency. The results showed a significant increase in HK-2 expression levels in the HK-2 stably transfected HD11 cell line following selection. Functional assays confirmed that HK-2 overexpression promoted glucose consumption and G6P production. Viral infection experiments demonstrated that HK-2 stable overexpression significantly enhanced ALV-J replication in HD11 cells， accompanied by a concurrent increase in viral Env protein expression. Furthermore， in ALV-J-infected HK-2-HD11 cells， the expression of key glucose metabolism proteins， including HK-2， PKM2， and LDHA， was correspondingly increased. In conclusion， this study successfully constructed an HD11 cell line stably expressing the chicken HK-2 gene， providing preliminary evidence that HK-2 overexpression promotes ALV-J replication and enhances the expression of glucose metabolism-related proteins. This work lays a foundation for further in-depth research on the role of HK-2 in the interplay between chicken metabolic regulation and viral infection.

African swine fever （ASF）， caused by the African swine fever virus （ASFV）， has inflicted substantial losses on the swine industry in our country. Flavonoids exhibit broad-spectrum antiviral activities. Myricetin， a type of flavonoid， has demonstrated antiviral activities against various RNA/DNA viruses. This study evaluates the role of myricetin against genotype II ASFV infectivity to porcine alveolar macrophages （PAMs）. During the infection of PAMs by ASFV， which were treated with varying concentrations of myricetin. The impacts of myricetin on the infectivity of ASFV to PAMs were evaluated using methods such as real-time quantitative PCR， Western blotting， immunofluorescence， and hemadsorption assays. Myricetin exhibits minimal toxicity to PAMs， with a CC₅₀ value of （152.270±1.091） μmol·L^-1； Myricetin can significantly reduce the viral yield of ASFV， with an IC₅₀ value of （31.670±1.489） μmol·L^-1， and it markedly inhibits the transcription of the viral early gene CP204L and the expression of its encoded protein p30； The inhibitory effect is most significant when myricetin is used to pretreat the virus and when added during the virus invasion of PAMs， whereas pretreatment of PAMs with myricetin and addition of myricetin post ASFV infection of PAMs have no significant impact on the viral yield of ASFV； Further results show that myricetin significantly inhibits the transcription of the viral early gene CP204L and the expression of its encoded protein p30 during the adhesion and internalization of ASFV into PAMs. This study is the first to reveal that myricetin exhibits a certain inhibitory effect on ASFV infection in PAMs.

To address the limitations of rapid resistance development and insufficient bactericidal persistence in single-phage therapy， this study investigated the synergistic bactericidal effects and underlying mechanisms of probiotic Bacillus combined with bacteriophage against enterohemorrhagic Escherichia coli O157：H7， providing support for the development of novel antibacterial strategies. Bacteriophage killing curves were plotted by enumerating host bacterial counts at different multiplicity of infection （MOI） gradients. Based on the in vitro antibacterial activity of probiotic Bacillus， a co-culture system was established to evaluate synergistic effects. Phage titers were determined using the double-layer agar method to assess potential impacts on phage proliferation. Host bacteria tolerant to phage lysis were collected and analyzed for phage-mediated killing characteristics. Probiotic Bacillus was further added to evaluate its synergistic inhibitory effect against the tolerant bacteria. Bacterial RNA was extracted after 24 h of culture， and the expression of host bacterial genes was detected by quantitative polymerase chain reaction （qPCR）. The O157：H7-specific phage exhibited rapid but non-persistent bactericidal activity， with host bacterial regrowth observed in later culture stages. The addition of probiotic Bacillus significantly prolonged the antibacterial effect during the late phage lysis phase without affecting phage proliferation. Notably， probiotic Bacillus also enhanced the phage-mediated killing of phage-tolerant host bacteria. qPCR analysis suggested that the synergistic mechanism may be closely associated with Bacillus-induced upregulation of host gene expression， including lacZ， lamB， hipA， sodA， and rpoS. Bacillus probiotica may significantly enhance the bacteriophage's bactericidal activity against O157：H7 by regulating bacterial energy metabolism， receptor absorption， and oxidative stress. This study provides a novel approach to address the issues of rapid resistance development and insufficient bactericidal persistence in phage applications， while also offering experimental evidence for the development of natural antimicrobial agents and the control of foodborne pathogens.

This study aimed to investigate the therapeutic efficacy of bioengineered cornea for full-thickness corneal injuries in canines， as well as the characteristics and effects of 1% tacrolimus ophthalmic solution versus 1% cyclosporine ophthalmic solution in suppressing postoperative immune rejection， thereby providing a theoretical foundation for the clinical application of bioengineered cornea in treating full-thickness corneal injuries in veterinary practice. Eighteen canines with alkali-burn-induced corneal models underwent penetrating bioengineered corneal transplantation and were randomly allocated into three groups： Group A （cyclosporine， n=6）， Group B （tacrolimus， n=6）， and Group C （normal saline control， n=6）. Postoperative clinical manifestations were observed and corneal rejection indices were recorded. Corneal optical coherence tomography （OCT） was performed on postoperative days 60 and 120. On day 120， normal and operated corneal tissues were randomly collected for histopathological examination and immunohistochemical detection of MMP-9 and CD34 protein expression. Postoperative immune rejection in all three groups showed an initial increase followed by a decline， with Group B exhibiting significantly lower rejection indices than the other two groups （P<0.01）. Fluorescein sodium staining demonstrated an overall decreasing trend， with Groups A and B showing significantly smaller stained areas than Group C on day 35 （P<0.05）. Intraocular pressure remained within the normal range in Groups A and B， while Group C showed a significant increase on day 35 （P<0.01）. Tear production in Groups A and B remained within normal physiological levels and was significantly higher than in Group C on day 35 （P<0.05）. At 77 days post-surgery， the positive rates of the dazzle reflex and menace reflex in groups A and B were significantly higher than those in group C （P<0.05）. Postoperative blood biochemical indicators related to liver and kidney function， as well as white blood cell-related parameters， were all within the normal range in each group. OCT revealed that corneal thickness in Group B most closely resembled normal thickness on day 120. Histopathological results indicated that Group B had the most regular corneal structure， with no significant neovascularization or inflammatory cell infiltration. The expression levels of CD34 and MMP-9 in Group B showed no significant difference compared to normal corneas （P>0.05）， but were significantly different from those in Group C （P<0.01）. Penetrating bioengineered corneal transplantation is feasible for treating full-thickness corneal injuries in canines. 1% tacrolimus ophthalmic solution demonstrates stronger inhibition of neovascularization and superior anti-rejection efficacy compared to 1% cyclosporine ophthalmic solution following penetrating bioengineered corneal transplantation in canines.

Previous studies have shown that polysaccharides of Atractylodes macrocephala Koidz （PAMK） can suppress the secretion of pro-inflammatory cytokines induced by various stimuli， suggesting a potential role in regulating lymphocyte homing to inflamed sites. However， the precise molecular mechanisms remain unclear. To investigate the effect of PAMK on LPS-induced splenic lymphocyte homing in goslings， one-day-old goslings were randomly divided into four groups： control group （CON）， PAMK group， LPS group， and LPS+PAMK group. The feeding trial lasted for 24 days. Hematoxylin-eosin （HE） staining was used to observe histomorphological changes in spleen tissue. Real-time quantitative PCR was performed to detect mRNA expression levels of inflammation-related factors， adhesion molecules， and chemokines； Western blot analysis was conducted to assess protein expression. In addition， transcription sequencing was carried out after lymphocyte isolation. LPS treatment led to a relative expansion of the red pulp area in the spleen， with significant increases in the areas of periellipsoidal lymphoid sheaths （PELS） and periarteriolar lymphoid sheaths （PALS） （P<0.05）， whereas PAMK treatment reversed this trend. PAMK downregulated LPS-induced expression of pro-inflammatory cytokines （e.g.， TGF-β， IL-18， IFN-γ）， adhesion molecules （e.g.， VCAM-1， ICAM-1， MAdCAM-1， Integrinβ1）， chemokines （e.g.， CXCL13， CXCR5， CCR7， CXCR4）， and endothelin-1， while upregulating the endothelial marker VE-cadherin and the chemokine CXCL12. Further investigation revealed that PAMK blocked LPS-induced adhesion molecule expression via the PI3K/AKT signaling pathway. KEGG pathway analysis indicated that focal adhesion plays an important role in the mechanism by which PAMK inhibits lymphocyte homing. Based on these findings， we further validated the RHO/ROCK2 signaling pathway and demonstrated that PAMK significantly modulated the expression of genes related to this pathway. These results indicate that PAMK suppresses LPS-induced splenic lymphocyte homing by multi-target regulation of adhesion molecules， chemokines， and inflammation-related factors， and through modulation of the PI3K/AKT and RHO/ROCK2 signaling pathways.

This study aimed to investigate the protective effects of taraxasterol on acetaminophen （APAP）-induced damage in AML12 cells and its underlying mechanisms. The AML12 cells were revived， cultured， and passaged by DMEM/F12 medium. The cytotoxicity of taraxasterol on AML12 cells was determined by MTT. An AML12 cell damage model induced by APAP was established and intervened by taraxasterol. The levels of intracellular reactive oxygen species （ROS）， malondialdehyde （MDA）， superoxide dismutase （SOD）， and reduced glutathione （GSH） were determined by kits. The levels of the inflammatory factors including interleukin-1β （IL-1β）， interleukin-6 （IL-6）， tumor necrosis factor-α （TNF-α）， and interferon-γ （IFN-γ） were measured by ELISA. The cell membrane and mitochondrial structure of AML12 cells were observed by transmission electron microscopy. TUNEL staining was used to detect the level of apoptosis of AML12 cells.Results showed： 1） Taraxasterol at concentrations ranging from 0~40 μg·mL^-1 exhibited no significant toxicity on AML12 cells. When APAP was administered at a concentration of 10 mmol·L^-1， the viability of AML12 cells was between 70%-80%. Therefore， concentrations of 5， 10， and 20 μg·mL^-1 were determined to be the therapeutic concentrations of taraxasterol， while 10 mmol·L^-1 was established as the modeling concentration of APAP. 2） Compared to the APAP group， taraxasterol at concentrations of 5， 10， and 20 μg·mL^-1 could increase the level of GSH and the activity of SOD in AML12 cells to varying degrees， reduced the concentrations of ROS and MDA， and inhibited the expression of inflammatory factors IL-1β， IL-6， TNF-α， and IFN-γ in the supernatant of AML12 cells. 3） Observations of cell morphology indicated that taraxasterol alleviated APAP-induced apoptosis as well as damage to the cell membrane and mitochondria in AML12 cells. Taraxasterol alleviates oxidative stress， inhibits excessive inflammatory response， decrease apoptosis， and improves APAP-induced damage to the hepatocyte membrane and mitochondria.

This study aims to explore the effects of nuciferine liposome （NUC-Lip） on the gene expression profile of subcutaneous adipose tissue in obese dogs using RNA sequencing technology （RNA-Seq）. Twelve obese beagle dogs induced by a high-fat diet were randomly divided into two groups： the high-fat diet group （HFD group） and the HFD+NUC-Lip group. The HFD+NUC-Lip group received NUC-Lip orally at a dosage of 125 mg·kg^-1 body weight， twice daily， for a duration of 8 weeks， while the HFD group received an equivalent volume of saline. Throughout the experiment， body weight， food intake， subcutaneous fat thickness， and blood lipids （TG， TC） were measured. HE staining was performed to observe the morphology of adipose tissue， and RNA-Seq was conducted on subcutaneous adipose tissue. The results indicated that： 1） Compared to the HFD group， the HFD+NUC-Lip group exhibited significant reductions in body weight， subcutaneous fat thickness， serum TG and TC levels， and average adipocyte area （P<0.05）. 2） The HFD+NUC-Lip group identified a total of 152 up-regulated differentially expressed genes （DEGs） and 339 down-regulated DEGs in comparison to the HFD group. Metabolism-related genes such as PPARA， CPT2， SLC2A4， and ACSS3 were significantly up-regulated， while inflammation-related genes such as CCL2， MMP9， TNF， and CD163 were significantly down-regulated. The qRT-PCR validation results were consistent with those from RNA-Seq. 3） Gene Ontology （GO） and KEGG pathway enrichment analyses revealed that differentially expressed genes （DEGs） were significantly enriched in pathways related to lipid metabolism and inflammatory responses. 4） Correlation analysis indicated that the expression of metabolic genes was negatively correlated with body weight and fat thickness （P<0.05）， while inflammatory genes showed a positive correlation （P<0.05）. In conclusion， NUC-Lip may exert anti-obesity effects by synergistically regulating metabolism and inflammatory pathways in adipose tissue， providing a scientific basis for the development of novel anti-obesity drugs and targeted interventions in adipose tissue.

This study aimed to analyze the genomic characteristics and genetic evolution of the Senecavirus A （SVA） strain SVA-CADC01-2024. Seven pairs of primers were designed based on the whole genome sequences of SVA strains in the NCBI database. The whole genome sequence of SVA-CADC01-2024 was obtained through RT-PCR amplification and sequencing， followed by sequence analysis. The results revealed that the whole genome of SVA-CADC01-2024 comprises 7 264 nucleotides and belongs to Genotype Ⅲ. It clustered within the same subclade as the domestic Chinese isolates SVA/ICR-F10 （2019） and SVA/CHN/07/2017 （2018）， exhibiting high nucleotide identities of 99.92% and 99.29%， respectively. The identities with the US isolate SVA/US-NC/NC2015/2015 （2020） and the Colombian isolate Colombia/2016 （2017） were 98.43% and 98.28%， respectively. Compared to other domestic and international isolates， this strain exhibited an amino acid mutation at position 277 of the VP2 protein （T→K）. This study successfully obtained the complete genome sequence of the SVA-CADC01-2024 strain， providing crucial data for a deeper understanding of SVA’s molecular epidemiological characteristics and evolutionary patterns. These findings offer important guidance for SVA prevention and control， as well as vaccine development.

This study aimed to combine recombinase-aided amplification （RAA） with the CRISPR/Cas12a system to establish a rapid visual detection method for BNoV， designated as the RAA-CRISPR/Cas12a assay. The complete genome of BNoV was sequenced and subjected to bioinformatics analysis， leading to the identification of a conserved Motif C （YDGG） site within the ORF1 RdRp region. Based on this， the RAA primers， reaction temperature， and reaction time were optimized， and the optimized RAA system was integrated with the CRISPR/Cas12a system to develop the RAA-CRISPR/Cas12a detection method. Using the p-CE2BNoV plasmid standard as a template， the sensitivity， specificity， and repeatability of the method were evaluated. The positive agreement rate of the method was compared against RT-PCR and RT-qPCR using 100 clinical samples. The optimal reaction conditions determined in this study were as follows： RAA primer pair F2R2， amplification at 35 ℃ for 15 minutes， and optimal final concentrations of 12.5 nmol·L^-1 for both crRNA and Cas12a in the CRISPR/Cas12a system. The detection limit of this method for p-CE2BNoV was 2.14×10^-1 copies·μL^-1. The assay showed no cross-reactivity with BVDV， BCoV， BAstV， BRV， BKV， or BNEV， and demonstrated good repeatability. When tested on clinical samples， the positive agreement rates of the RAA-CRISPR/Cas12a assay compared to RT-PCR and RT-qPCR were 100% and 93.33%， respectively. In summary， the RAA-CRISPR/Cas12a detection method established in this study is simple to operate， rapid， and highly sensitive， offering potential value for the rapid on-site diagnosis of BNoV in basic laboratories and livestock farms.

Feline infectious peritonitis （FIP） caused by feline infectious peritonitis virus （FIPV） is highly lethal， posing a serious threat to the health of cat populations， especially kittens and young cats. To develop a FIPV diagnostic method with high sensitivity， low cost and easy operation， this study combined RT-RPA with the CRISPR detection system. Based on laboratory isolation， sequencing and NCBI database alignment， the N gene sequence of FIPV strains was selected and cloned into the pUC57 vector to construct a positive control plasmid； specific RPA primers and reaction temperature were screened， CRISPR reaction parameters were optimized， and the specificity and sensitivity of the method were evaluated； LwCas13a protein was induced， expressed and purified by the Escherichia coli （E. coli） prokaryotic expression system. The active LwCas13a protein was successfully obtained； the F1R1 primer pair showed the optimal amplification efficiency， and the established FIPV-Cas13a detection method could specifically recognize FIPV nucleic acid without cross-reaction； the optimal reaction conditions were 37 ℃， crRNA dosage of 7.5 ng， LwCas13a protein dosage of 250 ng， the fluorescence was stable after 40 min of reaction， the limit of detection reached 10¹ copies·μL^-1， and visual detection could be achieved by combining with test strips. The detection of 38 clinical samples of anal swabs showed that the positive detection rates of both the fluorescence method and the test strip method were 50% （19/38）， which was highly consistent with the industry standard RT-PCR at 42% （16/38）. In conclusion， the FIPV-Cas13a detection system provides a robust and visual alternative detection method， which is suitable for on-site FIPV monitoring.

To achieve accurate differentiation between the vaccine strain and wild-type strains of duck hepatitis A virus genotype 3 （DHAV-3）， specific MGB probes and primers were designed based on the double base substitution （DBS） sites in the 3C gene of the DHAV-3 SD wild-type strain， and the SD70 live attenuated vaccine strain. A dual TaqMan-MGB probe real-time fluorescent quantitative PCR assay was established and systematically validated for the discrimination of the two strains. The results showed that the linear correlation coefficients （R²） of the standard curves for the SD wild-type strain and the SD70 vaccine strain were 0.998 4 and 0.990 5， respectively. The limit of detection reached 10 copies， and the sensitivity was approximately 100-fold higher than that of conventional RT-PCR. No cross-reactivity was observed with nine common duck pathogens. The assay could distinguish 17 DHAV-3 wild-type strains isolated in East China after 2022 from the SD70 vaccine strain. The intra-assay and inter-assay coefficients of variation were both <2%， and the detection accuracy for artificially simulated samples was 100%. This assay is sensitive， specific and reproducible， enabling the quantitative differentiation of DHAV-3 vaccine strains and wild-type strains. It provides a reliable technical tool for immune efficacy evaluation and epidemic surveillance in the prevention and control of DHAV-3.

This study aimed to construct a GADD45a gene-targeted A549 cell line and investigate its effects on the replication of H13 subtype avian influenza virus （AIV） and host innate immune response. shRNA sequences targeting GADD45a were designed and screened， and the recombinant lentiviral vector pLKO.1-CMV-copGFP-PURO was constructed. Lentiviruses were packaged using a three-plasmid system and used to infect A549 cells， followed by puromycin selection to establish an A549 cell line with stable GADD45a knockout. After infecting cells with H13N8 subtype AIV， the mRNA and protein expression levels of the viral NP gene were detected by qRT-PCR and Western blot， respectively， and the mRNA level of IFN-β was measured by qRT-PCR. The A549 cell line with stable GADD45a knockout was successfully constructed. Silencing of GADD45a significantly inhibited the replication of H13N8 subtype AIV and markedly enhanced virus-induced IFN-β production. This study preliminarily demonstrates that silencing the host factor GADD45a can promote IFN-β production and thereby suppress the replication of H13 subtype AIV， laying a solid foundation for further exploration of the molecular mechanism by which the host factor GADD45a regulates AIV replication.

Endometrial dysfunction and fibrosis induced by bovine endometritis is one of the main causes of embryo implantation and intrauterine adhesion， which has a certain impact on current livestock production. The aim of this study was to investigate the effect of curcumin on alleviating endometrial fibrosis caused by endometritis in cows by promoting autophagy. Endometrial tissues from healthy cows and those from cows with endometritis were collected for HE and Masson staining to assess fibrosis. The protein expression levels of Beclin-1， LC3， N-cadherin， E-cadherin and Vimentin in uterine tissues were detected by Western blot. The fibrosis model of cow endometrial epithelial cells （BEND） was established by adding transforming growth factor-β （TGF-β1）， and autophagy inhibitor Chloroquine （CQ） was used to detect the expression levels of autophagy and epithelial-mesenchymal transition （EMT） related proteins. Subsequently， a mouse model of lipopolysaccharide （LPS） -induced endometritis was established to investigate the therapeutic effect of curcumin on endometrial fibrosis. The results showed that endometritis induced fibrosis in the uterine tissue of dairy cows， and there were significant differences in the expression of autophagy and epithelial-mesenchymal transition related proteins in fibrosis （P<0.05）. Curcumin can activate autophagy in endometrial cells in a dose-dependent manner and improve the EMT process， thereby alleviating the fibrosis of endometrial tissue caused by inflammation. This study confirmed that curcumin could alleviate uterine fibrosis induced by endometritis in dairy cows by activating autophagy， which has guiding significance for bovine breeding practices.