The OMEGA editing system is considered the ancestor to the CRISPR-Cas system and has become a hotspot in gene editing tool research due to its compact size and RNA-guided DNA cleavage capability. This article provides a detailed introduction to the basic structure and function of the OMEGA editing system, and reviews the strategies for improving the editing efficiency and specificity of the OMEGA system nuclease through optimized design and engineered modification of guide RNA. By fusing deaminases to achieve base editing, it expands its application scope, providing references for the development and optimization of new tools.

Genetic improvement of carcass appearance and meat quality traits in broilers is pivotal for enhancing production efficiency and meeting market demands. With the rapid expansion of chilled chicken markets and increasing consumer emphasis on product quality, traits such as carcass appearance (eg., skin color, pore density, body size) and meat quality (e.g., meat color, tenderness, intramuscular fat) have become critical determinants of commercial competitiveness. This review systematically summarizes the application of genome-wide association studies (GWAS) in poultry breeding, highlighting the candidate genes identified through GWAS, transcriptomics, and multi-omics approaches. Key genes include LCORL and LDB2 (affecting body size), BCO2 and TYR (affecting skin pigmentation), Wnt3A (affecting pore development), ATP5L and COL1A2 (affecting meat color), FASN and PLA2G4F (affecting intramuscular fat deposition), as well as CAPN1 and CAST (affecting tenderness). By synthesizing current findings, this paper provides a theoretical foundation for marker-assisted breeding and proposes future research directions in functional genomics and precision breeding.

Preimplantation genetic testing (PGT) not only plays a significant role in human reproductive medicine but also has important applications in animal husbandry. In the process of breeding superior livestock, PGT can be used to introduce desirable traits, enhance genetic diversity, and improve the overall quality of the herd. By selecting embryos with specific genetic characteristics, traits such as disease resistance, productivity, and lifespan can be enhanced, thus promoting progress in animal breeding programs. Embryo selection based on genomic evaluation increases selection intensity and shortens generation intervals, thereby increasing genetic gain. However, current PGT mainly employs invasive methods, which can damage the structural integrity of embryos. Therefore, preserving the structural integrity of embryos to the greatest extent can better maintain their viability. Currently, non-invasive PGT (ni-PGT) methods are being explored to assess the developmental potential of embryos before implantation, including the detection of cell-free DNA (cfDNA) in blastocoel fluid and embryo culture medium. This article summarizes the methods of PGT and non-invasive PGT for embryos, and compiles the application progress of existing PGT in animal husbandry and human assisted reproduction, with the aim of providing theoretical references for the wide application of ni-PGT in the field of animal husbandry.

Intestinal flora is an important part of the host defense mechanism, and plays a crucial role in animal health and its host defense against pathogenic microbial infections, and an imbalance of intestinal flora can lead to a variety of intestinal diseases. Fecal microbiota transplantation (FMT) is one of the most effective methods to regulate intestinal microbiota and treat intestinal diseases. Intestinal dysfunction caused by intestinal microbiota imbalance can be improved by FMT. In this paper, the potential mechanism of FMT in improving intestinal barrier function and the development prospect of FMT from the perspective of intestinal microbial barrier, chemical barrier, physical barrier and immune barrier of piglets were reviewed, in order to provide reference for promoting the application of FMT specification in pig production.

Brucella spp. is the pathogen that causes zoonotic brucellosis. Brucellosis is one of the important diseases that threaten public health safety in China. The direct and indirect economic losses caused by brucellosis in our country reach billions of dollars every year. According to the statistics of World Organization for Animal Health and World Animal Health Information System, the epidemic situation of brucellosis in most regions is still severe except Western Europe, parts of North America and Australia. The clinical treatment of brucellosis has always been based on antibiotic therapy. In recent years, researches have shown that Brucella spp. has an obvious trend of resistance to commonly used antibiotics, which has brought challenges to clinical treatment. This article mainly summarizes the researches on antibiotic resistance of Brucella spp. from 2004 to 2024, analyzes the current situation and mechanism of antibiotic resistance of Brucella spp., provides reference for the prevention and control of brucellosis, and reduces the direct and indirect losses caused by chronic brucellosis due to antibiotic resistance.

The objective of this study was to estimate and compare genetic parameters of different general resilience traits using longitudinal daily feed intake data of Large White and Landrace pigs. Growth and feed intake records data from 1 692 Large white and Landrace finishing pigs were collected via automatic feeders, and used to analyze average daily gain (ADG), average daily feed intake (ADFI), average daily feeding duration (ADFD), average daily visit times (ADVT) and 8 general resilience traits were analyzed. Daily variability in feed intake (Var_FI), daily variability in feeding duration (Var_FD), daily variability in visit times (Var_VT), the log-transformed variance of deviation for the daily feed intake (LnVar_FI), the log-transformed variance of deviation for the daily feeding duration (LnVar_FD), the log-transformed variance of deviation for the daily visit times (LnVar_VT), off-feed days for corresponding feed intake (OFF_FI), and off-feed days for corresponding feeding duration (OFF_FD) were calculated as general resilience traits. The heritability and genetic correlation of the above traits were calculated using the single and multiple traits models, respectively. The results showed that the heritability of ADG, ADFI, ADFD and ADVT were 0.25, 0.49, 0.53 and 0.43, respectively. The heritability of general resilience traits Var_FI, Var_FD, Var_VT, LnVar_FI, LnVar_FD, LnVar_VT, OFF_FI and OFF_FD were 0.33, 0.26, 0.38, 0.39, 0.29, 0.37, 0.14, and 0.16, respectively. The genetic correlation was positive and high between ADG and ADFI with value of 0.83. The genetic correlation between ADG and general resilience traits ranged from -0.85 to 0.83. Specifically, there was a high positive correlation (r=0.83) between both Var_FI and ADG and between LnVar_FI and ADG, while a high negative correlation (r=-0.85) was observed between OFF_FD and ADG. There was a high negative correlation both between LnVar_FD and ADVT, and between LnVar_VT and ADG, with values of -0.89 and -0.76, respectively. In conclusion, we calculated and compared the genetic parameters of different general resilience traits, and found that the heritability of these general resilience indicators was low to moderate. Among them, LnVar_FI and Var_FI exhibited high heritability and high genetic correlations with production traits, and can be considered as evaluation indicators of general resilience traits in pigs for individual selection. The results of this study provide important theoretical basis for the breeding of general disease resistance or resilience-related traits in pigs.

This study aimed to investigate the lipid and volatile organic compound (VOC) profiles in the longissimus dorsi muscle of Ningxiang pigs across different growth stages and to analyze the correlation between lipids and VOCs.Thirty-six healthy one-day-old Ningxiang pigs (average live weight: 0.98±0.09 kg) were randomly allocated to 6 groups (n=6 per group, 1∶1 of male: female ratio). Each group was raised for 60, 120, 180, 240, 300, or 360 days prior to slaughter, respectively. Post-slaughter, samples of the left longissimus dorsi were collected for meat quality assessment. Lipid profiles were analyzed using high-performance liquid chromatography-mass spectrometry (HPLC-MS), and VOC profiles were analyzed using gas chromatography-ion mobility spectrometry (GC-IMS). The results indicated: Meat pH at 60 and 120 days was significantly lower than at 360 days (P < 0.05). The L* value (lightness) of meat color significantly decreased after 240 days (P < 0.05), while the b* value (yellowness) gradually decreased after 180 days. The a* value (redness) at 60 and 120 days was significantly lower than at other ages (P < 0.05). Shear force (indicating tenderness) was significantly lower at 60 days compared to other ages (P < 0.05), with no significant differences among other age groups (P>0.05). Drip loss significantly decreased at 240 days (P < 0.05) and remained stable thereafter. Intramuscular fat (IMF) content increased with age, showing a significant increase at 240 days (P < 0.05), with no significant differences observed in subsequent age groups. Lipidomics analysis identified 55 lipid subclasses comprising 1 841 lipid molecules. Principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) revealed clear differentiation of lipid profiles among age groups. During the growth period, the peaks or troughs in the percentage contents of major differential lipid subclasses (triglycerides (TG), ether-linked phosphatidylcholine (EtherPC), ether-linked phosphatidylethanolamine (EtherPE), phosphatidylcholine (PC), diglycerides (DG), acylcarnitine (CAR), and sphingomyelin (SM)) all occurred at 240 d. Fifty potential lipid biomarkers (variable importance in projection (VIP) >1, P < 0.05) were identified, primarily phospholipids (PC, phosphatidylethanolamine (PE)), along with TG, fatty acids (FA), and DG. Enrichment analysis highlighted 7 lipid metabolic pathways, with glycerophospholipid metabolism being the most significant, followed by sphingolipid and ether lipid metabolism. Flavoromics analysis identified 24, 31, 34, 57, 37, and 36 known VOCs in the 60-, 120-, 180-, 240-, 300-, and 360-day groups, respectively. VOC fingerprinting and PCA demonstrated distinct VOC profiles for each age group. Nineteen key volatile flavor compounds were identified, including decanal, nonanal, hexanal, 1-penten-3-one, 1-octen-3-ol, ethyl acetate, and methional. Correlation analysis revealed strong associations between VOCs and phospholipids/glycerides. As Ningxiang pigs aged, their meat color became increasingly bright red, and water-holding capacity improved significantly from 240 days. TG, PC, and PE were identified as key lipids potentially involved in VOC formation. The 240-day stage was determined to be the "window period" for optimal muscle flavor quality. This study provides a scientific basis for regulating pork flavor quality and optimizing the slaughter age of Ningxiang pigs.

This study aimed to investigate the differences between porcine white and beige adipocytes at the three-dimensional (3D) genome level, identify cell type-specific promoter-enhancer interactions (PEIs) in both adipocyte types, and screen for critical enhancers that promote the differentiation of porcine beige adipocytes. This study differentiated porcine white and beige adipocytes, and employed high-throughput chromosome conformation capture (Hi-C) to compare differences in chromatin compartments, topologically associating domains (TADs), and chromatin loops between the two adipocyte types. We comprehensively resolved the 3D genome architecture of porcine adipocytes. Using genome-wide H3K27ac and H3K4me3 ChIP-seq data, the study identified promoter-enhancer interactions (PEIs) in both adipocyte types and screened for potential enhancers that promote the differentiation of pig beige adipocytes. The activity of these enhancers was further validated through the construction of dual-luciferase reporter vectors. The Hi-C data revealed divergent 3D genome architectures between the two adipocyte types, with chromatin compartment switching occurring in 5% of genomic regions. A total of 1 923 white adipose-specific TADs (topologically associating domains) and 12 920 white adipose-specific loops were identified, while 2 010 beige adipose-specific TADs and 9 526 beige adipose-specific loops were also detected. ChIP-seq results revealed the identification of 795 white adipose-specific PEIs and 737 beige adipose-specific PEIs. By integrating transcriptomic data, we identified gene enhancers that are specifically highly expressed in beige adipocytes and exhibit cell type-specific PEIs, which were prioritized as critical enhancers promoting beige adipocyte differentiation. Furthermore, dual-luciferase reporter plasmids carrying 525 bp (TMEM100 gene) and 200 bp (AMCF-Ⅱ gene) candidate enhancer fragments were constructed. Luciferase activity assays demonstrated that these putative enhancers upregulated luciferase activity significantly (P < 0.05). This study constructed three-dimensional genome maps of porcine white and beige adipocytes, revealing differences in chromatin spatial structures between the two types of adipocytes. We validated the enhancer activity of the TMEM100 and AMCF-Ⅱ genes preliminarily, suggesting that the enhancers of both genes may promote the differentiation of porcine beige adipocytes through PEIs regulation.

This study aimed to clarify the fat deposition characteristics and lipid composition of the new black pig strain "Jinwu pig", providing a theoretical basis for its further breeding and promotion. Backfat tissue of 7-month-old "Jinwu pigs" was used as the experimental group, with commercial Duroc×Landrace×Large White pigs of comparable slaughter weight ((117±5)kg) as the control. Frozen sections were prepared to analyze adipocyte size and morphology. Non-targeted lipidomics combined with a Waters UPLC coupled to a Q Exactive high-resolution mass spectrometer was applied to determine lipid composition. Data annotation was performed using Lipid Search software, and multivariate statistical analyses including principal component analysis (PCA), partial least squares discriminant analysis (PLS-DA), and variable importance in projection (VIP) were conducted. Lipids were considered differentially expressed when VIP>1, FC>1.5 or FC < 0.65, and P < 0.05. Histological analysis showed that the adipocyte diameter of "Jinwu pigs" ((199±14)μm) was significantly larger than that of Duroc×Landrace×Large White pigs ((120±15)μm) (P < 0.05). Lipidomics revealed 95 lipids with significantly different abundance between the two groups (34 upregulated, 61 downregulated), mainly enriched in phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and triacylglycerol (TG). Functional enrichment analysis indicated that these differential lipids were involved in cell membrane components, glycerophosphocholine (neurotransmitter synthesis), glycerophospholipids (membrane structure and signaling), endoplasmic reticulum and unsaturated fatty acids. Correlation analysis showed that adipocyte diameter was negatively correlated with PC lipids, but positively correlated with TG, PE, and PS lipids. Compared with Duroc×Landrace×Large White pigs, "Jinwu pigs" exhibited larger adipocytes and greater lipid deposition in backfat tissue. Lipidomic profiling revealed higher abundance of TG, PE, and PS, but lower abundance of PC. These findings elucidate the lipid composition characteristics of "Jinwu pigs" at the lipidomics level and provide a theoretical basis for understanding the breed-specific traits of black pigs.

This study aimed to generate XIST-knockout fibroblast cell lines in Chinese Huaxi cattle using CRISPR-Cas12i^HIFI-mediated gene editing. Ear-edge fibroblast cells from a healthy adult bull (No. 22284#) were used as donors. Multiple sgRNAs were designed targeting the bovine XIST gene, and 6 highly efficient sgRNAs were selected and grouped into 3 editing strategies. A total of 15 ninety-six-well plates of cells were collected per strategy and subjected to electroporation. Off-target analysis confirmed the specificity of the selected sgRNAs. As a result, we successfully established XIST-knockout fibroblast models, obtaining five homozygous and one heterozygous mutant cell line. This results demonstrates the high efficiency of Cas12i in editing GC-rich repetitive sequences. Our work provides essential experimental material and theoretical basis for elucidating the mechanism of X-chromosome inactivation (XCI) and improving the developmental efficiency of cloned bovine embryos.

The aim of this study was to investigate the effect of interfering with MAPK6 (extracellular signal regulated kinase 3, ERK3) gene on the differentiation of Qinchuan cattle myoblasts and its action mechanism. The expression profile of the MAPK6 gene in different tissues of Qinchuan cattle was detected using qRT-PCR technology. siRNA technology was used to interfere with the expression of MAPK6 gene in Qinchuan cattle myoblasts. The interference efficiency and expression levels of myotube differentiation related genes were detected by qRT-PCR and Western blot, and changes in myotube phenotype were observed. Subsequently, transcriptome sequencing was performed to screen differentially expressed genes, followed by GO functional annotation and KEGG pathway enrichment analysis. Additionally, preliminary validation of myogenic differentiation-related pathways was conducted. The results indicated that MAPK6 was highly expressed in the longissimus dorsi muscle tissue of Qinchuan cattle. Interfering with MAPK6 significantly promoted myotube differentiation and fusion in Qinchuan cattle myoblasts (P < 0.01), significantly increased the expression of myogenic differentiation-related genes MEF2C and MYH7 (P < 0.01) and the protein expression of MEF2C and MYL2 (P < 0.01), and significantly increased the protein expression of MYH7 (P < 0.05). The transcriptome results showed that, compared with the control group, there were 3 224 differentially expressed genes after interfering with MAPK6, of which 1 707 were upregulated and 1 517 were downregulated. The KEGG enrichment results showed that differential genes were mainly enriched in the PI3K-AKT and MAPK signaling pathways. Interfering with the MAPK6 gene could significantly reduces the expression of ERK1/2 and p-ERK1/2 proteins (P < 0.01) in the MAPK signaling pathway. This study preliminarily elucidates the role and mechanism of MAPK6 gene in beef muscle production, laying a foundation for improving beef quality and guiding molecular breeding strategies in beef cattle.

This study aimed to investigate the effect of PFN1 on the myogenic differentiation of bovine skeletal muscle satellite cells (BSMSCs) through its interaction with PTEN. Small interfering RNA (siRNA) and overexpression plasmids were transfected into primary bovine skeletal muscle satellite cells isolated from fetal bovine leg muscles, with 3 biological replicates for each treatment. The differentiation process of the cells was observed under an optical microscope. RNA and protein samples were collected at specific time points during the differentiation phase for analysis by quantitative real-time PCR (qRT-PCR) and Western blot. PFN1 knockdown promoted the formation of more robust myotubes, with significantly increased MyHC mRNA and protein levels. Conversely, PFN1 overexpression reduced myotube formation (thinner and fewer) and significantly decreased MyHC expression. Co-IP confirmed the interaction between PFN1 and PTEN, and PFN1 overexpression increased PTEN protein levels. Under optical microscope observation, PTEN knockdown resulted in fewer myotubes, with elevated MyHC and MyOG mRNA levels but reduced MyHC protein levels. Key proteins in the PI3K/AKT/mTOR pathway were downregulated, while p-mTOR levels increased. PFN1 knockdown upregulated key proteins in the PI3K/AKT/mTOR pathway, whereas PFN1 overexpression downregulated them. PFN1 interacts with PTEN to inhibit the PI3K/AKT/mTOR pathway, thereby negatively regulating BSMSCs myogenic differentiation. This study provides new insights into the molecular regulatory network of muscle development.

This study aimed to evaluate the call rate, genotype concordance, imputation accuracy, and genomic prediction accuracy of the self-developed 13K and 40K liquid-phase SNP chips for dairy cattle. Genotyping was performed on 203 individuals in the validation population using the 13K, 40K, and existing commercial 126K and 150K SNP chips. Call rates and genotype concordances were then compared. Beagle software was used to impute the genotypic data of the 4 chips to a 50K density level based on the Chinese Holstein genomic selection reference population. The GBLUP method was employed to assess the genomic prediction accuracy for 9 traits in the validation population. Imputation accuracy and genomic prediction accuracy were then compared among the 4 chips. In the validation population, the call rates of the 13K and 40K chips were 97.70% and 97.20%, respectively, while those of the 126K and 150K chips were 99.30% and 98.27%. The 13K and 40K chips shared 11 331 and 11 125 SNPs and 42 504 and 40 881 SNPs with the 126K and 150K chips, respectively, with genotype concordance rates of 96.23%, 96.50%, 97.18%, and 97.43%. The 13K chip (MAF>0.01) achieved an accuracy of 95.90%, slightly lower than that of high-density commercial chips but higher than the international low-density Illumina 7K (94.30%) chips. Meanwhile, The imputation accuracy of the 40K chip (MAF>0.01) reached 99.07%, comparable to the 126K (99.23%) and 150K chips (99.05%).The genomic prediction accuracies for 9 traits—milk yield, fat percentage, protein percentage, fat yield, protein yield, udder system score, final score, somatic cell score, and feet and legs score—using the 13K, 40K, 126K, and 150K chips ranged from 0.619 0 to 0.735 2, 0.642 0 to 0.758 7, 0.620 8 to 0.742 8, and 0.633 4 to 0.751 4, respectively, with no significant differences observed. The findings indicate that the self-developed 13K and 40K liquid-phase SNP chips demonstrate robust performance and are suitable for early genetic evaluation and elite selection in Holstein dairy cattle.

To screen for molecular markers of the eyelid-colobomus trait and the dark/light yellow feather trait in geese, in this study, the nucleotide polymorphic loci in the coding sequences (CDS) of candidate genes (FREM1, SLC45A2, TYRP1, and PTPRM) were detected using the skin samples of different phenotypic individuals (n=10, totally 40, regardless of genders) in 1-day-old goslings. The genomic DNA of 95 goslings was used to detect the key polymorphic loci identified on the CDS, and the association of the loci with the traits was analyzed. This study also carried out a similar analysis on the intron sequence of the candidate gene PRLR gene. In addition, to explore the molecular basis underlying the formation of the traits, this study screened out differentially expressed genes (DEGs) and their enriched pathways through transcriptome sequencing analysis of skin samples. The results showed that a total of 15 single nucleotide polymorphisms (SNPs) loci (SNP1-SNP15) were identified over the CDS of FREM1 gene, of which 7 were missense mutations and 8 were synonymous mutations, and SNP10, SNP14 and SNP15 loci were significantly associated with the eyelid-colobomus trait. A total of 7 SNPs loci (SNP1-SNP7) were identified over the CDS of PTPRM gene, of which 2 were missense mutations and 5 were synonymous mutations, and SNP5 was significantly associated with the eyelid-colobomus trait at the allele level. A total of 11 SNP loci (SNP1-SNP11) were identified over the CDS of SLC45A2 gene, of which 10 were missense mutations and 1 was synonymous mutation, and no locus was significantly associated with the dark/light yellow feather trait. A total of 2 SNPs loci (SNP1 and SNP2) were identified over the CDS of TYRP1 gene, both of which were synonymous mutations, and their association with the dark/light yellow feather trait was close to the significant level at the allele level. Deletion or insertion mutation (INDEL), namely insertion mutation 1 (INS1), insertion mutation 2 (INS2) and deletion mutation (DEL), were identified over the intron of PRLR gene, and were significantly associated with the dark/light yellow feather trait. In addition, a total of 131 DEGs (62 up-regulated and 69 down-regulated) related to the eyelid-colobomus trait were identified by transcriptome sequencing analysis. These genes were mainly enriched in the ECM-receptor interaction pathway and the cell adhesion molecules pathway. A total of 1 280 DEGs (92 up-regulated and 1 188 down-regulated) related to the feather color trait were identified. These genes were mainly enriched in the neuroactive ligand-receptor interaction pathway, the tyrosine metabolism pathway, the phenylalanine metabolism pathway and the calcium signaling pathway. In summary, the results of association analysis have not only identified some molecular markers significantly correlated with the eyelid-colobomus trait and the dark/light yellow feather trait, but also provided more scientific evidence that FREM1, PTPRM, PRLR, and TYRP1 are the causative genes for these traits. In addition, the transcriptomic sequencing analysis suggested that the pathways including ECM-receptor interaction, cell adhesion molecules, neuroactive ligand-receptor interaction and tyrosine metabolism are involved in the formation of these traits.

The study aimed to identify the candidate genes associated with breast muscle rate in 28-day-old squabs by the analysis of the genome-wide SNPs and breast muscle transcriptomic data from two populations of pigeons. Ten pigeons from each of the M and B lines were selected for whole genome sequencing. Selective signature analysis was performed based on genomic wide SNPs with extracting and filtering. Ten 28-day-old squabs were selected from two populations with significant differences in breast muscle rate. The transcriptomic data were collected from breast muscles. The expression levels of genes and identification of differentially expressed genes (DEGs) were performed in the pectoral muscle. The key genes associated with breast muscle rate in 28-day-old squabs were identified and screened by combining selection signal analysis. The expression levels of 9 DEGs associated with muscle development were analyzed using real-time fluorescent quantitative PCR (RT-qPCR) and compared with transcriptome data for validation. M-line and B-line squabs were distinctly clustering into separate groups through principal component analysis (PCA) of 11 577 846 SNPs. Twenty-nine genes associated with muscle development were identified through sliding window Fst and pFst analysis. In the breast muscle of 28-day-old squabs, 192 DEGs were identified through transcriptome analysis, including 68 up-regulated and 124 down-regulated genes. The DEGs were mainly enriched in the nuclear component, apoptosis pathway, Foxo signaling pathway, and PI3K-Akt signaling pathway. Four genes were identified with the integrated analysis of whole-genome and transcriptome data. Among these genes, LTBP1, DPP4, and TNFSF10 are associated with muscle development. The data foundation was provided for deciphering the differences in the breast muscle rate among squabs. The expression levels of 9 DEGs validated by RT-qPCR showed a correlation coefficient of 0.50-0.95 with transcriptomic data, indicating that the two datasets were highly consistent. In this study, genes associated with breast muscle rate differences in squabs were revealed by analyzing genome-wide combined with transcriptome data, providing a new perspective on the molecular mechanism of breast muscle development in pigeons.

In order to investigate the dynamic transcriptional differences of round spermatids, the present study was conducted on adult Guanzhong black pigs (10-12 months), based on 10×single cell RNA sequencing (scRNA-seq) data from testicular tissues, to analyze germ cell clustering and round spermatids reclustering, and to functionally annotate and enrich differentially expressed genes in round spermatids, with a view to investigating the developmental changes of round spermatozoa. We analyzed germ cell clustering and round spermatids reclustering, as well as functional annotation and enrichment of differentially expressed genes in round spermatids, in order to investigate the developmental changes of round spermatids. The results identified 5 porcine round spermatids types, mapped the cellular trajectories of early round spermatids from development to the spermatid stage, and found that the differentially expressed genes in round spermatids and spermatozoa were related to the process of protein binding and endoplasmic reticulum activity, mainly enriched in pathways such as spermatid development through GO and KEGG enrichment analyses; histone variant analyses showed that the fischerin family and the convertin family might also play an important role in the development of porcine round spermatids. Candidate genes were verified by immunohistochemical assays. Based on the results, it was hypothesized that SUN5, SPATA3, and APOC4 could be used as regulatory genes for round spermatids development. This study can provide theoretical basis for the subsequent injection of round spermatozoa into the cytoplasm of the egg by biotechnology, and also provide reference information for porcine round spermatids monocytomics.

The present study was designed to examine the effects of L-malic acid (L-MA) on the cell proliferation of porcine ovarian granulosa cells (PGCs) and to uncover the potential mechanism based on transcriptomic analysis. In this study, primary PGCs were isolated, cultured and randomly assigned to either a control group or L-MA treatment groups (10, 25, 50, 100, and 200 μmol·L^-1). CCK-8 assay, EdU incorporation assay (5-ethynyl-2'-deoxyuridine) and qPCR analysis were used to assess the cell viability and cell proliferation. Furthermore, RNA sequencing (RNA-seq) was employed to delineate L-MA-induced the transcriptional landscape alterations in PGCs. Compared with the control group, 25, 50, 100 and 200 μmol·L^-1 L-MA significantly increased cell viability of PGCs (P < 0.05). PGCs exhibited the highest cell viability at an L-MA final concentration of 50 μmol·L^-1. Treatment with 50 μmol·L^-1 L-MA markedly upregulated the mRNA expression of proliferating cell nuclear antigen (PCNA) (P < 0.01), a key proliferation marker, and enhanced the proportion of EdU-positive cells in PGCs. Transcriptomic analysis revealed that under the threshold of |FC|≥1.5 and P < 0.05, 169 significantly upregulated and 118 downregulated genes were identified in PGCs with L-MA exposure. GO enrichment analysis demonstrated that differentially expressed genes (DEGs) were predominantly enriched in cellular components related to cell cycle complexes and nucleases, with molecular functions and biological processes related to genetic material replication, epigenetic modification, substance transport and metabolism, as well as cell proliferation and differentiation. KEGG and GSEA analyses further indicated that DEGs were primarily involved in signaling pathways associated with cellular immunity, genetic material replication, substance metabolism, and reproductive functions. Notably, predictions of transcription factor (TF) and binding sequences between TF and TF's target genes suggested that ELF3 was identified as the most likely candidate transcriptional factor mediated by L-MA, with potential binding sites within the PCNA promoter region. In summary, the findings demonstrate that L-MA significantly enhances the proliferation of porcine ovarian granulosa cells. This proliferative effect may be attributed to L-MA-mediated regulation of gene expression involving cell cycle complexes and nucleases, enhancement of molecular functions and biological processes related to genetic material replication, epigenetic modification, and substance transport, as well as improvement of cell health. This study provides important scientific evidence for the potential application of L-malic acid-based products in modulating granulosa cell proliferation and ovarian function.

The aim of this study was to explore the reproductive toxicity mechanism of gossypol-induced testicular tissue injury and TM4 cells(mouse testicular sertoli cells) apoptosis in mice. A gossypol intragastric administration model in male ICR(improved castle road) mice (0, 30 (mg·kg) ·d^-1) for 30 consecutive days) and a gossypol exposure model in TM4 cells (0, 15, 20, 25 μmol·L^-1) were established. Subsequently, the sperm quality of mice was assessed by an automatic sperm analysis system; the histopathological alterations of testicular tissue were observed via HE staining; and the expression levels of apoptosis, autophagy, ferroptosis, tight junction, and spermatogenesis-related genes in testicular tissue, as well as apoptosis-related genes in TM4 cells, were detected using real-time fluorescence quantitative PCR, and the expression of apoptosis-related proteins in testicular tissue was detected by Western blot. The findings revealed that gossypol significantly decreased the weight gain rate, sperm motility, and sperm density of mice (P < 0.01), and elicited histopathological changes in testicular tissue (such as atrophy of seminiferous tubules, destruction of some sertoli cells, and reduction in sperm count). The expression trends of genes related to the three death modes of ferroptosis, autophagy, and apoptosis in testicular tissue all changed, and the expression trend of apoptosis-related genes changed most significantly. The expression of the apoptosis inhibitory gene Bcl-2 was significantly downregulated (P < 0.05), while the expressions of the apoptosis promoting genes Bax, Tnf-α, and the Bax/Bcl-2 ratio were upregulated (P < 0.01, P < 0.05, P < 0.01). The expressions of apoptosis proteins Bax, Tnf-α, and the Bax/Bcl-2 ratio in testicular tissue were significantly increased (P < 0.05). Additionally, the expressions of tight junction and spermatogenesis-related genes in testicular tissue were significantly downregulated. In the in vitro experiment with TM4 cells, exposure to different concentrations of gossypol significantly influenced the expression of apoptosis-related genes within TM4 cells, and the expressions of apoptosis promoting genes Bax, Tnf-α, and the Bax/Bcl-2 ratio were Extremely significantly upregulated (P < 0.01), which was consistent with the quantitative results of testicular tissue. In summary, the weakened spermatogenic ability of the testis in gossypol-exposed mice is closely related to the apoptosis of TM4 cells, and this apoptosis is associated with the increased expressions of Bax, Tnf-α, and Bax/Bcl-2.

This study aims to investigate the effects of different short-chain fatty acids (SCFAs) on the growth, behavior, microstructure of brain and liver, and the expression of inflammatory cytokines in young mice. The experimental subjects were newborn C57BL/6J pups (7 groups, 8 mice in each group). The pups in each group were from eight litters of different female mice, and were given gavage with different SCFAs concentrations for 28 consecutive days. The treatment groups were: CTL (0.9% saline), SF-900 group (900 mg·kg^-1 BW sodium formate), SF-1400 group (1 400 mg·kg^-1 BW sodium formate), SP-2 (20 mg·kg^-1 BW sodium propionate), SP-200 (200 mg·kg^-1 BW sodium propionate), SV-5 (5 mg·kg^-1 BW sodium valerate), and SV-20 (20 mg·kg^-1 BW sodium valerate). Body weight, brain index, and liver index were recorded, and spontaneous activity was assessed using the open-field test. Additionally, the expression levels of relevant genes in brain and liver tissues were measured. The results indicated that propionate (SP-2) and valerate (SV-5) significantly enhanced weight gain in young mice, with the propionate group (SP-2) showing the most significant weight increase by 12.83%. HE staining revealed that the microstructure of brain and liver tissues remained intact across all groups, with no significant pathological changes. Behavioral tests showed that formate-treated mice (SF-1400) and valerate-treated mice (SV-5, SV-20) exhibited significantly increased spontaneous activity and exploratory behavior, suggesting that these SCFAs might modulate behavioral responses through the central nervous system. Furthermore, the valerate-treated groups (SV-5, SV-20) regulated the expression of neurotrophic factors BDNF and TRKB in the brain, potentially promoting neuronal survival and synaptic plasticity through increased levels of neurotrophic factors and receptor signaling pathways (such as GPR41). RT-qPCR results revealed that propionate treatment significantly upregulated the expression of anti-inflammatory cytokines IL-4 and IL-10 in liver tissues, while downregulating pro-inflammatory cytokines IL-6, TNF-α, and IFN-γ, indicating that propionate has a positive regulatory effect on liver immune function. In conclusion, SCFAs, particularly propionate and valerate, positively influence the growth and behavior of young mice by regulating immune responses and neurodevelopmental pathways. These findings support the potential role of SCFAs in improving immune function and promoting neurodevelopment.

This experiment aimed to investigate the effects of adding quercetagetin (QG) to the diet on the slaughter performance, meat quality, serum antioxidant and immune functions, and gut microbiota of broiler chickens challenged with hydrogen peroxide (H₂O₂). A single-factor completely randomized design was adopted in the experiment. A total of 240 one-day-old Cobb broiler chickens were selected and divided into three groups: the control group (fed a basal diet+intraperitoneal injection of 1.0 mL·kg^-1 BW of normal saline at 37 days of age), the H₂O₂ group (fed a basal diet+intraperitoneal injection of 1.0 mL·kg^-1 BW of a 10% H₂O₂ solution at 37 days of age), and the QG group (fed a basal diet supplemented with 100 mg·kg^-1 QG+intraperitoneal injection of 1.0 mL·kg^-1 BW of a 10% H₂O₂ solution at 37 days of age). Each group had 8 replicates, with 10 chickens in each replicate. The experimental period lasted for 42 days in total. The results showed that: 1) Compared with the control group, the slaughter rate and leg muscle rate of broiler chickens in the H₂O₂ group were decreased (P < 0.05); The addition of QG to the diet led to no significant difference in the slaughter rate and leg muscle rate of broiler chickens compared with the control group (P>0.05). 2) Compared with the control group, the redness (a^*) of the leg muscle in the H₂O₂ group decreased significantly (P < 0.05), and the cooking loss increased significantly (P < 0.05); The addition of QG to the diet resulted in no significantly difference in the a^* value and cooking loss of the leg muscle compared with the control group (P>0.05). 3) Compared with the control group, the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), total antioxidant capacity (T-AOC), and catalase (CAT), as well as the contents of immunoglobulin A (IgA), immunoglobulin G (IgG), and complement 3 (C3) in the serum of broiler chickens in the H₂O₂ group were decreased significantly (P < 0.05), while the malondialdehyde (MDA) level and the contents of immunoglobulin M (IgM) and tumor necrosis factor-α (TNF-α) were increased significantly (P < 0.05); However, compared with the H₂O₂ group, the addition of QG to the diet significantly increased the activities of SOD, GSH-Px, T-AOC, and CAT, as well as the contents of IgA, and C3 in the serum of broiler chickens (P < 0.05), and significantly decreased the MDA level (P < 0.05). 4) Firmicutes and Bacteroidota were the dominant phyla at the phylum level in the cecum of broiler chickens. Compared with the control group, the relative abundance of Firmicutes in the H₂O₂ group decreased significantly (P < 0.05), and the relative abundance of Bacteroides in the QG group increased significantly (P < 0.05). In conclusion, the addition of 100 mg·kg^-1 of QG to the diet can mitigate the adverse effects of H₂O₂-induced oxidative stress on the slaughter performance and meat quality of broiler chickens by enhancing antioxidant and immune functions and optimizing the structure of the cecal microbiota. This provides a theoretical basis for the scientific application of QG in the healthy farming of broiler chickens.

This study aimed to explore the arginine (Arg) requirement model of WOD 188 broilers aged 1-14 days under the condition of low-protein diets. A total of 600 1-day-old WOD 188 broilers were selected for the experiment, with half male and half female, and they were reared separately. Each gender was randomly divided into 5 treatments, including 1 control group with normal protein level diet and 4 low-protein diet (crude protein was reduced by 1.5 percentage points) groups with different Arg levels. The ratios of standard ileal digestible Arg (SID Arg) to SID Lys levels (SID Arg/SID Lys) were 92% (LP92), 104% (LP104), 113% (LP113), and 124% (LP124) respectively. Each treatment had 6 replicates, with 10 chickens per replicate. The results showed that: 1) A quadratic regression model for the Arg requirement of 1-14-day-old WOD 188 broilers was obtained by fitting the pattern of body weight gain (BWG) and the SID Arg/SID Lys level ratio in low-protein diets (Female broilers: BWG/g=-0.041X²+9.197X-140.429, R²=0.998; Male broilers: BWG/g=-0.067X²+14.87X-399.52, R²=0.910); A dynamic Arg requirement model was derived with metabolic body weight (BW^0.70) and BWG as independent variables, and SID Arg intake (Y) as the dependent variable (Female broilers: Y=1.772BW^0.70+2.699BWG; Male broilers: Y=23.243BW^0.70+24.067BWG). The optimal production performance of female broilers was achieved when the SID Arg/SID Lys in the low-protein diet was 111%-112%; The optimal production performance of male broilers was achieved when the SID Arg/SID Lys in the diet was 108%-112%. 2) For female broilers, compared with the control group, supplementing Arg in the low-protein diet group significantly reduced the bursa of Fabricius index. The bursa of Fabricius indexes in the LP92, LP104, LP113, and LP124 groups were reduced by 39%, 28%, 33%, and 28% respectively (P < 0.05); For male broilers, compared with the conventional diet group, the spleen indexes in the LP92, LP104, LP113, and LP124 groups were reduced by 20%, 10%, 30%, and 20% respectively (P < 0.05). 3) Compared with the control group, the fecal nitrogen content in the low-protein diet groups were significantly reduced. The fecal nitrogen contents in the LP92, LP104, LP113 and LP124 groups of female broilers were reduced by 8.3%, 8.8%, 10.2% and 10.7% respectively (P < 0.01), while no significant difference was obtain in the index of nitrogen retention rate. The fecal nitrogen contents in the LP92, LP104, LP113, and LP124 groups of male broilers were reduced by 8.8%, 8.8%, 7.2%, and 12.3% respectively (P < 0.01), and the nitrogen retention rate in the low-protein diet group of LP92 was reduced by 4.9% (P < 0.05). 4) The contents of serum total protein (TP) and albumin (ALB) in the LP92 group were significantly lower than those in other groups (P < 0.01), and there was no significant difference between other low-protein diet groups and the control group. In conclusion, this study established the Arg requirement model of 1-14-day-old WOD 188 broilers under the condition of low-protein diets. Adding an appropriate amount of Arg to low-protein diets can improve the growth performance of WOD 188 broilers and reduce nitrogen emissions.

The intestinal development and health of early-weaned piglets play a critical role in determining their future growth performance. During this period, the maturation of intestinal physiological functions not only supports current nutritional requirements but also establishes a solid foundation for subsequent rapid growth. This study was designed to investigate the effects of supplementing isotonic protein solution during the suckling period on growth performance, intestinal morphology, and jejunal transcriptomic profiles in suckling piglets. A total of 120 suckling piglets (Duroc×(Landrace×Large White) three-way cross) from 12 litters with comparable birth weights (1.48±0.22 kg) and sows of the same parity were selected and randomly assigned to either a control group (Con group) or an experimental group (Px group), with 6 litters per group, based on similarity in body weight. From postnatal days 2 to 8, piglets in the Con group received 500 mL of distilled water per litter daily, whereas those in the Px group received 500 mL of 3% isotonic protein solution per litter daily. Three days prior to weaning, both groups were supplemented with 250 g of starter feed paste per day; The paste for the Con group was prepared using distilled water, while that for the Px group was prepared using the 3% isotonic protein solution. The experimental period lasted 24 days. Results indicated that isotonic protein supplementation significantly improved weaning weight, average daily gain during days 1-24 and days 16-24, and enhanced jejunal villus height (P < 0.05). Transcriptomic analysis of jejunal tissues identified 163 significantly differentially expressed genes, including 105 up-regulated and 58 down-regulated genes. GO functional annotation and KEGG pathway enrichment analyses revealed that these genes were primarily involved in key biological processes and signaling pathways, such as ion transport (SLC46A1, SLC40A1, SLC26A6), mineral absorption (S100G), ferroptosis regulation, amino acid metabolism, and vitamin digestion, also include specific genes such as ICA and TRPM6. These findings suggest that isotonic protein supplementation during the suckling period enhances growth performance and promotes intestinal development in suckling piglets. The underlying mechanism may be associated with alterations in the expression of genes involved in mineral and vitamin absorption and transport in the jejunum, thereby influencing intestinal development and nutrient utilization efficiency.

Babesia is a kind of zoonotic pathogen transmitted by ticks, surviving in human or animal red blood cells (RBCs). After infection, it can cause clinical symptoms such as fever, anemia and hemoglobinuria in humans or animals, and even death, posing a severe threat to the health of humans, especially those with asplenia, malignant tumors, and immunosuppression. Previous epidemiological surveys demonstrated the significant differences in the infection rates of Babesia in different domestic animals, but the specific pathogenic mechanisms remain unclear. This study aims to provide a basis for further elucidating the pathogenic mechanisms of Babesia, through comparing the infectivity, changes in blood indices, and immune responses of Babesia microti in different common laboratory animals. In this study, mice, rabbits and sheep were inoculated with B. microti respectively. Then parasitemia, blood parameters and anti-parasite specific protein antibodies were monitored from 0 to 36 d post-infection. The results showed that the parasitemia in mice reached the peak (62.9%) on the 4th day after innoculation, with a copy number of 6.05×10⁷ copies·μL^-1, while the parasitemia in New Zealand rabbits and sheep was about zero. Hematology tests demonstrated that the number of RBCs, hemoglobin concentration (HGB), and hematocrit (HCT) in the mice blood were significantly decreased during the early and middle monitoring term (0-20 d post-inoculation). During 0-8 d post-infection, the number of platelets (PLT) and platelet packed volume (PCT) were prominently lower than those in the control group, but with a backup later to a normal level. ELISA test showed that anti-B. microti secreted antigen 1 (BmSA1) antibody in mice increased continuously to the highest level at 16 d post-infection and then kept at high level until the end of the tested term. Anti-BmSA1 antibody in New Zealand white rabbits speedly rose up to the highest level at 8 d post-infection and persisted to the end of the mornitoring time, while sheep showed low immune response. The results suggest that mice were susceptible to B. microti which led to abnormal changes in the blood, while rabbits and sheep were resistant to the B. microti infection. This specificity may be associated with the genetic diversity of the recognition site in hosts. Rabbits and sheep demonstrated different anti-BmSA1 antibody levels, which hinted the innate differences in the anti-Babesia mechanisms of the two species. The present study laid a base for further research on the Babesia-host interaction mechanism, as well as vaccine and drug exploration.

The aim of this study is to investigate the immune protection effect of recombinant Eimeria tenella RON2_L2ecto protein on chicks. The full-length sequence encoding EtRON2_L2 protein from Eimeria tenella cDNA was amplified and the extracellular domain of EtRON2_L2 was expressed through prokaryutic recombination in this study. A hundred and twenty 7-day-old healthy Roman Grey male chicks with similar body weight (raised in clean and coccidia-free environment, fed with diet without coccidiostats or artibiotic) were randomly allotted into 5 groups, including negative control group (without immune or coccidia infection), positive control group (without immune but infected with coccida), and 50 μg, 100 μg, 200 μg recombinant protein immune groups. Recombinant protein was mixed with Freund's adjuvant as a subunit vaccine to immunize chicks in three immune groups. The immune protection effect of recombinant protein was evaluated by measuring the body weight gain, cecal lesion score, number of oocysts per gram of feces (OPG), calculating the anticoccidial index, observing the histopathological changes in the cecum, detecting serum cytokines (IL-2, IL-10, IFN-γ) and serum IgG, and the secreting levels of cecal mucosal sIgA. The results showed that the extracellular domain of EtRON2_L2 was successfully expressed, with a size of 107 ku. The immune protection experiment indicated that compared with the non-immunized group, the average body weight gain of the 100 μg protein immunization group and the 200 μg protein immunization group significantly increased (P < 0.05), while the OPG significantly decreased (P < 0.05), the cecal lesion scores of each dose of immune group showed a significant decrease (P < 0.05), pathological histology observation shows that the degree of cecal lesions has been alleviated to some extent. The anti-coccidiosis index of the 100 μg protein immunization group and the 200 μg protein immunization group is greater than 160, indicating moderate anti-coccidiosis activity. The spleen index of three protein immune groups were also significantly increased (P < 0.05). At 28 days of age (the 7th day after infection), the serum cytokines IL-2, IFN-γ, and IL-10 in the protein immune groups were significantly higher (P < 0.01) than those in the two control groups, and the serum IgG levels were significantly higher than those in the negative control group (P < 0.05). The sIgA secretion levels in the 50 μg protein immune group were significantly higher (P < 0.05) than those in the other immune groups and the two control groups. The above results indicate that the extracellular domain of RON2_L2 has an immunoprotective effect on chicks infected with Eimeria tenella.

This study aims to develop a blocking ELISA method for detecting antibodies against Lawsonia intracellularis. The recombinant MltA protein of L. intracellularis, expressed in a prokaryotic system, was used to immunize mice. Monoclonal antibodies were successfully prepared, and their specificity was confirmed through Western blot and indirect immunofluorescence assay (IFA). A blocking ELISA method for detecting L. intracellularis antibodies was developed, utilizing the recombinant MltA protein as the coating antigen and the test serum as the primary antibody. The optimal reaction conditions for the blocking ELISA method were established, and the method's sensitivity, specificity, repeatability, and coincidence rate were thoroughly evaluated. The analysis of the sensitivity and specificity of the established method demonstrated that positive results could be consistently detected at a 1∶4 dilution of L. intracellularis-positive serum. No cross-reactivity was observed with porcine epidemic diarrhea virus, pseudorabies virus, or Escherichia coli positive sera. The repeatability test results revealed that the coefficient of variation (CV) for intra-assay repeatability ranged from 1.4% to 9.3%, while the CV for inter-assay repeatability ranged from 1.0% to 9.6%. Compared with the IFA result, the method developed in this study achieved a sensitivity of 95.91%, a specificity of 80.65%, and an overall agreement rate of 85.92%.This study conducted the prokaryotic expression and purification of the L. intracellularis outer membrane protein MltA. Monoclonal antibodies targeting MltA were successfully prepared. A blocking ELISA method for detecting L. intracellularis antibodies was developed, which can be effectively applied to the clinical diagnosis of porcine proliferative enteropathy.

Staphylococcus aureus is a common pathogen in the poultry industry, causing significant economic losses due to its multiple virulence factors and multidrug resistance. To investigate the epidemiological characteristics of Staphylococcus aureus from chicken arthritis sources in Shenyang, Liaoning, a total of 78 strains of Staphylococcus aureus isolated from chicken arthritis sources between 2023 and 2024 were analyzed for virulence and resistance genes. The distribution of virulence genes and antibiotic resistance genes was detected using polymerase chain reaction (PCR). Antibiotic susceptibility was determined by the Kirby-Bauer (K-B) method, and biofilm formation capacity was assessed via crystal violet staining. Among the 15 virulence genes tested, the adhesion gene clfA had the highest detection rate (100%), followed by the adhesion gene clfB (98.7%) and the hemolysin gene hlA (82%). In contrast, the toxic shock syndrome toxin gene tssT (12.8%), enterotoxin gene seA (3.8%), and exfoliative toxin gene etA (1.3%) showed low detection rates. Antibiotic resistance testing against 10 antibiotics revealed that all 78 strains exhibited varying degrees of resistance. High resistance rates were observed for tetracycline (100%), penicillin G (98.7%), norfloxacin (96.2%), clindamycin (89.7%), trimethoprim-sulfamethoxazole (84.6%), gentamicin (82.1%), and erythromycin (79.5%). However, the strains were highly susceptible to cefoxitin and rifampicin (97.4%). Among the 15 antibiotic resistance genes tested, the aminoglycoside resistance genes aac(6') /aph(2″) and ant(4') were detected at rates of 92.3% and 80.8%, respectively. The macrolide resistance genes ermC and ermB were detected at 53.8% and 50.0%, respectively, while the tetracycline resistance gene tetM was only detected in 19.2% of strains. Biofilm formation assays demonstrated that 73 strains (93.6%) exhibited biofilm-forming capacity. Correlation analysis revealed significant associations between aminoglycoside resistance genes (aac(6') /aph(2″) and ant(4')), macrolide resistance genes (ermB and ermC), and the multidrug resistance gene cfr with resistance to gentamicin, erythromycin, and penicillin G, respectively (P < 0.05). For the correlation between biofilm phenotype and antibiotic resistance phenotypes, the results indicated that the development of erythromycin- and chloramphenicol-resistant strains was significantly correlated with biofilm formation (P < 0.05). The 78 strains of Staphylococcus aureus isolated from chicken arthritis exhibited strong drug resistance and a high propensity for biofilm formation, providing valuable insights for the prevention and control of chicken arthritis caused by Staphylococcus aureus in this region.

In this study, a combination of network pharmacology, molecular docking, and experimental validation was used to jointly investigate the therapeutic mechanism of Bidens pilosa L. against bacterial diarrhea in chickens. Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) was used to identify the pharmacological components and targets of Bidens pilosa L., while the target genes related to bacterial diarrhea were analyzed in GeneCards, OMIM and DisgeNET databases, the intersecting targets were identified using the Venny database. Then, the 'Bidens pilosa L.-Component-Target-Diarrhea Network Diagram' was drawn by Cytoscape 3.10.2 software. STRING database was used to construct the protein-protein interaction (PPI) network and identify core genes; DAVID database was used to analyze GO enrichment and KEGG pathway enrichment; AutoDock was used for molecular docking validation; A chicken E. coli diarrhea model was established to observe the effect on intestinal tissue morphology and validate the target protein content using ELISA. The results showed that, the main active ingredients of E. coli for the treatment of chicken bacterial diarrhea were luteolin, coreopsin, 6, 7-dihydroxybenzofuran, and quercetin, corresponding to 215 target genes; After removing the duplicates, a total of 82 targets were intersected with the disease, and the main target proteins were interleukin-6 (IL-6), interleukin-1 beta (IL-1β), Caspase-3 (CASP3), Matrix metalloproteinase-9 (MMP9), and Hypoxia-inducible factor 1-alpha (HIF-1alpha); GO Functional enrichment analysis showed that the main targets of Bidens pilosa L.were related to apoptosis and inflammation; KEGG pathway enrichment analysis showed that the main targets of Bidens pilosa L. were concentrated in the signaling pathways such as HIF-1α, C-type lectin receptor, IL-17, TNF, and so on; Molecular docking results showed that Bidens pilosa L. had a good connectivity between the main active components and the key targets. Tissue section and ELISA results showed that Bidens pilosa L. significantly alleviated intestinal tissue damage and significantly reduced the levels of IL-6, IL-1β, CASP3, MMP9 and HIF-1α proteins in the jejunum (P < 0.05 or P < 0.000 1). The network pharmacology and experimental validation revealed that Bidens pilosa L. may regulate the development of intestinal inflammation by modulating the expression of IL-6, IL-1β, CASP3, MMP9 and HIF-1α, which in turn affects the signaling pathways such as HIF-1α, IL-17, TNF, etc., providing new ideas and methods for in-depth research on the mechanism of action of Bidens pilosa L. in the treatment of bacterial diarrhea in chickens.

This experiment aims to study the immunogenicity of Brucella antigen btpA and its immune protective effect on mice. In the experiment, the btpA gene of Brucella strain was amplified by PCR and cloned into the pET-22b prokaryotic expression vector to obtain the recombinant plasmid pET-22b-BtpA. After double enzyme digestion and identification, it was induced with IPTG in E.coli BL21 (DE3). The target protein was expressed and purified and then identified by SDS-PAGE and Western blot. 100 μg per mouse of rbtpA protein was intraperitoneally injected into 6-week-old SPF grade BALB/c mice, and the mice were boosted with 50 μg per mouse on the 14th day. Blood was collected from the tail vein of mice on 7 d, 14 d, 21 d and 28 d, respectively. Brucella-specific IgG antibodies and antibody subtypes (IgG1 and IgG2a) in mouse sera were detected using an indirect ELISA. The number of IFN-γ-releasing lymphocytes in mouse spleens was detected using the ELISpot method. The mice were challenged with the Brucella epidemic strain (including 2×10⁵ CFU per mouse) for 28 days, and necropsy was performed 15 days later. The protective efficacy of the subunit vaccine was evaluated based on the Brucella load in the spleen of the mice.The results showed that 30 ku purified recombinant BtpA protein (rBtpA) was obtained; Mice produced Brucella-specific antibodies 7 days after rBtpA immunization, with the highest levels at 28 days, while undetectable in the PBS group; After rBtpA-immunized mice 14 days and 28 days, the IgG2a/IgG1 values were all greater than 1, and were more significant at 28 days, indicating that rBtpA induced a Th1-biased immune response in mice. Within 28 days, the number of IFN-γ-releasing splenic lymphocytes in mice was significantly higher than that in the PBS control group, indicating that mice can produce a higher level of Th1-type cellular immune response after rBtpA immunization. The spleen index SI of mice in the rBtpA group was significantly lower than that of the PBS group (P < 0.001), and the spleen bacterial load was also significantly lower than that of the PBS group (P < 0.01), indicating that the spleen index and spleen bacterial load of mice in the rBtpA subunit vaccine group were much lower. The results indicate that the rBtpA subunit vaccine has the potential to be a candidate vaccine for brucellosis. This experiment laid the foundation for the development of Brucella rBtpA subunit vaccine.

The aim of this study was to investigate the characteristics of ferroptosis induced by recombinant Candida krusei 14-3-3 (rCK14-3-3) protein in bovine mammary epithelial cells (MAC-T), providing a theoretical basis for revealing the mechanism of damage to bovine mammary epithelial cells by Candida krusei virulence factors. The optimal concentration and action time of rCK14-3-3 protein on MAC-T were screened by CCK-8 method. MAC-T cells were divided into blank control group, positive control group (10 μmol·L^-1 Erastin), experimental group Ⅰ (different concentrations of rCK14-3-3 protein (25, 50, 75 μg·mL^-1)), experimental group Ⅱ (different concentrations of rCK14-3-3 protein (25, 50, 75 μg·mL^-1) +ferroptosis inhibitor Ferrostatin-1 (20 μmol·L^-1)), and experimental group Ⅲ (different concentrations of rCK14-3-3 protein (25, 50, 75 μg·mL^-1) +Nrf2-specific inhibitor ML385 (25 μmol·L^-1)). The morphological characteristics of ferroptosis were detected by transmission electron microscopy. The expression levels of Nrf2-SLC7A11/GPX4 signaling pathway proteins induced by rCK14-3-3 in MAC-T were detected by Western blot. The expression levels of glutathione peroxidase 4 (GPX4), reactive oxygen species (ROS), and mitochondrial membrane potential (JC-1) were detected by immunofluorescence. The contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH), and ferrous ions (Fe²⁺) in MAC-T were detected by colorimetry. The results showed that compared with the blank control group, the positive control group and experimental group Ⅰ showed obvious ferroptosis characteristics. The expression levels of Nrf2, GPX4, SLC7A11 proteins and GSH content in the positive control group and experimental group Ⅰ were significantly decreased (P < 0.05), while the contents of ROS, MDA, and Fe²⁺ were significantly increased, and the content of JC-1 was significantly decreased (P < 0.05). Compared with experimental group Ⅰ, the expression levels of Nrf2, GPX4, SLC7A11 proteins and GSH content in experimental group Ⅱ were significantly increased (P < 0.05), while the contents of ROS, MDA, and Fe²⁺ were significantly decreased, and the content of JC-1 was significantly increased (P < 0.05). In experimental group Ⅲ, the expression levels of Nrf2, GPX4, SLC7A11 proteins and GSH content were significantly decreased (P < 0.05), while the contents of ROS, MDA, and Fe²⁺ were significantly increased, and the content of JC-1 was significantly decreased (P < 0.05). This study demonstrated that rCK14-3-3 protein could induce ferroptosis in MAC-T through the Nrf2-SLC7A11/GPX4 signaling pathway, providing data support for future targeted intervention in ferroptosis to prevent and treat Candida-induced bovine mastitis.

In recent years, bovine respiratory diseases have been increasingly reported, posing significant threats to the cattle industry. Large-scale outbreaks can result in substantial and often irreversible economic losses for livestock operations. This study aims to investigate the prevalence of key respiratory pathogens—Mycoplasma bovis (M. bovis), Mannheimia haemolytica (Mh), Pasteurella multocida (Pm), and Klebsiella pneumoniae (K.pn) —in calves from the Ningxia Hui Autonomous Region. A total of 457 nasal swab and tissue samples were collected from cattle across various counties and districts in Ningxia between 2022 and 2024. Pathogen identification was conducted using a combination of morphological examination and molecular diagnostic techniques. The infection rates of M. bovis, Mh, Pm, and K.pn were systematically analyzed based on year, geographic region, season, and developmental stage of the cattle. Among the four pathogens studied, Pm exhibited the highest overall detection rate (38.73%), a difference that was highly significant compared with the other three pathogens (P < 0.001). In the central arid zone, the prevalence of M. bovis was significantly greater than in both the Yellow River irrigation area (P < 0.01) and the southern mountainous region (P < 0.001). Seasonally, winter yielded peak detection rates for M. bovis (50%) and K.pn (70.5%), both of which exceeded summer and autumn rates (P < 0.001). Conversely, Pm reached its highest autumn prevalence (45.3%), significantly surpassing its winter (P < 0.01) and spring (P < 0.001) levels. During the growing phase, Pm detection was markedly higher than during both the lactation and mature phases (P < 0.001). Finally, single Pm infections were the most common (16.85%), significantly outnumbering mixed infections (P < 0.01), and the most frequent mixed co-infection occurred with Mh+Pm at 7.22%. This comprehensive epidemiological analysis delineates how temporal, geographical, seasonal, and developmental variables affect bovine respiratory pathogen prevalence in Ningxia, thereby furnishing a scientific framework to guide targeted prevention and control strategies for bacterial respiratory infections in the region.

Mycoplama synoviae (MS) is a significant avian pathogen, and its FtsZ protein (filamentous temperature-sensitive protein Z) has good immunogenicity. This study aimed to establish an indirect enzyme-linked immunosorbent assay (ELISA) for detecting MS antibodies based on the FtsZ gene of the MS-FJ01 strain, a conserved cell division protein with high immunogenicity. The FtsZ gene from the MS-FJ01 strain was cloned into pET-28a vector and expressed in Escherichia coli. The purified recombinant protein was used as a coating antigen to optimize ELISA conditions. Specificity and reproducibility was evaluated using sera from MS-infected and other pathogen-infected chickens. The established MS indirect ELISA antibody detection method demonstrated high specificity, and only had specific reaction with the positive serum of MS. The method showed no cross-reactivity with serum of MG, NDV, AIV H9, E. coli and Salmonella. The cutoff value (S/P≥0.498) was determined based on 30 negative sera (${\bar x}$=0.132, s=0.122). Intra-and inter-assay coefficients of variation (CV) were 1.32%-4.78% and 2.81%-4.72%, repeatability. Compared with IDEXX, the positive, negative, and total coincidence rates were 82.1%, 95.8%, and 82%, repeatability. This study successfully established an indirect ELISA antibody detection method based on the FtsZ protein of MS. The method demonstrates high specificity and reproducibility, offering a cost-effective tool for MS diagnosis and epidemiological surveilance.

Since the autumn and winter of 2018, a newly emerged infectious disease characterized by decreased egg production and diarrhea in laying hens has been observed in China's poultry farming regions. To investigate the etiology of this condition. Intestinal tissue samples from affected chickens were collected and pathogenic agents were isolated through 10 consecutive blind passages in specific pathogen-free (SPF) chicken embryos. The isolated viral strain was characterized for physicochemical and hemagglutination properties. Suspected virus-specific primers were designed to perform PCR detegtion, gene sequencing and nucleotide homology analysis on the extracted viral nucleic acid. Based on the alignment result, primers were designed to obtain the whole genome nucleotide sequence of the virus. The novel avian coronavirus strain AvCoV/CH/SD/24 was used to challenge SPF chickens, AA+ white-feathered broilers, and Hy-Line laying hens. Intestinal tissues from infected SPF chickens were processed for histopathological sections and serum antibody titers against infectious bronchitis virus (IBV) in different chicken groups post-challenge were measured using enzyme-linked immunosorbent assay (ELISA). Four novel avian coronaviruses (AvCoV/CH/BZ/24, AvCoV/CH/JS/24, AvCoV/CH/SD/24 and AvCoV/CH/HB/24) were successfully isolated, these viruses exhibited no hemagglutinating activity.The results of nucleic acid detection showed that four samples tested positive for both the N and S1 genes of avian infectious bronchitis virus (IBV). The N gene sequences showed the highest homology with avian infectious bronchitis virus strains in the GenBank database, while the S1 gene sequences exhibited maximum identity with the avian coronavirus AvCoV/TZ/CA127/19. Whole genome amplification and sequencing revealed that the four novel avian coronaviruses demonstrated 97.1%-98.6% nucleotide homology with two previously reported avian coronavirus strains: one isolated from Anhui Province, China in 2016 and another from Arusha, Tanzania in 2019. Phylogenetic analysis revealed that the S gene of the novel avian coronavirus is closely related to a turkey coronavirus-like, forming distinct subclades within the same evolutionary branch, while its other genes clustered within the evolutionary branch of the GⅠ-19 lineage infectious bronchitis virus. Animal experiments demonstrated that the AvCoV/CH/SD/24 strain induced diarrhea in SPF chickens, accompanied by duodenal villus detachment, congestion and hemorrhage at the villus tips and lamina propria and granular degeneration of renal tubular epithelial cells with interstitial congestion. The virus was primarily excreted through the cloaca with the highest viral loads detected in the intestines and bursa of Fabricus among tested organs. Infection did not affect the weight gain rate of chickens. ELISA results indicated serological cross-reactivity between the recombinant novel avian coronavirus and infectious bronchitis virus. In laying hens, infection led to reduced egg production and diarrhea, which was consistent with the disease in the field. Four novel avian coronaviruses were isolated from the intestines of laying hens exhibiting egg production decline and diarrhea, these newly isolated avian coronaviruses are recombinant viruses capable of inducing egg production decline and diarrhea in chickens.

To construct the immunogen of the S1 protein of avian infectious bronchitis virus (IBV), the amino acid sequences of the published S1 neutralizing epitopes were compared with the S1 protein of ck/CH/LDL/091022 (LDL091022), current prevalent GI-19 representative strain in China. The amino acid sequences at positions 22-100 and 410-492 of the S1 protein were recognized as principal neutralizing epitope areas, named as B1 and B2, respectively. Plasmids encoding LDL091022 B1 and B2 epitope peptides and plasmids with chicken IgY Fc fragment fused at the C-terminus of B1 and B2 were constructed. The significant increase of protein expression caused by IgY Fc fragment was demonstrated by indirect immunofluorescence assay (IFA) and Western blot assay. The recombinant plasmids were transfected into Expi 293F cells, and the recombinant proteins B1-Fc and B2-Fc with IgY Fc fragments were purified. Western blot and ELISA were used to assess the reactivity of B1-Fc and B2-Fc with LDL091022 serum, the results showed that B2-Fc had significantly better reactivity than B1-Fc. SPF chickens were immunized with the plasmid pCAGGS-B2-Fc and the recombinant protein B2-Fc. The serum antibody levels were detected by ELISA and IFA, the results demonstrated that B2-Fc booster immunization effectively induced antibodies against the B2 epitope. And the IFA result revealed the serum could react with LDL091022-S1 protein and B2-Fc. Moreover, the neutralization test showed that the serum neutralization titer was 16. These results demonstrated that the IBV S1 protein neutralizing epitope-Fc fusion protein prepared in this study showed good expression level and immunogenicity. It could induce certain levels of neutralizing antibodies, providing insights for the development of novel IBV subunit vaccines.

The objective of this study is to develop a quality-controled product that can be used in the RNA detection of infectious bronchitis virus (IBV). The relatively conserved N protein gene of IBV was selected as the target sequence, and it was cloned into the vector pET-28a(+) with the MS2 phage capsid protein gene and packaging site sequence, and the RNA containing the N protein gene sequence of IBV was obtained by induction expression and purification, and its physical characteristics were observed by electron microscopy, and the copy number was accurately quantified by digital PCR. The real-time RT-PCR method introduced in the national standard GB/T23197—2022 was used to verify the detection of purified armor RNA, and the RNase enzyme resistance and storage stability were verified as well. The armored RNA containing IBV N gene was successfully constructed, which was resistant to the degradation of RNase enzyme and could be stably stored at 37℃ for above 15 days. The developed armor RNA has the potential to become a positive control product for IBV, which can realize the quality control of the whole process of RNA virus detection.

This study aimed to construct a viral vector vaccine expressing the Cap protein of porcine circovirus type 3 (PCV3). In this study, pseudorabies virus (PRV) SX1910 strain was designated as the parental strain. Using CRISPR/Cas9 and homologous recombination technologies, a recombinant virus SX1910-ΔgE/gI-GFP was constructed, which lacked the gE and gI genes and harbored a LoxP site and GFP fluorescent marker. Then, the GFP expression cassette of SX1910-ΔgE/gI-GFP was replaced with the double expression cassette of the PCV3 Cap gene by using Cre enzyme, generating the recombinant virus rSX1910-ΔgE/gI-2Cap_PCV3. Then its in vitro biological characteristics and immunological effects were analysed. The results showed that rSX1910-ΔgE/gI-2Cap_PCV3 exhibited significantly reduced in vitro replication capacity and plaque size compared to the parental strain SX1910. Preliminary evaluation of immunogenicity in 6-week-old female BALB/c mice demonstrated that rSX1910-ΔgE/gI-2Cap_PCV3 effectively stimulated the production of Cap protein-specific antibodies in mice, alleviated PCV3-induced lung tissue damage, and significantly reduced viral loads in tissues. Therefore, rSX1910-ΔgE/gI-2Cap_PCV3 can elicit a good immune response in mice, providing a scientific foundation for the development of novel vaccines for PCV3.

This study aimed to establish a rapid and convenient triplex fluorescent quantitative PCR assay for simultaneous detection of porcine circovirus (PCV) types 2, 3, and 4, and to apply this method for the detection and molecular epidemiological analysis of PCV2, PCV3, and PCV4 in clinically suspected PCV-infected samples. Three pairs of specific primers and three TaqMan probes labeled with distinct fluorescent groups were designed and synthesized based on the Rep gene sequences of PCV2 and PCV3 and the Cap gene sequence of PCV4. The reaction system and conditions were optimized, and recombinant plasmids were constructed as positive controls to generate standard curves. The specificity, sensitivity, and reproducibility of the assay were systematically evaluated. The developed triplex fluorescent quantitative PCR assay demonstrated high specificity with no cross-reactivity among different PCV genotypes or common swine pathogens. The lower detection limit was 1.0×10² copies·μL^-1 for all targets, with intra-and inter-assay coefficients of variation both below 2.0%, indicating excellent reproducibility. Cap gene amplification, cloning, and sequencing of positive samples yielded 41 sequences, including 11 PCV2a, 8 PCV2b, 15 PCV2d, and 7 PCV3a strains. Sequence analysis revealed 96.7%-99.7% homology among 34 PCV2 Cap proteins, 97.2%-99.9% homology among 7 PCV3 Cap proteins. This study successfully established a triplex fluorescent quantitative PCR assay capable of detecting co-infections with different PCV genotypes. The results confirmed PCV2d and PCV3a as the predominant circulating genotypes in China. The assay provides a rapid and accurate diagnostic tool for clinical detection of PCV infections.

This study aims to systematically evaluate and analyze the prevalence of porcine reproductive and respiratory syndrome virus (PRRSV) in pig populations within the Yangtze River Delta region of China, providing a scientific basis for effective prevention and control strategies. A comprehensive literature search was conducted across five major databases: China National Knowledge Infrastructure (CNKI), Wanfang Data, VIP Chinese Science and Technology Journals Database, PubMed, and Science Direct. Studies on the prevalence of PRRSV in pig populations in the Yangtze River Delta region, collected since the outbreak of PRRSV in China in 1995, was systematically reviewed and subjected to Meta-analysis. A total of 36 articles were included in the Meta-analysis, involving 28 767 pigs, of which 8 277 tested positive for PRRSV. The overall prevalence of PRRSV in pig populations in the Yangtze River Delta region was 35.8% (95% CI: 0.266, 0.456). Among the provinces, Anhui exhibited the highest prevalence at 51.5% (95% CI: 0.321, 0.706), followed by Jiangsu at 45.0% (95% CI: 0.315, 0.589) and Shanghai at 41.2% (95% CI: 0.211, 0.629). Zhejiang had the lowest prevalence at 31.0% (95% CI: 0.213, 0.416). The highest infection rate was observed in suckling piglets (34.1%; 95% CI: 0.181, 0.521), followed by nursery pigs (30.4%; 95% CI: 0.077, 0.598), and finishing pigs (21.5%; 95% CI: 0.115, 0.333). The subgroup analysis and meta-regression analysis revealed that the heterogeneity in the studies was not associated with sampling location, sampling time, diagnostic methods, clinical symptoms, pig developmental stage, or study quality (P>0.05). However, factors such as sample type, feeding practices, and health status were found to significantly affect the prevalence of PRRSV (P < 0.05). The prevalence of PRRSV in pig populations within the Yangtze River Delta region was high. It is essential to maintain continuous monitoring of PRRSV infections, especially the free-range pig and suckling piglets. Additionally, the development and implementation of targeted prevention and control strategies is critical for reducing the risk of viral transmission.

The aim of this study was to establish an indirect ELISA antibody detection method based on the ORF002 protein of the Lumpy Skin Disease Virus (LSDV). By constructing the recombinant plasmid pET-30a(+) -ORF002 and inducing its expression, a protein with a molecular mass of approximately 13 ku was obtained. The ORF002 protein, verified by SDS-PAGE and Western blot, was used as the encapsulation antigen to establish an indirect ELISA antibody detection method for LSDV and to optimize the conditions. The optimized conditions for the indirect ELISA were as follows: antigen encapsulation concentration of 2.5 μg·mL^-1, serum dilution of 1∶100, secondary antibody dilution of 1∶120 000, and the OD_{450 nm} value of the sample≥standard positive value×20.34% was determined as positive, conversely, OD_{450 nm} value below this threshold were considered negative. The coefficient of variation from the repeatability test was < 10%, and there was no cross-reactivity observed with positive sera from various viruses. This method demonstrates good sensitivity, specificity, and reproducibility, making it suitable for the detection of LSDV in clinical serum samples and for evaluating the effectiveness of vaccine immunization.

The aim of this study was to explore the effects of Bidens pilosa L. (B.pilosa) on cecal injury, immune function and oxidative stress in chicks infected with E. tenella. Bidens pilosa methanol extract (BP) was prepared by alcohol extraction method. A total of 200 1-day-old healthy male chicks were randomly divided into negative control (Control) group, positive control (Et) group, Chicken Coccidiosis Powder (CCP) group, 1 mL·kg^-1 BP group, 5 mL·kg^-1 BP group and 10 mL·kg^-1 BP group. At the age of 14 days, except for the negative control group, each chick in the other five groups was infected with 5×10⁴ E. tenella sporulated oocysts, and were treated with different doses of medicine. All chicks were sacrificed at the age of 21 days, and cecal tissues were taken to make pathological sections of cecum; The changes of cecal flora, intestinal barrier related gene mRNA transcription level, immune indexes and oxidative stress related factors were detected. The results showed that compared with the positive control group, 10 mL·kg^-1 BP reduced the number of cecal parasitic oocysts, reduced cecal lesions and injuries; The number of Lactobacillus in cecum was increased, the number of Clostridium and Salmonella was significantly reduced, and the number of cecal flora was restored; The mRNA transcription levels of Zona Occludens 1 (ZO>-1), Occludin, Claudin-1, Mucin-2 (MUC-2), the contents of interleukin 10 (IL-10), immunoglobulin G (IgG) and the activities of catalase (CAT), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) were significantly increased, and the contents of interleukin 2 (IL-2), interleukin 6 (IL-6) and malondialdehyde (MDA) were significantly decreased. The results indicated that 10 mL·kg^-1 BP in this test could repair the damaged divesity of cecal flora caused by E. tenella infection, improve the immune function of infected chicks, and reduce intestinal injury and oxidative stress.

The purpose of this study is to better explore the interaction between host innate immunity and classical swine fever virus (CSFV) by constructing a stable expressing CSFV NS3/NS4A and pCMV-CBH-GFP-2A-Puro-CSFV NS3^pro-NS4A, pCMV-CBH-GFP-2A-Puro-CSFV NS3-NS4A and pCMV-CBH-GFP-2A-Puro recombinant expression vector were separately infected with HEK-293T cells packaged out of the lentivirus. Then, packaged lentivirus was used to infect porcine alveolar macrophages (3D4/21), monoclonal screening of infected cell lines was performed to obtain stable cell lines, and the protein expression of stable cell lines was verified by Western blot. Proteomic analysis was conducted on it to screen out the interacting proteins of MAPK and PI3K pathways, and functional analysis was carried out. Detection of CDK4, CDC37, CASP3, IKBKG, and NFκB1 Protein Expression Levels via Western blot Analysis. The results showed that the amino acid sequences of NS3-NS4A and NS3^pro-NS4A lentiviral vectors constructed by MegAlign analysis were correct without frameshift. After the recombinant expression vector infected 293T cells for 24 hours, the infection efficiency could reach more than 90%, the lentvirus was successfully packaged, and the stable expression of CSFV NS3-NS4A and NS3^pro-NS4A macrophage cell lines were successfully obtained by Western blot analysis. Through proteomic analysis, 11 up-regulated and 24 down-regulated proteins in the MAPK and PI3K pathways were screened out in the NS3-NS4A/mock group. Western blot analysis revealed that CSFV NS3-NS4A significantly reduced the expression of CASP3, NFκB1, and IKBKG in the MAPK pathway, as well as CDK4 and CDC37 in the PI3K pathway. In this study, 3D4/21 cell lines stably expressing NS3-NS4A and NS3^pro-NS4A proteins were successfully constructed, and conducted functional analysis on the key proteins screened by proteomics, which provides a material basis for studying the mechanism of CSFV NS3-NS4A regulating the host innate immune responses.

This study aimed to investigate whether porcine epidemic diarrhea virus (PEDV) infection induces ferroptosis and its impact on viral replication. Proteomic analysis of differentially expressed proteins in IPEC-J2 cells (porcine intestinal epithelial cells) post-PEDV infection, combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses, initially suggested ferroptosis occurrence. Subsequent validation included measuring malondialdehyde (MDA) and glutathione (GSH) levels, assessing protein expression of ferroptosis-related markers (TFRC, xCT, GPX4, ACSL4, LPCAT3, and Ferritin), and observing Fe²⁺ accumulation, lipid reactive oxygen species (ROS), and mitochondrial membrane potential via inverted fluorescence microscopy. Further, by adding the ferroptosis inhibitor Fer-1, the expression changes of N protein and the changes of viral titer during the infection process of PEC-J2 cells caused by PEDV infection were investigated. Proteomic analysis revealed significant downregulation of Ferritin in PEDV-infected cells. GO and KEGG analyses highlighted prominent enrichment in iron metabolism dysregulation and lipid metabolism disruption, indicating ferroptosis induction. PEDV infection markedly elevated MDA levels (P < 0.01) and reduced GSH content (P < 0.01). Protein expression of Ferritin, GPX4, ACSL4, and LPCAT3 was significantly downregulated (P < 0.01), while TFRC was upregulated (P < 0.01). Fluorescence assays demonstrated intensified Fe²⁺ and lipid ROS signals (P < 0.01) and diminished mitochondrial membrane potential (P < 0.01). After the addition of Fer-1, both the N protein level and the gene level of PEDV were extremely significantly downregulated (P < 0.01), and the viral titer also decreased extremely significantly after Fer-1 treatment (P < 0.01). PEDV infection induces ferroptosis in IPEC-J2 cells, a process that may mechanistically contribute to enhanced viral replication by disrupting cellular iron homeostasis and lipid peroxidation signaling pathways.

The objective of this research is to investigate the function of recombinant porcine chemokine CCL25 protein and develop polyclonal antibodies targeting CCL25 from rabbits. The amino acid sequence of truncated CCL25 without the transmembrane region was obtained, a recombinant vector was constructed, and the protein was expressed in Escherichia coli expression system. Protein expression was confirmed by SDS-PAGE, western blot, and ELISA after nickel column purification. The biological activity of the protein was validated through cell proliferation assays, scratch tests and Transwell cultures using flow cytometry and real-time fluorescence quantification. New Zealand white rabbits were immunized subcutaneously with the protein to produce polyclonal antibodies. The functionality and titer of the polyclonal antibodies were confirmed using indirect ELISA, confocal microscopy, and western blot. The soluble expression of the protein was verified by SDS-PAGE and western blot, and the target protein was obtained after purification. Experimental results demonstrated that CCL25 promoted the growth and migration of IPEC-J2 cells, upregulated cell AKT and p-AKT₄₇₃ expression, and significantly enhanced chemotaxis of porcine peripheral blood lymphocytes. Additionally, CCL25 inhibited LPS-induced IL-8 and caspase-3 expression, as well as 7-AAD⁺ cell death ratio. In addition, this study successfully prepared polyclonal antibodies with high titer and good specificity. The CCL25 protein was obtained by prokaryotic expression and purification. It was preliminarily identified that CCL25 protein had biological functions such as promoting cell proliferation, migration, chemotaxis, anti-inflammation and anti-apoptosis, which provided an effective experimental tool and basic data for the subsequent functional study of CCL25.

Studies have shown that long non-coding RNA (lncRNA) is differentially expressed in dairy cow mastitis, but the molecular mechanisms by which it regulates dairy cow mastitis are unknown. The aim of this study was to investigate the role of lncRNA TCONS_00062289 in the inflammatory response of bovine mammary epithelial cells (bMECs). The lncRNA was named lncRNA PFN1-AS1 because of its sequence identity with the actin monomer-binding protein antisense (PFN1-AS1). LncRNA PFN1-AS1 was cloned by RT-PCR and its function was predicted. Inflammatory cell modeling with lipopolysaccharides (LPS) induced bMECs, in order to explore the effect of interfering lncRNA PFN1-AS1 on cell inflammation, proliferation and apoptosis in the inflammatory model. The lncRNA PFN1-AS1 is 811 bp in length, may be involved in the regulation of apoptosis and inflammatory processes. LncRNA PFN1-AS1 mainly distributed in the cytoplasm. In the LPS-induced inflammation model of bMECs, the expression level of lncRNA PFN1-AS1 was significantly down-regulated (P < 0.05). After interfering with lncRNA PFN1-AS1, the mRNA expression and protein secretion level of pro-inflammatory cytokines interleukin (IL) -6, IL-8 and IL-1β were significantly up-regulated (P < 0.05). The expression level of cyclin dependent kinase2 (CDK2), CDK4 and proliferating cell nuclear antigen (PCNA) were significantly down-regulated (P < 0.05), and the cell viability value and proliferation rate were significantly decreased (P < 0.05); the expression level of cysteinyl aspartate specific proteinase (CASP3), CASP8 and bcl-2-associated X protein (BAX) were significantly up-regulated (P < 0.05), and the rate of apoptosis was highly significantly increased (P < 0.01). Interference with lncRNA PFN1-AS1 promoted the transcription and protein secretion of IL-6, IL-8, and IL-1β, promoted the level of apoptosis, and decreased the cell viability and proliferation. These results reveal the role of lncRNA PFN1-AS1 in the inflammation of bMECs, and provide a scientific basis for further investigation of its role in mastitis of dairy cows.

Lung neuroendocrine cells (PNEC) are located in the airway epithelium, which can secrete active amines and peptides, and have the characteristics of both nerve cells and endocrine cells, which are closely related to the lung development and adaptation to the external environment of terrestrial mammals. In order to explore the distribution of PNEC in the lungs of yaks of different ages and its possible role in the formation of hypoxic adaptive structure, six yaks were selected from newborn (1-6 days old), juvenile (1-3 years old), adult (4-6 years old) and elderly (7-10 years old). Grimelius silver staining, immunohistochemical staining and immunofluorescence staining were used to study the distribution characteristics of PNEC and its related factors, such as chromogranin A(CGA), S100, neuron-specific enolase (NSE) and neurogenic differentiation factor (NEUROD1). At the same time, Western blot and qRT-PCR were used to detect the expression of these related factors at the protein level and the transcription at the gene level. The results showed that the number of PNEC in the airway epithelium of yak lung was the highest in the newborn group. PNEC-related factors are mainly distributed in airway epithelial cells, type Ⅱ alveolar epithelial cells, pulmonary artery endothelial cells and tracheal glandular epithelial cells. The expression trends of CGA, S100A1 and NEUROD1 were basically the same, with the highest expression in the lungs of newborn yaks, and then showed a decreasing trend, with significant differences in protein levels among groups (P < 0.05). Although the transcription trend of each group at the gene level is the same, there are great differences among the four factors. The expression of NSE in the lungs of adult yaks is the highest, and it is significantly higher than that of newborn yaks, young yaks and old yaks, showing a trend of increasing first and then decreasing. This study shows that PNEC and its related factors may participate in the adaptive regulation of yak lungs to hypoxia environment through dynamic expression regulation, and provide key energy support for yak survival and reproduction, which provides data for further study on the adaptive mechanism of yak lungs to hypoxia.

This study aims to investigate the effects of Artemisia argyi essential oil on the in vitro replication of Marek's disease virus and elucidate the underlying molecular mechanisms involved. The cytotoxicity of AAEO on chicken embryo fibroblast (CEF) cells was determined by the CCK-8 method, and the optimal concentration range of AAEO was screened; The cells were treated and samples were collected according to the selected optimal concentration. The effect of AAEO on the proliferation of MDV in vitro was detected by plaque analysis and virus titer assay.The mechanism of AAEO inhibiting MDV proliferation was analyzed at the molecular level by RT-qPCR and Western blot. The results showed that AAEO had no cytotoxic effect on CEF cells in the range of 60-360 μg·mL^-1.In the AAEO treatment group, the virus plaque was significantly reduced and reduced, the virus titer was decreased, and the expression of virus at the gene level and protein level were decreased, indicating that AAEO can inhibit the replication of MDV in a dose-dependent manner. Through the screening of intracellular signaling pathways of AAEO, it was found that AAEO could down regulate the expression levels of phosphorylated ERK and STAT3 proteins, and down regulate the expression levels of IL-6 mRNA. Treatment of cells with the ERK inhibitor U0126 reduced viral replication when the intracellular ERK pathway was inhibited. AAEO effectively inhibits the in vitro proliferation of MDV in chickens. The antiviral mechanism may be associated with its suppression of IL-6/STAT3 signaling pathway and ERK pathway activation. This study demonstrates that AAEO exhibits significant anti-MDV activity, and the findings provide novel evidence for developing therapeutic agents against herpesviruses. The research outcomes offer new insights into the exploration of antiviral drug candidates targeting herpesvirus infections through natural product derivatives.

Chronic heat stress seriously affects the production performance of poultry. The purpose of this study was to investigate the mechanism of exogenous melatonin (Mel) on the synthesis and secretion of corticosterone in broilers under chronic heat stress, and to provide a theoretical basis for melatonin to alleviate chronic heat stress. Ninety 2-week-old broilers were randomly divided into 6 groups: normal control group (CON), chronic heat stress (HS) group, melatonin intervention group: CONH (2.0 mg·kg^-1) group, HSL (0.5 mg·kg^-1) group, HSM (1.0 mg·kg^-1) group, HSH (2.0 mg·kg^-1) group. The experiment lasted for 21 days. Results: Chronic heat stress resulted in a decrease in broiler performance and a significant increase in serum corticosterone content, while the performance of broilers treated with 0.5 mg·kg^-1 Mel was significantly improved (P < 0.001), and the feed-to-weight ratio of broilers in the HSH group was significantly decreased (P < 0.05). Dietary supplementation with 0.5-2.0 mg·kg^-1 Mel significantly reduced serum corticosterone content in broilers under chronic heat stress (P < 0.000 1). In addition, Mel could effectively improve the decrease of reduced glutathione (GSH) content, superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity caused by chronic heat stress. The network pharmacology analysis showed that Mel intervention in chronic heat stress was enriched in the cAMP signaling pathway. It was detected that the mechanism of Mel was related to the melatonin receptor MT1, and by inhibiting the cAMP/PKA/CREB signaling pathway, the synthesis and secretion of adrenocortical ketone were weakened, and the stress response was alleviated.

The aim of this study was to investigate the antibacterial activity of natural plant essential oils and the synergistic effect combined with potassium monopersulfate. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of eight essential oils and potassium monopersulfate against Staphylococcus aureus ATCC 29740 and Escherichia coli CVCC 1450 were detected by the micro broth dilution method. In vitro inhibitory activities were detected by using the disc diffusion method. The dynamic inhibitory effects of different essential oil concentrations on bacterial growth were analyzed by time-inhibition curves.The synergistic antimicrobial effects of essential oils and potassium monopersulfate were investigated using the checkerboard method combined with the FIC index.The results indicated that the essential oils of domestic lavender and Australian lavender exhibited the lowest MIC (1.95 μg·mL^-1) against Staphylococcus aureus, while the essential oils of domestic chamomile and domestic lavender demonstrated the lowest MIC (15.63 μg·mL^-1) against Escherichia coli. The evaluation of the joint antibacterial effect indicated that the essential oil of domestic chamomile, in combination with the potassium monopersulfate, exhibited a synergistic antibacterial effect, with an FIC index of 0.5. In this study, the four highly bacteriostatic plant essential oils under examination exhibited promising inhibitory activity against animal pathogens. Potassium monopersulfate's combination effectively reduced the MIC of Staphylococcus aureus ATCC 29740 and Escherichia coli CVCC 1450, and the combination of domestic chamomile essential oil and potassium monopersulfate exhibited a synergistic effect. These findings provide a valuable reference for the development of natural and safe disinfectant preparations.

This study aims to develop a multiplex fluorescent quantitative PCR method for the rapid and simultaneous detection of Bordetella bronchiseptica (Bb), Glaesserella parasuis (GPS) and Streptococcus suis (SS). Specific primers and probes were designed based on the conserved sequences of the fla gene (Bb), infB gene (GPS), and gdh gene (SS). After optimizing the reaction conditions, a TaqMan-based multiplex fluorescent quantitative PCR method for detecting these three pathogens was established. The method demonstrated good specificity, with no positive signals for nucleic acids from Actinobacillus pleuropneumoniae, Pasteurella multocida, Pseudorabies virus, Klebsiella pneumoniae, and Escherichia coli from pigs. It exhibited high sensitivity, with the minimum detection limits of 8.06, 7.11, and 7.15 copies·μL^-1 for the standard plasmid templates of Bb, GPS, and SS, respectively. The method also showed good reproducibility, with intra-assay and inter-assay coefficients of variation both < 2%. Analysis of 513 clinical porcine lung samples collected between 2023 and 2025 revealed that the proportion of mixed infections was 16.57%. The developed multiplex fluorescent quantitative PCR method exhibited superior specificity, sensitivity, and stability, providing a reliable tool for the rapid detection and diagnosis of these three pathogens in clinical settings.