Gene editing technology is a novel molecular tool for modifying specific genes, which can effectively introduce new mutations into gene sequences, providing effective means for analyzing gene function and the phenotype-related DNA sequences. Since its discovery, gene editing technology has been successfully applied in various animals, plants, and other organisms, providing important technical support for gene therapy and precision crop breeding. This article aims to review the development of gene editing tools in recent years, elucidate the progress of classic editing tools, base editing tools, and other new editing tools, in order to provide reference for researchers in related fields to further understand and optimize editing systems.

Wool is an important livestock product that develops from wool follicles. wool follicles is a micro-organ derived from the skin tissue, which consists of more than 20 types of cells, including hair follicle stem cells, dermal papillae, hair matrices, outer root sheaths and inner heel sheaths, with complex structure and intercellular communication. Therefore, in order to improve wool yield and quality, comprehensive understanding of the developmental characteristics of hair follicles under various cell interactions was needed in advance. Currently, cell culture techniques are mainly categorized into 2D cell culture and 3D cell culture. Conventional 2D cell culture techniques for culturing wool follicle-associated cells are characterized by a large gap with in vivo growth conditions and a gradual loss of hair-inducing ability. 3D cell culture is the cultivation of cells in 3D structures that mimic the specificity of tissues and organs. By mimicking the natural environment, it allows cells to aggregate into a certain morphology and facilitates cell-cell communication, as well as cell-extracellular matrix communication, thus restoring a certain in vivo function, which makes up for the shortcomings of traditional 2D cell culture. This paper briefly introduces 3D cell culture models and types, construction methods, applications in various cell and wool follicle development studies, and looks forward to the development of 3D cell culture technology in hair follicle development studies and other fields.

Semen quality is an important reference index for selecting good breed male livestock and poultry. Intestinal microorganisms not only can regulate gonadotropin-releasing hormone (GnRH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone (T) and other reproductive hormones through the gut-testis axis, but also regulate the synthesis and metabolism of butyric acid, amino acids, vitamins, bile acids, gas signaling molecules (H₂S、NH₃ and NO), gamma-aminobutyric acid (GABA) and 5-hydroxytryptamine (5-HT) in the intestine, thereby regulating semen quality.Therefore, increasing the level of suitable microorganisms or their metabolites in the gut can improve the semen quality of male animals. This paper reviews the research progress on the regulation of semen quality by animal gut microbes and their metabolites, to guide production practice.

Cryopreservation of oocytes is one of the important strategies for long-term storage of genetic resources in both humans and animal-assisted reproduction. However, due to the low surface-volume ratio and poor cell permeability, oocytes are more susceptible to cryoinjuries during vitrification, including ice crystals formation, osmotic shock, oxidative stress, and cryoprotectants toxicity, which seriously decreases their developmental ability after thawing. In contrast to other animals, porcine oocyte cryopreservation is more challenging owing to its abundant lipid droplets and poor hypothermia tolerance. Although live piglets have been produced from vitrified immature oocytes, the developmental competence of vitrified-oocytes remains to be further promoted compared with fresh oocytes. This paper mainly reviews the injury types, the mechanisms of damage, as well as the alleviating strategies of porcine vitrified-oocytes. It will provide further reference for establishing and improving the vitrification protocol of porcine oocytes.

Metabolic associated fatty liver disease is a chronic liver disease associated with metabolic disorders, usually caused by disturbances in the body's metabolism of fats, carbohydrates and proteins, and its pathogenesis is related to inflammatory response, oxidative stress, insulin resistance, and intestinal microbiota dysbiosis. This disease seriously affects the liver health of humans and animals, leading to liver injury, hepatic failure and even the occurrence of liver cancer, increasing the risk of complications such as diabetes, cardiovascular disease and even death. Natural active substances derived from vegetables, fruits, grains and plant medicinal herbs that regulate sugar and lipid metabolism, resist antioxidant stress, relieve inflammatory response, and improve intestinal microbiota balance can regulate liver metabolic function, reduce inflammatory response, and repair liver injury, and have unique benefits in the prevention and treatment of metabolic associated fatty liver disease. This article summarizes natural active substances that can regulate glucose and lipid metabolism, resist oxidative stress, relieve inflammatory response, improve intestinal flora and bile acid metabolism disorders, and discusses potential preventive and therapeutic effects and possible mechanisms of these agents in human and animal metabolic associated fatty liver disease, with the aim of providing a theoretical reference for the development of new drugs for the prevention and treatment of metabolic associated fatty liver disease in clinical medicine or animal husbandry practice.

Symbiotic fungi in gastrointestinal tract of livestock and poultry refer to fungi that have been colonized in gastrointestinal tract persistently, and they maintain the stability of gastrointestinal microecosystem together with bacteria and other microbial flora. Many studies have shown that there are a large number of symbiotic fungi in the gastrointestinal tract of livestock and poultry, which not only will not cause diseases, but also have beneficial effects to the host. There are different kinds of gastrointestinal symbiotic fungi in different species. On the whole, the symbiotic fungi in the gastrointestinal tract of livestock and poultry mainly include Ascomycota, Basidiomycota and Neocallimastigomycota. The composition of symbiotic fungi community in gastrointestinal tract of livestock and poultry can be affected by many factors, such as diet, age, breed and health condition. Similar to bacteria, the research on symbiotic fungi is mainly carried out by traditional culture medium method and high-throughput sequencing method currently. The emerging cultureomics technologies can separate and identify more kinds of symbiotic fungi. Studies have shown that symbiotic fungi, as an important part of gastrointestinal microorganisms in livestock and poultry, directly or indirectly participate in various physiological processes, such as promoting the digestion of nutrients ingested by livestock and poultry, participating in host immune regulation, promoting the glycolysis, etc. In this paper, the symbiotic fungal probiotics or potential symbiotic fungal probiotics in gastrointestinal tract of livestock and poultry were reviewed on the types of symbiotic fungi, the main methods of identifying symbiotic fungi, the factors affecting the abundance of symbiotic fungi in gastrointestinal tract, the beneficial effects and mechanism of symbiotic fungi, in order to provide new ideas and references for regulating the gastrointestinal health of livestock and poultry.

The colostrum of ruminants encompasses abundant bioactive components, of which microRNAs are kind of non-coding RNAs that can regulate gene expression at the post-transcriptional level and play important roles in regulating development of the mother's mammary gland and gastrointestinal tract of young ruminants. In recent years, studies on the profile and function of microRNAs derived from ruminant colostrum have attracted attention. In this review, the source, composition, influencing factors, and function of microRNA in ruminant colostrum as well as the limitations of current studies and future research directions were summarized, with an aim to provide theoretical support for the production of high-quality colostrum and improve integrated mother-neonate cultivation level.

As a popular pet companion, cats have become an indispensable member of countless families, and their feeding number is increasing. Food intolerance is a non immune abnormal physiological reaction of the body to non immune food or food additives, which can affect cats of any age, sex and breed. Its clinical characteristics are mainly gastrointestinal, respiratory and skin symptoms. Digestive system diseases caused by food intolerance are the most common, which can lead to wasting, loss of appetite and mental depression in cats. It has the characteristics of long treatment cycle and easy to repeat. Recent, in view of the limitations of the diagnostic technology of cat food intolerance in clinical practice and the lack of attention to this problem, there is a significant deviation between its detection rate and the actual prevalence, which brings great challenges to the accurate diagnosis and timely treatment of cat food intolerance. Therefore, this review comprehensively considered the physiological characteristics, nutritional needs and feeding strategies of cats, combined with the clinical manifestations, immunological mechanisms, diagnostic methods and treatment strategies of food intolerance, summarized the current situation and treatment methods of food intolerance in cats. This review aims to attract the attention of clinical pet physicians and provide ideas and references for the clinical diagnosis and treatment of food intolerance.

African swine fever (ASF) is a highly contagious disease characterized by severe bleeding in both domestic pigs and various wild boars caused by infection with the African swine fever virus (ASFV). The global pig breeding industry has suffered significant economic losses due to the high morbidity and mortality rates associated with ASF. ASFV contains over 150 different proteins, with its structural proteins playing a crucial role in facilitating the virus's attachment to and entry into host cells, as well as in the replication, assembly, and release of new viral particles. Research has demonstrated that these viral structural proteins can enhance virus invasion and replication, impact viral virulence, and counteract the host immune response through interactions with host proteins. This review summarizes the interactions between ASFV structural proteins and host proteins, along with their underlying mechanisms, in order to contribute to the understanding of ASFV pathogenesis and the development of strategies for its prevention and treatment.

Bacteria biofilm refers to the bacterial aggregates formed by embedding the bacterial into the protective matrix which self produces. The survival mode based on biofilms helps microorganisms resist external threats such as immune system and drug treatments. Therefore, biofilms of bacterial organisms can exacerbate bacterial infections and lead to serious animal diseases. Biofilm plays a role in the pathogenesis of animal pathogens, including inducing and regulating local inflammatory responses and promoting intracellular invasion. The physical barrier function can provide protection for pathogens, reduce the treatment effectiveness, and increase the risk of chronic and subclinical infections. Bacteria and their biofilms can infect husbandry animals, wild animals, and companion animals. The review shows the role of biofilms in the pathogenesis of bacterial infections in animal cardiovascular, central nervous, digestive, reproductive, respiratory, urinary, and skin systems, aiming to provide reference for accurate detection and effective treatment of animal bacterial biofilms.

Acute kidney injury (AKI) is a kidney disease with rapid onset, complex etiology and high mortality, which poses a serious threat to the life and health of animals. In recent years, mesenchymal stem cells (MSCs) have been widely recognized as an effective method for the treatment of acute injury. However, due to the limitations of cell therapy, mesenchymal stem cell-derived exosomes (MSC-exos), a new cell-free treatment method that has attracted much attention, has attracted great attention in the treatment of acute kidney injury. MSC-exos can cure acute kidney injury through a variety of mechanisms such as inhibiting inflammation, reducing apoptosis, and regulating autophagy. This review summarizes the progress of MSC-exos in the treatment of AKI in animals, and aims to provide a theoretical reference for the clinical management and application of stem cell-derived exosomes in animals.

Isoxazoline drugs have a variety of biological effects. In recent years, they have been widely used in pet to combat ectoparasites due to their excellent effectiveness and high safety against various types of ectoparasites. Isoxazoline drugs selectively inhibits glutamate and gamma-aminobutyrate gated chloride ion channels in invertebrates, resulting in nervous system dysfunction, paralysis and death. This review focuses on the structural characteristics, mechanism of action, pharmacokinetics, pharmacodynamics and clinical anthelmintic application of isoxazoline anthelmintic drugs in recent years. The application and potential toxicity of different drugs in veterinary medicine are analyzed, and presents the future prospect of isoxazoline anthelmintics in animals.

The present study aimed to explore whether the structural variation in CUL2 affected the gene expression and wrinkle severity on skin of Xiang pigs. The 200 skins and blood samples were collected from Xiang pigs with wrinkled skin and common Xiang pigs in 6 months old. The polymorphic distribution of the CUL2-I3-sv300 site was examined using PCR method. Based on the prediction of potential functional elements within the structural variation region, and the interaction between CUL2 gene and other related genes by softwares online such as UCSC, the expression levels of CUL2 and those collagen formation-related genes were determined using RT-qPCR and Western blot methods. The results showed that the structural variation CUL2-I3-sv300 exhibited abundant polymorphism within the pig populations. Genotypes of the mutant (MM), the heterozygous (WM) and the wild (WW) were detected from skins of the common Xiang pigs, while type WW was not detected from the Xiang pig with wrinkled skin. Furthermore, the allele M was significantly more abundant in Xiang pigs with wrinkled skin compared with that in the common Xiang pigs (P < 0.05). Based on the bioinformatics analysis, several elements were screened from the structural variation region including a short interspersed nuclear element (SINE), three intronic splicing silencers (ISS) and four RNA binding protein sites (RBPs). And CUL2 might regulate HIF-1α, indirectly change VEGFA, MMP-1, and MMP-9, and eventually affect the formation of skin collagen. After detection by using both of the wrinkled skin and common skin, the mRNA and protein levels of CUL2 expression were higher in the wrinkled skin with MM genotypes than that with other genotypes in wrinkled and common skins. Moreover, the expression of CUL2, MMP-1 and MMP-9 were up-regulated, while mRNA levels of HIF-1α, VEGFA, COL1A1, COL3A1 and COL6A3 genes were down-regulated in Xiang pig with wrinkled skin compared with samples from the common Xiang pig.It is suggested that the loss of those functional elements in the structural variation region might promote the expression of CUL2 while inhibit collagen formation, thereby aggravating the severity of skin wrinkles on Xiang pig body.

This study aimed to identify the candidate genes associated with important economic traits of Rongchang pigs in the process of artificial and natural selection by selection signal analysis based on the imputed sequence data. In this study, a total of 591 Rongchang pigs were genotyped by porcine 50K SNP chip, among these, 120 pigs were randomly selected to perform whole genome sequencing. The whole genome resequencing data was used as reference template to imputed 50K SNP data. Then genetic structure analysis, Tajima'D and CLR tests were conducted based on the imputed sequence data. After genotype imputation, the imputation accuracy was 0.942, and 8 823 367 high-quality SNPs were retained after quality control (the imputation accuracy was 1.00). The genetic structure analysis showed that there was no obvious population stratification in Rongchang pig, and the genetic distance among most individuals was greater than 0.1, and the numerator relationship was less than 0.1. The CLR test detected 226 potential selected regions and found candidate genes (CDK9, SLC2A8, IGF1R, GSK3B) related to reproduction, growth and carcass traits through gene annotation. The Tajima'D test detected 225 potential selected regions, and gene annotation found that genes associated with hair length (CFAP299), hair color or deafness (MITF, ZNF532), fat deposition (GSK3B), and reproduction (FOXP1). Further analysis, a total of 11 same potential selected regions between the results of CLR and Tajima'D test were found, and 123 candidate genes related to important economic traits such as reproduction (ZNF532 and GSK3B), deafness and albinism(MITF)were screened. Moreover, a significant GO term and KEGG pathway (P < 0.05) related to melanin generation and ocular vision development was found. In this study, according to the results of enrichment analysis and molecular biological functions of genes, 8 important candidate genes, including MITF, ZNF532, GSK3B, FOXP1, SLC2A8, CDK9, IGF1R and TBC1D4, were suggested as the candidate genes for hair color and deafness, fat deposit, reproductive performance and growth performance in Rongchang pigs. These results explored the genetic structure and selection signature characteristics of Rongchang pigs at the whole genome level, the identified candidate genes associated with important economic traits provided an important theoretical reference for future breeding and genetic mechanism analysis of characteristic traits in Rongchang pigs.

The study aimed to identify SNP sites of the candidate gene G3BP1 that influence intramuscular fat deposition using a population of 721 Gushi F2 chickens, to develop molecular markers for meat quality traits, and to further investigate its function using intramuscular preadipocytes from Gushi chickens. First, the study identified SNPs in G3BP1 gene and found 3 mutation sites in strong linkage disequilibrium, especially the different genotypes of the 13588667G>A site were significantly associated with the meat quality traits such as sebum, sebum rate and intramuscular fat in the F2 resource group of Gushi chicken (P < 0.05). Therefore, the 13588667G>A site of the G3BP1 gene could be used as an important molecular marker affecting intramuscular fat deposition. Additionally, qRT-PCR, CCK-8, flow cytometry, triglyceride content determination and Oil Red O staining were used to evaluate the effect of G3BP1 gene on the proliferation and differentiation of intramuscular preadipocytes in Gushi chickens. The results showed that after overexpression of G3BP1, the expression levels of proliferation marker genes CDK1, CCNB2, PCNA and CCND1 in intramuscular preadipocytes were significantly increased (P < 0.001), and the cell cycle process was significantly promoted. G3BP1 overexpression also significantly upregulated the expression of the differentiation marker gene LPL (P < 0.05) and dramatically increased the expression of CEBPA (P < 0.001), with a significant increase in lipid droplet formation and triglyceride content in intramuscular preadipocytes (P < 0.01). This study clarifies the role of G3BP1 in promoting the proliferation and adipogenesis of intramuscular preadipocytes and identifies a molecular marker affecting intramuscular fat deposition, providing valuable theoretical and practical insights for breeding high-quality meat chickens in China.

To investigate the selection and breeding indexes and molecular markers for melanin deposition in early growth stages of Muchuan black-bone chickens, 54 Muchuan black-bone chickens at 35 days of age were selected as research subjects to determine the melanin content of the breast muscle and skin color difference, the relative expression of PMEL17 gene in different tissues by using real-time fluorescence quantitative PCR, the SNPs sites of PMEL17 gene by using Sanger sequencing, and statistics of genotypes frequency of the mutation sites, and their association analysis was performed with melanin content of the breast muscle. The results showed that there was a highly significant positive correlation between melanin content of breast muscle and breast skin yellowness b value, and a significant positive correlation with leg skin brightness L value; the PMEL17 gene was expressed highest in skin tissues; a total of 5 mutation sites were found in the PMEL17 gene, among which, the SNP G-553A locus was associated with melanin deposition in Muchuan black-bone chickens and the AG genotype was the dominant genotype at this mutation site. In conclusion, at 35 days of age, the yellowish b value of the breast skin and the brightness L value of the leg skin of Muchuan black-bone chickens could be used as indicators for the preliminary estimation of the melanin content of the breast muscle; the PMEL17 gene was associated with the melanin deposition in the breast muscle of Muchuan black-bone chickens, and the significantly related SNP G-553A could be used as a molecular marker for the melanin deposition in the breast muscle. This study provides a theoretical basis for the selection and breeding of Muchuan black-bone chickens and enriches the germplasm characteristics of local breeds.

The study aimed to explore the effect of overexpression of TRIM39.2, localized in the chicken major histocompatibility complex (MHC), on the transcription expression of chicken macrophages. The tissue expression profile of chicken TRIM39.2 gene was constructed using RT-qPCR.The chicken TRIM39.2 gene was cloned and a recombinant expression vector carrying the tag was constructed. HD11 cells were transfected with either an empty vector or a TRIM39.2 eukaryotic expression vector.RNA was harvested to prepare a library, and transcriptome sequencing and bioinformatics analysis were performed. Differentially expressed genes were screened, followed by GO and KEGG enrichment analysis, and RT-qPCR method was used to further verify the transcriptome sequencing results. The results showed that the expression of TRIM39.2 was the highest in the spleen compared to other tissues. The chicken TRIM39.2 gene was successfully cloned, and a recombinant expression vector carrying a Myc tag, pCAGGS-Myc-TRIM39.2, was constructed, and confirmed to express TRIM39.2 recombinant protein in chicken HD11 cells.Transcriptome sequencing analysis identified 33 significantly differentially expressed genes. GO analysis was primarily enriched in the active characteristics of cytokines and their involvement in signal transduction pathways.KEGG analysis mainly focused on the interactions between cytokines and receptors, as well as pathways such as RIG-I-like receptor signaling pathway. The chicken TRIM39.2 gene plays a significant regulatory role in macrophage-mediated immune responses and signal transduction, particularly in positively regulating the type Ⅰ interferon signaling pathway mediated by RIG-I-like receptors. This study lays the foundation for further exploration of the regulatory mechanism of RIG-I-like receptor signaling pathway in chickens.

The aim of this study was to investigate the regulation role of miR-137 in coat color formation in black goats, in order to provide a new perspective in investigating the molecular mechanism of melanogenesis. Melanocytes were isolated from skin tissues of 4-day-old black goats at about 5 cm behind the shoulder, the melanocytes were localized by immunohistochemistry and immunofluorescence assays, the goat melanocytes were identified by Dopa staining and immunofluorescence, the content of melanin were determined after melanocytes lysed, and miR-137 regulation mechanism of melanogenesis by targeting melanocyte inducing transcription factor (MITF) was explored by cell transfection, real-time fluorescence quantitative PCR, and Western blot. The results demonstrated the presence of melanocytes above the dermal papilla and at the outer hair root sheath, melanocytes were bipolar or tripolar, Dopa staining identified the melanocytes as brownish-black positive, and immunofluorescence identified the expression of marker genes related to hair color generation as positive. The melanin content of transfected goat melanocytes was reduced compared with that of the NC group (P < 0.05), meanwhile the trend of the inhibitor group was opposite (P < 0.05). The expression levels of melanocyte generation-related genes were analysed by RT-qPCR and Western blot, and it was found that overexpression or inhibition of miR-137 did not have a significant effect on the mRNA expression levels of the target gene MITF (P>0.05). While overexpression of miR-137 inhibited the expression of mRNA of TYR, TYRP-1 and TYRP-2, which are marker genes for melanogenesis (P < 0.05), meanwhile, the trend was opposite in the inhibitor group (P < 0.05). Regarding the protein expression level, the protein expression level of MITF in the mimic group was significantly lower than that in the mimic NC group (P < 0.05), the protein expression level of MITF in the inhibitor group was significantly higher than that in the inhibitor NC group (P < 0.05).The protein expression levels of TYR, TYRP-1 and TYRP-2 were significantly lower in the mimic group than that in the mimic NC group (P < 0.05), and their expression in the inhibitor group was higher than that in the inhibitor NC group (P < 0.05). In this study, we found that miR-137 did not affect the mRNA expression of MITF, but indirectly inhibited melanin production in goat melanocytes by targeting MITF, which provides a theoretical basis for studying the role of miR-137 in the formation of skin and coat colour in goats.

The aim of this study was to analyze the molecular mechanism of resveratrol (RES) regulating myofiber type transformation by mediating PROX1 gene in goat skeletal muscle. In vivo component, twelve 180-day-old depopulated Nubian goats ((28.25±0.26) kg) were randomly divided into two groups, with 6 replicates each, the PROX1 gene expression levels of in dorsal longest muscle tissues supplemented with 0 and 150 mg·kg^-1 RES were detected by real-time fluorescence quantitative PCR (RT-qPCR) and Western blot. In vitro test, based on the previous research results, 20 μmol·L^-1 RES was identified as the optimal concentration for cell culture. PROX1 interference fragments and a combination of interference fragments with 20 μmol·L^-1 RES were transfected into goat skeletal muscle myoblasts. The expression levels of key genes in the CaN/NFAT pathway and the marker genes for the four muscle fiber types were measured at both mRNA and protein levels. Meanwhile, protein-protein docking technology was used to detect whether SIRT1 interacts with the PROX1 protein interact, and the regulatory relationship between SIRT1 and the CaN/NFAT pathway was verified by co-treatment of goat myoblasts with the SIRT1 inhibitor EX-527 and RES. The results showed that, compared with the control group, the expression of the SIRT1 gene and key genes in the CaN/NFAT pathway at the mRNA level in goat myoblasts treated with 150 mg·kg^-1 RES was significantly upregulated (P < 0.001), the expression of MYH7 protein was significantly upregulated (P < 0.01), and the expression of MYH4 protein was significantly downregulated (P < 0.01). After PROX1 gene interference, the mRNA and protein expression levels of key genes in the CaN/NFAT pathway, MYH7 and MYH4 showed opposite trends, which were altered when the interference fragments and RES co-treated the myoblasts. The protein-protein docking results indicated that SIRT1 and PROX1 proteins could interact. After myoblasts treated with the SIRT1 inhibitor, the mRNA expression levels of key genes (NFATc1, TNNC1, TNNI1, MYOZ2, SLN) in the CaN/NFAT pathway were significantly downregulated compared to the control group (P < 0.01). Following RES intervention, the mRNA expression levels of TNNC1 and TNNI1 were significantly upregulated (P < 0.01). This study initially revealed the mechanism of RES regulating the transformation of muscle fiber types through SIRT1/PROX1/CaN/NFAT pathway, which provides a theoretical basis for the subsequent improvement of livestock and poultry meat quality.

The study aimed to compare the prediction accuracy and computational efficiency of different genomic prediction models, explore the application value and prospects of support vector machine (SVM) regression and RandomForest regression in genomic prediction. In this study, the 50K liquid chip was used to predict the reproductive traits of 1 001 Landrace pigs by GBLUP, BayesB, BayesLASSO, support vector machine regression and RandomForest regression. It was found that support vector machine regression radial basis function kernel had the highest predictive accuracy of reproductive traits such as total litter size, live litter size and litter weight. The predictive accuracy of live litter size and litter weight reached the maximum value when the parameter C value was 1, and the predictive accuracy of total litter size reached the maximum value when the parameter C value was 2. When using RandomForest regression to evaluate parameters such as ntree, mtry and nodesize in reproductive traits such as total litter size, number of live litter and litter weight, it was found that the predictive accuracy showed a certain randomness with the change of parameters. The RandomForest regression model showed the highest accuracy in total litter size, live litter size and litter weight, followed by support vector machine regression. The predictive accuracy of GBLUP, BayesB, BayesLasso models was poor and consistent. In the correlation of cross-validation of different models, it can also be found that there is a strong correlation between the results of different models ranging from 0.806 to 0.995. Non-parametric machine learning models such as support vector machine regression and RandomForest regression have certain advantages in pig reproductive trait genome selection, but the running time limits the use of this machine learning algorithm to some extent. With the optimization of the algorithm, non-parametric machine learning models such as support vector machine regression and RandomForest regression will have a good application prospect.

This study aimed to investigate the effect of vitrification on genome methylation level of porcine parthenogenetic activation (PA) blastocysts. The porcine PA blastocysts on day 6 were used for vitrification-thawing experiments using conventional vitrification solution (vitrification group Ⅰ) and improved vitrification solution (vitrification group Ⅱ). Three blastocysts with good morphology were selected from each group for single cell whole-genome bisulfite sequencing (scWGBS) to analyze the genomic methylation levels changes in blastocysts. The results showed that compared with the fresh group, the genomic methylation levels of porcine PA blastocysts in the vitrification group Ⅰ were significantly reduced (18% vs. 20.9%, P < 0.01), while the vitrification group Ⅱ were significantly increased (21.5% vs. 20.9%, P < 0.05). Compared with the fresh group, a total of 2 717 differential methylation regions (DMRs) were identified in the vitrification group Ⅰ, and a total of 1 964 DMRs were identified in the vitrification group Ⅱ. Gene function enrichment analysis showed that vitrification caused disturbances in the methylation levels of genes related to biological processes such as embryonic development, ATP metabolism, MAPK signaling pathway, and Wnt signaling pathway. In the vitrification Ⅱ group, biological processes such as the Hippo signaling pathway, PI3K-Akt signaling pathway, and mTOR signaling pathway were more active. In summary, vitrification changes the genomic methylation level of porcine PA blastocysts, which may damage the development of porcine PA blastocysts by disrupting the methylation levels of genes related to embryonic development, ATP metabolism, MAPK signaling pathway, and Wnt signaling pathway, etc. In addition, the Hippo signaling pathway, PI3K-Akt signaling pathway, and mTOR signaling pathway can be used as candidate pathways to alleviate vitrification injury.

This study aimed to identify the candidate genes related to egg production in Wulong geese through a combined analysis of transcriptome and proteome, which will provide theoretical basis for understanding of the molecular mechanisms of egg production performance in goose. A total of 200 healthy 36-week-old Wulong geese with similar weight were selected for this study, whose egg production from 36 to 54 weeks of age was recorded. At the end of the experiment (54 weeks of age), according to the egg laying level, the 30 geese with the highest egg production were designated as the high yield group, and the 30 geese with the lowest egg production were set as the low yield group, geese with high (H) and low (L) laying performance were selected for 3 ovarian tissue collection, respectively. RNA and proteins were extracted from 3 geese of each group. And, differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were identified through RNA-Seq and 4D-DIA quantitative proteomic analysis. RT-qPCR validation, functional enrichment analysis, and protein-protein interaction (PPI) network construction and analysis were performed to identify the candidate genes related to egg production performance in Wulong geese. The results showed that 450 DEGs and 333 DEPs were identified by transcriptome and proteome analysis, respectively. And, AFAP1, INHA, FNDC1 and INHBB genes showed the same difference trend in proteome and transcriptome. Enrichment analysis indicated that these DEGs and DEPs were significantly enriched in 364 GO terms and 75 KEGG pathways, involving gland development, sex differentiation, hormone activity, TGF-β signaling pathway, oocyte meiosis, GnRH signaling pathway, progesterone-mediated oocyte maturation and other pathways and key biological processes related to egg production. Nine potential candidate genes were selected for RT-qPCR validation in 8 sample of each group, their expression trends were consistent with the sequencing results. In conclusion, the candidate genes (PTTG1, LRP2, TNFSF10, INHA, INHBB, FST) were identified to be related to egg production performance in geese. This study will provide new insights into the molecular mechanisms of egg production performance in geese.

The purpose of this study was to evaluate the amplification efficiency of different single cell whole genome amplification systems for different numbers of embryonic cells by second generation sequencing technology. In this study, healthy Huaxi cows under the same feeding and management conditions were selected as donors, and the 7th day embryos were collected after superovulation by FSH method. Samples containing 1C(cell), 5C and 10C embryos were obtained by biopsy technique for whole genome amplification, and then the amplification efficiency of single cell genome amplification system was evaluated by whole genome resequencing techniques. The results showed that the amplification success rate of multiple displacement amplification (MDA) system was better than that of the multiple annealing and looping-based amplification cycles (MALBAC) system in 1C and 5C samples (100% vs. 60%, 100% vs. 80%, P < 0.05). In addition, the total amount of amplification products of MDA system was significantly higher than that of MALBAC system (P < 0.01). Based on the sequencing analysis index: correct mapping rate, dup rate, typing consistency rate, allele drop-out rate and false positive rate, etc., MALBAC system was better than MDA system. The typing consistency rate of full-sib samples was higher than that of half-sib samples, but there were great differences between different full-sib and half-sib embryo samples. To sum up, MDA and MALBAC systems have their own characteristics, and the combination of their advantages may be a way to improve amplification efficiency. This study provides a theoretical basis for the whole genome selection of early bovine embryos and promoting the development of bovine breeding.

The purpose of this study was to investigate the effects of steroid 5 alpha reductase 2 (SRD5A2) on proliferation, apoptosis of granulosa cells, and the expression of genes related to steroid hormone synthesis in granulosa cells (GCs) of New Zealand White female rabbits. Healthy sexually mature female New Zealand White rabbits aged 4 to 6 months were selected. GCs were isolated from the rabbit ovaries and identified using follicle-stimulating hormone receptor (FSHR). Overexpression and knockdown of the SRD5A2 gene were performed. The proliferation, apoptosis of GCs, and the expression of genes related to steroid hormone synthesis were detected using CCK-8, flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) methods. The full-length coding sequence (CDS) of the rabbit SRD5A2 gene was 765 bp, encoding 252 amino acids. After overexpressing/knocking down SRD5A2 in GCs, qRT-PCR analysis revealed that the expression level of HIF1A was extremely significantly decreased/increased, while the expression levels of INHBB, HSD17B1, GDF9, and BMP15 were extremely significantly increased/decreased (P < 0.01). Furthermore, overexpressing/knocking down SRD5A2 significantly promoted/inhibited GCs proliferation and inhibited/promoted apoptosis (P < 0.05). These findings indicate that the SRD5A2 gene affects the expression of genes related to steroid hormone synthesis, as well as the proliferation and apoptosis of GCs, and is involved in the follicular development process in New Zealand White rabbits.

This study was conducted to determine the relative bioavailability of tricalcium phosphate (TCP), which is produced by high-temperature sintering method, by using dicalcium phosphate (DCP) as the reference, and talking the growth performance, dietary digestibility, serum and bone indexes of weaned piglets as indicators. The experiment conducted a 2×3 factorial arrangement experimental design, DCP and TCP were added into basal diet at 3 phosphorus supplemental levels, which were 0.08%, 0.16% and 0.24%, respectively, and two kinds of phosphates shared one control treatment which fed the basal diet. One hundred and forty Duroc×Large White×Yorkshire weaned piglets with similar body weight were randomly allocated into 7 groups with 4 replicates in each group and 5 piglets in each replicate. The experimental period lasted for 38 days including a 3-day adaptation period and a 35-day normal test period. The results showed that the phosphorus source and supplemental level and their interaction had no significant effects on growth performance of weaned piglets (P>0.05). The level of phosphorus supplementation significantly affected the apparent digestibility of dietary calcium and phosphorus, phosphorus content in tibia and metacarpal bones, and fracture strength of femur and metacarpal bones in piglets and presented a dose effect with the increase of phosphorus (P < 0.05). Compared with DCP, TCP significantly increased the apparent digestible phosphorus intake, femoral fracture strength, and metacarpal phosphorus content of piglets (P < 0.05). Considering the sensitive indicators such as dietary apparent digestibility of calcium and phosphorus, apparent digestible phosphorus, femoral and metacarpal strength, as well as metacarpal calcium and phosphorus contents, the average relative bioavailability of TCP is 109% with a reference to the bioavailability of DCP (100%). In conclusion, the bioavailability of phosphorus in TCP is better than that in DCP, which is beneficial for promoting bone calcium and phosphorus deposition and enhancing the bone strength.

In this study, bamboo leaf flavonoids (BLF) was used as an additive to explore its protective effect on hydrogen peroxide (H₂O₂)-induced pyroptosis of bovine mammary epithelial cells (BMECs). In this experiment, the concentration of BLF was 80 μg·mL^-1 and the concentration of H₂O₂ was 800 μmol·L^-1. The BMECs without BLF treatment were set as the control group, and the treatment groups were BLF treatment group, H₂O₂ treatment group, BLF and H₂O₂ co-treatment group. The expression of pyroptosis-related genes and related proteins and the fluorescence intensity of apoptosis-related microparticle-like protein(ASC) were detected by RT-qPCR, Western blot, immunofluorescence and flow cytometry. The results showed that compared with the control group, 800 μmol·L^-1 H₂O₂ treatment of BMECs for 8 h significantly increased the pyroptosis of bovine mammary epithelial cells (P < 0.05). After H₂O₂ treatment, the relative expression levels of NLRP3, ASC, GSDMD, IL-18, IL-1β genes which related to pyroptosis genes were significantly increased (P < 0.05), especially the expression of NLRP3, indicating that H₂O₂ significantly increased the pyroptosis of BMECs. Compared with the H₂O₂ treatment group, BLF could reduce the pyroptosis level of normal cells, significantly reduce the mRNA up-regulation trend of NLRP3, Nek7, ASC, GSDMD, IL-18, IL-1β, and significantly alleviate the increase of intracellular pyroptosis protein expression of NLRP3, ASC, Nek7, pro-caspase1, caspase1 p20, GSDMD-N, IL-18 and IL-1β induced by H₂O₂ (P < 0.05), and the fluorescence intensity of ASC was significantly reduced (P < 0.05). In summary, H₂O₂ significantly up-regulated the expression levels of pyroptosis-related genes and proteins such as NLRP3, ASC, Caspase-1, GSDMD, IL-1β, and IL-18, and induced pyroptosis in bovine mammary epithelial cells. In conclusion, BLF reduced the level of inflammatory factors by reducing the expression of NLRP3 inflammasome pathway proteins and genes, thereby alleviating the pyroptosis of dairy cow mammary epithelial cells.

This experiment aimed to investigate the effects of three different kinds of soybean isoflavone (SIF) supplemented in diet on laying performance, egg quality, serum indexes, ovarian function and tibia quality of laying hens in late laying period. A total of 432 66-week-old Hyline brown laying hens with similar laying rate were randomly divided into 4 treatment groups with 6 replicates per group and 18 hens per replicate. The control group was fed a basal diet, the three experimental groups were fed the basal diet supplemented with one of the three soybean isoflavone products, respectively, namely SIF1, SIF2 and SIF3. The pre-trial period was 1 week and the trial period was 8 weeks. The results showed that: 1) Compared with the control group, dietary supplemented with SIF1 and SIF3 significantly increased the laying rate at weeks 1-4, 5-8 and 1-8 (P < 0.05); 2) Dietary supplemented with SIF1 and SIF3 significantly increased the Haugh unit at weeks 4 and 8 (P < 0.05) and dietary SIF1 significantly increased the egg protein height at week 8 (P < 0.05) compared to the control group; 3) Compared with the control group, dietary supplemented with SIF1 significantly decreased serum MDA content (P < 0.05), dietary SIF1 and SIF3 significantly increased the content of GSH-Px and E₂ in serum (P < 0.05), dietary SIF3 supplementation significantly increased serum P₄ content and serum Ca content (P < 0.05); 4) Dietary supplemented with SIF2 and SIF3 significantly increased the total number of follicles, the number of primary follicles, the number of secondary follicles, and the number of small white follicles (P < 0.05); 5) Dietary supplemented with SIF1, SIF2 and SIF3 significantly increased the content of calcium and phosphorus in tibia (P < 0.05). In conclusion, SIF may improve the performance and egg quality of laying hens in the late laying period by increasing the number of ovarian follicles, increasing the serum antioxidant capacity, the content of reproductive hormones and the content of calcium and phosphorus in tibia, and the improvement effects of different SIF products are different.

In order to study the potential of displaying foreign epitopes in the 3-fold axes region on the surface of PCV2 capsid, 3D structures of recombinant proteins after amino acid mutation are modeled, displayed and analyzed, combined with PCV2 capsid surface amino acid analysis. The constructed plasmids were expressed in E.coli (BL21), and the differences in soluble expression and purification difficulty of various mutant cap genes were analyzed, then the suitable region for displaying foreign epitope was determined. After the recombinant Cap protein inserted with PPV epitope was assembled in vitro, then the chimeric VLPs were immunized to mice, and the immunogenicity of chimeric VLPs was evaluated by antibody detection. The results showed that there were two amino acid regions (named Motif A and Motif B) suitable for displaying exogenous epitopes in 3-fold axes region on the surface of PCV2 capsid. Under the same expression conditions, mutant proteins related to Motif A region were mainly expressed in supernatant, and the target protein could be purified in one step, while most mutant proteins related to Motif B region were mainly in the form of precipitation. After PPV epitope was chimeric in Motif A region, and the purified recombinant protein could be assembled into cVLPs. IFA results demonstrated that cVLPs retained a similar capacity to enter PK15 cells as its wild type. The formed cVLPs could stimulate the mouse body to produce antibodies against the PCV2 VLPs, the B5-E1 epitope and PPV VP2 protein, respectively. The results showed that Motif A in the Loop GH region of PCV2 Cap protein, can display foreign epitopes on the outer surface of virus-like particles, and can be recognized by immune cells to produce specific antibodies.

To establish a rapid detection method for swine acute diarrhea syndrome coronavirus (SADS-CoV) serum antibodies, N gene was amplified by PCR, and then the purified fragment was cloned into pET-32a (+) prokaryotic expression vector to construct the recombinant plasmid pET-32a-N. The expression of recombinant N protein was determined by SDS-PAGE and western blot. The purified recombinant N protein was applied as the coating antigen. Rabbit anti-pig HRP-IgG was used as the secondary antibody. We used the square-array titration to optimize the reaction conditions of the indirect ELISA method targeting SADS-CoV N protein antibodies. In addition, we identified the sensitivity, specificity and repeatability of this method, and evaluated its clinical applicability. SDS-PAGE and western blot results showed that the recombinant N protein was about 70 ku and had good reactivity. The antibody titer of SADS-CoV positive serum detected by this method was 1: 3 200, and there was no cross reaction with PEDV, TGEV, PDCoV, PRRSV, PoRV or PCV positive pig serum, which exhibited good sensitivity and specificity. The coefficient of variation of intra-batch and inter-batch repeatability tests were both less than 10%, demonstrating favorable reproducibility of this method. Fifty randomly selected porcine clinical serum samples were tested by ELISA and immunofluorescence assay, and the coincidence rate of results was 98%. In conclusion, we established an indirect ELISA based on N protein to test SADS-CoV clinical serum antibodies, which is useful for epidemiological surveillance, and is of great significance for prevention and control of SADS-CoV.

In order to understand the biological characteristics of EHV-1/China/YL2023 isolated from Yili and its pathogenicity to Syrian golden hamsters, the isolates were purified by plaque assay, virus culture and proliferation, saturated ammonium sulfate concentration and sucrose density gradient centrifugation. To investigate the pathogenicity of EHV-1/China/YL2023 virus isolate, Syrian golden hamsters were infected with different doses of EHV-1/China/YL2023 virus by intranasal inoculation. The results showed that the titer of purified EHV-1/China/YL2023 strain reached 10^6.64 TCID₅₀·mL^-1. Syrian mice aged 4-5 weeks infected with EHV-1/China/YL2023 strain showed depression, decreased food intake, drooling, curled up, hunched back, unkily dropped fur and varying degrees of neurological symptoms within 14 days. The average body weight of the recovered hamsters decreased by 25.9%, and the survival rate of the 10⁷-10⁵ TCID₅₀·0.1 mL^-1 dose groups was 50%.The pathological results showed that different doses of virus caused different degrees of damage to the lungs and brains of Syrian mice, including alveolar wall thickening, brain neuron swelling, a large number of neutrophils and lymphocytes infiltration, hemorrhage and congestion. The viral load measurement results of different tissues showed that the Syrian rats contained viral DNA in the lungs, brain and lymph nodes, and the content was high in the lungs and brain. In conclusion, the EHV-1/China/YL2023 isolates were highly pathogenic to Syrian golden hamsters aged 4 to 5 weeks. This study provides a basis for the study of the biological characteristics and pathogenic mechanism of EHV-1, and provides support for the future development of EHV-1 vaccine.

The aim of this study was to establish a rapid diagnostic method of pigeon circovirus (PiCV), a real-time quantitative PCR based on Cap gene was established and a molecular epidemiological investigation on PiCV was conducted. Specific primers targeting the conserved sequence of the Cap gene of PiCV were designed and synthesized. The sensitivity, specificity, and repeatability of the developed quantitative PCR method (qPCR) were evaluated. One hundred and forty-seven clinical samples from Northern and Northwest China were detected by the qPCR, followed by genome sequencing and phylogenetic analysis on whole length of Cap gene of isolates from different regions. The established qPCR method showed a good linear response for PiCV standards ranging from 1×10⁴ to 1×10⁸ copies·μL^-1, with a minimum detection limit as low as 1×10¹ copies·μL^-1, substantially surpassing conventional PCR. The intra-assay and inter-assay demonstrated a good repeatability. Importantly, there was no cross-reactivity with other pigeon pathogens. Detection results of 147 clinical samples collected from Northern and Northwest China by this method showed that the positive rate for PiCV was 85.0%. Sequencing results and phylogenetic analysis revealed that Cap gene of six isolates shared homology within the range of 89.8% to 97.5%, and were closely related to PiCV HeBeiTS2021 isolate. The established real-time quantitative PCR method for PiCV demonstrated sensitivity, specificity and repeatability, making it available for rapid detection of PiCV. Epidemiological investigation results indicated a higher PiCV infection rate in Northern and Northwest China during 2022-2023, providing valuable data for prevention and control of PiCV.

This study evaluated the immunogenicity and protective efficacy of OmpD and LppB proteins from Actinobacillus pleuropneumoniae. Homology analysis revealed that all 19 serotypes of APP have ompD and lppB genes, with amino acid sequence identities of 98.2%-100% and 99.3%-100%, respectively. Recombinant OmpD (rOmpD) and LppB (rLppB) proteins from serotype 1 were expressed and purified using engineered E. coli and subjected to immunogenicity analysis. The results showed that both proteins were effectively expressed and exhibited obvious reactivity with swine convalescent serum. Mice immunized with rOmpD or rLppB leaded to significantly elevated ELISA antibody titers and protection rates of 70%-80% against challenge with prevalent serotype 1 and 7 strains. Further, an oil emulsion containing rOmpD and rLppB was prepared, and immunization of piglets elicited significantly elevated ELISA antibody titers and conferred partial protection and cross-protection against serotype 1 and 7 challenge. However, complete protection against infection and death was not achieved. These findings suggest that OmpD and LppB are promising candidates for subunit vaccine development against Actinobacillus pleuropneumoniae, and provide valuable data for the further development of vaccines with cross-protective efficacy.

This experiment was conducted to investigate the pathogenicity of highly virulent Klebsiella pneumoniae of sheep origin and the extraction of its outer membrane vesicles. In this study, sheep that died with cough and diarrhea symptoms were dissected and the diseased organs such as lungs, livers, and jejunum were collected. Morphological observation, biochemical characterization, molecular biological identification, and sequencing methods were used to isolate and identify the pathogenic bacteria; the pathogenic bacteria were identified by drug sensitivity test, pulling test, virulence gene test, pathogenicity test, and pathohistological observation, and analyzed for pathogenicity and resistance; and the modified precipitation method was used to Its outer membrane vesicles were extracted and identified by transmission electron microscopy, nanoparticle size determination and SDS-PAGE. The results showed that the pathogen was isolated and purified, and the microscope showed oval-shaped gram-negative bacteria, and the biochemical test and 16S rRNA identification results indicated that the pathogen was Klebsiella pneumoniae; the results of drug sensitivity test showed that the pathogen was sensitive to four drugs, namely, amikacin, ceftazidime, imipenem, and piperacillin; the results of the pulling test showed the positive characteristics, and the virulence gene amplification showed that the pod polysaccharide gene wzy-K1 and the metabolism gene peg-344 were positive; the results of mouse pathogenicity test showed that the pathogen was positive to the LD₅₀ of mice. metabolic gene peg-344 was positive, the pathogen was identified as highly virulent Klebsiella pneumoniae; the results of the mouse pathogenicity test showed that the pathogen had a concentration of 1.8×10⁵ CFU in the mouse LD_50, and the liver and spleen were bruised with a large number of inflammatory cell infiltration; transmission electron microscopy and nano-particle sizing showed that the morphologic structure of the sediment and the particle size were consistent with the characteristics of the outer membrane vesicles of the bacteria. Transmission electron microscopy, nanoparticle size determination showed that the morphology and structure of the precipitate and the size of the particles were consistent with the characteristics of bacterial outer membrane vesicles, and SDS-PAGE showed the presence of characteristic bands. In this experiment, highly pathogenic Klebsiella pneumoniae was successfully isolated from sick and dead sheep and an improved method of extracting its outer membrane vesicles was established to provide reference for the prevention and treatment of Klebsiella pneumoniae disease of sheep origin and help for basic research on the outer membrane vesicles of Klebsiella pneumoniae.

MgtC protein is an important virulence factor in a variety of intracellular parasites and possesses ability of resistance to low Mg²⁺ environment. Brucella is an important intracellular parasite, and there are few reports on the biological function of MgtC protein in Brucella. To explore the biological characteristics of MgtC in Brucella abortus, the adaptability to low Mg²⁺ environment and other possible biological functions were studied in this study. Brucella abortus S2308 mgtC deletion mutant (S2308ΔmgtC) and the complementary strain S2308ΔmgtC (pBBR1MCS-mgtC) were constructed. Subsequently, studies on the three strains in low/high Mg²⁺ environment with growth curve determination, ATP content determination, intracellular survival, environmental stress, biofilm detection and mouse challenge test were conducted to comprehensively analyze the biological function of MgtC in Brucella abortus. The deletion strain S2308ΔmgtC and its complementary strain S2308ΔmgtC (pBBR1MCS-mgtC) were successfully constructed. In Brucella abortus S2308, expression of mgtC gene was significantly up-regulated in low Mg²⁺ environment(P < 0.05). The growth curve(P < 0.001), membrane structure integrity(P < 0.05)and biofilm formation ability(P < 0.01)of S2308ΔmgtC in low Mg²⁺ environment were lower than those of the parent strain and the complementary strain, while the ATP content in the strain was higher than that of the parent strain and the complementary strain(P < 0.001). In addition, the deletion of mgtC gene did not affect the survival of Brucella abortus to macrophages and the pathogenicity of mice. In low Mg²⁺ environment, Brucella abortus employed MgtC to ensure the normal hydrolysis of ATP and maintain its normal physiological activities and the integrity of membrane structure. However, MgtC had no significant effect on the virulence and pathogenicity of Brucella abortus. This study provides data support for further understanding of Brucella abortus.

The objective of this study is to construct a surface-displayed Saccharomyces cerevisiae EBY100/PYD1-EgM123 for Echinococcus granulosus, and to evaluate its immunogenicity. The EgM123 gene of E. granulosus was cloned, and a surface display yeast strain EBY100/PYD1-EgM123 was constructed. The S. cerevisiae strain EBY100/PYD1-EgM123 was examined using immunofluorescence assay, and BCA protein quantitative analysis to determine its localization and quantity. Additionally, experimental mice were immunized with the EBY100/pYD1-EgM123. The immune efficacy of the EBY100/pYD1-EgM123 was evaluated by measuring antibody and cytokine in the serum of the mice. The results showed that the ORF of EgM123 was successfully cloned and its length was 594 bp. The results of PCR and restriction endonuclease analysis showed that the surface-displayed S. cerevisiae EBY100/PYD1-EgM123 was successfully constructed. The results of immunofluorescence showed that the EBY100/PYD1-EgM123 protein exhibited green fluorescence on the surface of the S. cerevisiae. The expression level of EBY100/pYD1-EgM123 was quantified at 50 OD_{600 nm} using the BCA protein quantitative assay. At 72 h, the expression level was found to be 15.59 μg and 0.31 μg/OD_{600 nm}, respectively. The levels of specific IgG, IgA, and IgM in the EBY100/pYD1-EgM123 group were found to be significantly higher than those in the PBS Group, EBY100 Group, and EBY100/PYD1 group (P < 0.05). The results also indicated that the EBY100/PYD1-EgM123 effectively enhances the secretion of IL-17 and IFN-γ (P < 0.001). In conclusion, the present study successfully constructed the surface-displayed S. cerevisiae EBY100/PYD1-EgM123, and demonstrating its ability to stimulate specific immune responses in mice. It is further indicated that it has the potential to be a candidate vaccine against E. granulosus in the final host.

This study was designed to study the evolutionary trend of structural genes of high pathogenicity island (HPI) of Escherichia coli (E. coli) and its effect on TNF/NF-κB pathway. In this study, MEGA and other software were used to analyze the genetic evolution of HPI structural genes, and R language function package was used to mine the gene expression omnibus (GEO) of E. coli infected cells, and differential gene enrichment analysis was performed. Molecular docking was used to predict the binding of HPI-Ybt to TNF/NF-κB pathway related proteins. E. coli and the previously constructed HPI deficient strain E. coliΔHPI were infected with IPEC-J2 cells, and qPCR, ELISA and Western blot were used to detect the key regulatory points in TNF/NF-κB pathway. Phylogenetic analysis showed that the intb gene of isolate YUNNAN001 was most closely related to GQ891729.1, but far from CQ903045.1. KEGG enrichment found that TNF and NF-κB pathways were both involved in E. coli infection, and there were 10 common genes. Volcano plot and heat map enrichment showed that E. coli infection significantly promoted the expression of PTGS2, NF-κB, TRAF6 and other proteins in TNF/NF-κB pathway (P < 0.05). Molecular docking showed that HPI-Ybt could bind to 10 proteins belonging to TNF/NF-κB pathway, among which MAP3K14 and TNFAIP3 were the strongest. The mRNA levels of NF-κB, PTGS2, TNF-α, TRAF6, TRIF, ATF4, cxcl2 and IL-6 in E. coli group were significantly higher than those in E. coliΔHPI group (P < 0.01), and compared with E. coliΔHPI group, E. coli group significantly activated the expression of TNF/NF-κB pathway proteins. In addition, E. coli infection significantly increased the levels of NF-κB and IL-6 compared with the E. coliΔHPI group (P < 0.01). The results suggest that E.coli-HPI activates TNF/NF-κB pathway by synthesizing Ybt and binding to key proteins in TNF/NF-κB pathway.

To detect the expression of Qa-1b/NKG2A in skins of Mongolia cattle for analyzing the potential roles in skin immunity and inhibition of development of melanoma cells of Qa-1b. The expression of Qa-1b/NKG2A in skins was quantitatively analyzed by quantitative real-time PCR, Western blot and immunohistochemistry, and by co-immunoprecipitation for interaction between Qa-1b and NKG2A. The sequence of Qa-1b nanoloody (Qa-1b-VHH) binding with Qa-1b was obtained by phage display technique from alpaca melanoma cell nanobady library using Qa-1b polypeptide as antigen, followed by the construction of pET28a-Qa-1b-VHH prokaryotic expression recombinant plasmid and expression through IPTG inducing and purification through nickel column. After the binding of Qa-1b nanobody to antigen was detected by ELISA, the Qa-1b nanobody was used as the primary antibody in immunohistochemistry and Western blot methods to detect Qa-1b expression in tissues. Qa-1b nanobody was added into B16 cell culture medium, and its effects on the proliferation and migration of B16 cells and the expression of proteins related to proliferation and migration were detected by CCK8, cell scratch and Western blot methods, respectively. The results showed that Qa-1b and NKG2A were expressed in the out root sheath of hair follicles and vascular endothelium in Mongolian cattle skins with significantly higher expression in skins of young cattle than that in skins of the adult cattle (P < 0.01 or P < 0.001). Moreover, there existed the interaction between Qa-1b and NKG2A. The Qa-1b nanobody with strong specificity was obtained, which could be used as a primary antibody for Western blot and immunohistochemistry to detect Qa-1b expression in other tissues. The supplement of Qa-1b nanobody in B16 culture medium resulted in the obvious inhibition of cell proliferation and migration, significant down-regulation of the expression of proteins such as Ras, MEK1, CyclinD1, and CDK4 during cell proliferation(P < 0.01 or P < 0.001). The results suggested that Qa-1b might play a role in the skin immunity to ensure the normal growth of hair follicles, especially in calves, and the Qa-1b nanobody could inhibit the proliferation and migration of B16 cells, which provided a theoretical basis for the strengthened skin immunity and anti-melanoma antibody drugs.

This study was to detect the effects of Daidzein (DZ) on the synthesis of lactose, milk protein, milk fat and cell proliferation of mouse mammary epithelial cells EpH4-Ev to investigate the associated regulatory roles. EpH4-Ev cells were treated with different concentrations of DZ (0 to 1 000 μmol·L^-1) for 6, 12, 24, 48 h and the concentration of DZ and time were determined by detecting cell viability; they were divided into a control group (0 μmol·L^-1 DZ treatment), a low-concentration group (10 μmol·L^-1 DZ treatment), a medium-concentration group (20 μmol·L^-1 DZ treatment), and a high-concentration group (40 μmol·L^-1 DZ treatment), and physiological dose of estradiol (E2) was used as a positive control, and the cells were incubated for 12 h at 37 ℃ and 5% CO₂ as follows experiments were performed: 1) Determination of triglyceride (TG) and glucose (GLU) content in cells and supernatants; 2) Cell proliferation and apoptosis-related proteins, milk component synthesis-related proteins and PI3K/AKT-mTOR signaling pathways were detected, and milk lipid synthesis was probed by combining with lipid droplet staining. The apoptosis rate and cell cycle distribution were analyzed by flow cytometry. The results showed that: 1) Compared with the control group, between 2.5-80.0 μmol·L^-1 DZ treatments could significantly improve the cell viability of EpH4-Ev cells (P < 0.01), and 20 μmol·L^-1 DZ treatment had the most significant improvement effect. Combined with the results of the pre-laboratory period, the low, medium and high concentrations of DZ action were selected to be 10, 20, and 40 μmol·L^-1. 2) Compared with the control group, medium and high concentration of DZ and E2 treatments significantly increased the content of TG and GLU in EpH4-Ev and supernatants and promoted the synthesis of lipid droplets (P < 0.01). 3) Compared with the control group, the expression of glucose transporter carrier 1 (GLUT1) and β-casein were both highly significantly elevated after treatment with different concentrations of DZ (P < 0.01); meanwhile, both DZ and E2 treatments increased the expression of fatty acid synthetase (FASN), cholesterol regulatory element-binding protein 1 (SREBP1), peroxisome proliferator-activated receptor γ(PPAR-γ), and acetyl coenzyme A carboxylase (ACC) expression. 4) Compared with the control group, the proportion of cells in G2/M phase and S phase, the expression of proliferating cell nuclear antigen (PCNA), cell cycle proteins D1 and D3 (CyclinD1 and D3), and anti-apoptotic protein Bcl-2 were increased after treatment with different concentrations of DZ and E2, and the medium concentration of DZ group highly significantly increased the Bcl-2/Bax ratio (P < 0.01) and decreased the apoptosis rate. 5) Compared with the control group, three concentrations of DZ and E2 treatment increased the phosphorylation levels of p-PI3K, p-mTOR, p-AKT and promoted the activation of the PI3K/AKT-mTOR signaling pathway. These results suggested that DZ can promote the proliferation of mammary epithelial cells and the synthesis of milk components. The mechanism is related to the up-regulation of the expression of cell proliferation proteins, the reduction of apoptosis rate, and the activation of PI3K/AKT-mTOR pathway.

The aim of this study was to investigate the inhibitory effects of epicatecin gallate (ECG) on nuclear factor κB (NF-κB) signaling pathway and NOD-like receptor 3 (NLRP3) inflammasome in bovine mammary epithelial cells. Lipopolysaccharide (LPS) was used to treat bovine mammary alveolar cells (MAC-T) and mouse mammary glands. The treatment concentration of ECG in MAC-T cell was screened by the CCK-8 method. The expression levels of proinflammatory factors, oxidative stress indicators and pyroptosis indicators were detected by ELISA. Hematoxylin-eosin staining and Annexin V-FITC/PI were used to detect the inflammatory changes in mouse mammary tissues and cell membrane rupture. The expression levels of NF-κB, NLRP3, apoptosis-associated speck-like protein (ASC), caspase-1 and Gasdermin NH₃ end (GSDMD-N) were detected by Western blot. Nuclear transfer of p65 was detected by immunofluorescence. The results showed that: 1) ECG at 10, 20 and 40 mg·kg^-1 could reduce LPS-induced inflammatory changes and inflammatory cell infiltration; 2) ECG significantly and dose-dependent decreased LPS-induced levels of pro-inflammatory factors (IL-1β, IL-6, TNF-α) and oxidative indicators (COX-2, iNOS) in both in vivo and in vitro systems (P < 0.05); 3) ECG dose-dependently reduced of LPS-induced cell death and the levels of ROS, IL-18 and LDH (P < 0.05); 4) ECG significantly inhibited the levels of NLRP3, ASC, GSDMD-N, caspase-1 and the phosphorylation and nuclear translocation levels of p65 (P < 0.05). The results indicate that ECG inhibits MAC-T cell and mouse mammary gland inflammation and pyroptosis as well as the NF-κB pathway and NLRP3 inflammasome. This study provides a new idea for the prevention and treatment of bovine mastitis.

With the continuous development of industrial and agriculture, more and more fluoride enters the production, life and environment through drinking water, medicines, pesticides and fungicides, which may have adverse effects on animals living in the area. In order to explore the related effects of fluoride poisoning on small intestinal injury in broilers, we established an animal experimental model through fluoride diet exposure for subsequent verification. Broilers were divided into control group C (0 mg·kg^-1), low-dose fluoride group L (500 mg·kg^-1), medium dose fluoride group M (1 000 mg·kg^-1) and high-dose fluoride group H (2 000 mg·kg^-1) according to the exposure dose level of sodium fluoride in feed. The effects of fluoride exposure on small intestine of broilers were analyzed. With the increase of sodium fluoride exposure concentration, Nrf2 increased (P < 0.001), Keap1 decreased (P < 0.001), HO-1 decreased (P < 0.001), and NQO1 decreased in the high concentration group (P < 0.01). The Nrf2/HO-1 pathway was activated, but its downstream antioxidant enzymes decreased, suggesting the oxidative stress may occur; the protein expression of p-AKT and p-mTOR increased significantly (P < 0.001), and finally returned to normal or even decreased (P < 0.05), while PI3K decreased significantly (P < 0.001), suggesting that the phosphorylation of PI3K/Akt/mTOR pathway was inhibited; Beclin-1 was significantly increased (P < 0.001), ATG12 was not significantly changed, and LC3 protein was significantly higher than the C group (P < 0.05), indicating the autophagosomes formation and autophagy flow were accelerated, p62 protein combined with autophagy substrate protein (P < 0.001), and the content decreased, indicating the authay pathway was not blocked. At the same time, FTH1 decreased (P < 0.01), NCOA4 increased (P < 0.01), and ferrous ions were released from the degradation of ferritin; ASCL4 and ALOX12 protein increased (P < 0.01), which promoted the lipid peroxidation process; SLC7A11 and GPX4 protein significantly decreased (P < 0.001), and the ability of scavenging lipid peroxide was reduced, which was prone to ferroptosis. Sodium fluoride interfered with the Nrf2 antioxidant pathway, leading to oxidative stress, also activate autophagy and ultimately lead to ferroptosis in the small intestine of broilers.

The aim of this study was to investigate the effects of salidroside-added complete nutrition food on blood and liver transcriptomics in dogs, with a view to provide experimental data to support liver conservation and prevent liver disease in dogs. Ten adult Chinese field dogs (8.67±1.00 kg) were selected and randomly divided into control (n=5) and test (n=5) groups. The control group was fed with control food, while the test group was fed with complete nutrition food supplemented with salidroside. The study evaluated the feeding effect and safety of complete nutrition food supplemented with salidroside through blood examination. Additionally, the potential regulatory mechanism of salidroside-added complete nutrition food on dogs was explored using transcriptomics technology. The trial period lasted 8 weeks. The blood test results showed that salidroside-added complete nutrition food significantly reduced the concentration of serum alanine aminotransferase in dogs (P < 0.05), and did not significantly affect most of the other indexes (P>0.05). However, compared to the control food, the complete nutrition food with added salidroside did not cause greater fluctuations in blood indexes and could maintain their stability. The transcriptomics analysis results revealed that differentially expressed genes were significantly enriched in the calcium signaling pathway, PI3K-Akt signaling pathway, and MAPK signaling pathway. The results of gene set enrichment analysis for phenotypes revealed that salidroside-added complete nutrition food played a regulatory role in lipid metabolism, glucose metabolism, inflammation, oxidative stress, and fibrosis. It showed a more significant regulation of glucose and lipid metabolism, with the related signaling pathways significantly up-regulated in the test group. In conclusion, salidroside-added complete nutrition food has been found to reduce serum alanine aminotransferase concentration in dogs and help stabilize the concentration of blood indexes in dogs, and regulate hepatic glucolipid metabolism in dogs. These effects may be primarily achieved by regulating the calcium signaling pathway, the PI3K-Akt signaling pathway, and the MAPK signaling pathway.

The aim of this experiment was to study the effects of Chinese medicine Jianpisiwei formulas on the growth performance, rumen fermentation and microbiota composition of weaned Hu sheep. A total of 100 healthy 2-month-old Hu sheep with average body weight of (14.55±0.21) kg were randomly divided into 5 groups with 4 replicates in each group and 5 sheep per replicate. The control group was fed with basal diet, A1 and A2 groups were fed basal diet supplemented with 0.3% and 0.5% Jianpisiwei formula A, the B1 and B2 groups were fed basal diet supplemented with 0.3% and 0.5% Jianpisiwei formula B. The results showed that compared with the control group, the average daily weight gain of the A2 group was significantly increased (P < 0.05), the feed-to-gain ratio was significantly decreased (P < 0.05), the content of ammonia nitrogen (NH₃-N) in the A1 and B2 groups was significantly increased (P < 0.05), the content of microbial protein (MCP) was significantly decreased (P < 0.05), the pH value of the the A2 group was significantly decreased (P < 0.01), and the content of propionic acid was significantly increased (P < 0.01). The ratio of acetic acid to propionic acid was significantly decreased (P < 0.01), the total volatile fatty acids were significantly increased (P < 0.01), the number of species and Chao 1 index in the the A1 and A2 groups were significantly increased (P < 0.05), the Shannon index was significantly increased (P < 0.01), the relative abundance of Spirochaetota was significantly decreased (P < 0.01), and the relative abundance of Euryarchaeota in the A2 group was significantly increased (P < 0.01);The relative abundance of Rikenellaceae_RC9_gut_group and Eubacterium_ventriosum_group in the A1 and A2 groups decreased significantly (P < 0.05), while the relative abundance of Bacteroidales_RF16_group increased significantly (P < 0.05). The relative abundance of Selenomonas in the A2 group was significantly decreased (P < 0.05), while the relative abundance of Quinella increased significantly (P < 0.05). The above results indicated that the addition of Chinese medicine Jianpisiwei formulas to the diet could promote the growth performance, improve rumen fermentation, and increase the diversity and richness of rumen flora in weaned Hu sheep.