The technique of in vitro embryo production is one of the crucial technologies to improve the reproductive efficiency of elite livestock breeds. However, its industrialized application has been severely restricted due to the environmental variation of in vivo and in vitro, which leads to poor quality of matured-oocyte and decreased developmental potential of in vitro-generated embryo. Microfluidics can be considered as the science and technology of systems that precisely manipulate structure-fluid at a sub-micrometer level in a microchannel of tens to hundreds of micrometers. They have been extensively applied to the field like chemistry, medicine, and life science due to numerous characteristics, such as low sample consume, fast analysis speed, high-throughput, and high integration. Microfluidics systems possess huge application potentials in several key technical aspects involved in in vitro embryo production procedure. Hence, this review intends to summarize the current knowledge about the applications of microfluidics in oocyte selection and cultivation, sperm sorting, in vitro fertilization, in vitro embryo culture, and gamete and embryo cryopreservation, providing novel conceptions for enhancing the efficiency of in vitro embryo production of domestic animals.

During mammalian early embryonic development, the fertilized egg undergoes a series of epigenetic reprogramming events, erasing the epigenetic memory of the parental nuclei and establishing the embryonic totipotency. However, the exact regulation of these reprogramming events by species-specific mechanisms calls for more research into the subject. Significant success has been made in understanding the regulatory network of epigenetic reprogramming during early mammalian embryonic development thanks to the ongoing development of molecular biology techniques, particularly the innovations in low-input whole-genome chromatin analysis. The goal of this paper is to outline recent developments in the mechanisms of epigenetic reprogramming in model animals and different livestock, including work on DNA methylation, histone modifications, chromatin accessibility, and chromatin 3D structure.

In recent years, with the deepening research on the intestinal microbiota and a clearer understanding of its relationship with organismal health, it has been found that the intestinal microbiota plays a dual role in regulating viral infections. The intestinal microbiota can inhibit viral infection by protecting the intestinal mucosal barrier, directly neutralizing viruses or preventing their effects on cell adhesion, indirectly influencing the establishment and development of the body's immune system, and secreting antiviral metabolites to play an antiviral role. Moreover, probiotics have played an increasingly prominent role in disease treatment, and it is expected that probiotics can overcome the disadvantages of current viral disease treatments. This paper reviews the research progress on the mechanisms of the intestinal microbiota's resistance to viral infection in recent years and discusses related issues and future directions for research.

Streptococcus suis (S. suis) infection has long plagued the health and sustainable development of our pig industry. In particular, the emergence of drug-resistant strains will block the original prevention and control strategy of streptococcosis. The summary of the prevalence of resistance to S. suis at China and abroad in recent years can provide effective measures for the prevention and treatment of S. suis infection. Therefore, we first summarized the S. suis infection situation in large-scale pig farms at home and abroad in the past five years (most positive rate>40%). Among them, the serotype 2 strain is the most widespread in all provinces and cities in China. Subsequently, we summarized the epidemic status and infection characteristics of drug-resistant S. suis in pig farms, and found that drug-resistant S. suis in large-scale pig farms concentrated in the Asian and Europe, and the drug resistance exceeded 80% in the most regions, mainly tetracycline resistant strains. However, resistance to S. suis in large-scale pig farms in our country is mainly tetracycline and macrolide resistance. In addition, S. suis is a zoonotic agent that can cause systemic infection through the respiratory tract and blood. Finally, in view of the characteristics of S. suis strain variation, we proposed the importance of accurate detection for the prevention and control of S. suis, summarized the rapid detection methods. The prevention and control measures based on vaccination, combined drug use and the development of new host-directed antibiotics are emphasized to provide data support for the subsequent prevention and control of resistant strains of S. suis.

Avibacterium paragallinarum (Apg) is the pathogen of infectious coryza in chickens. It induces symptoms such as nasal cavity mucus secretion, sneezing, infraorbital sinus swelling, facial edema, and conjunctivitis. These clinical manifestations lead to stunted growth and a significant decrease in egg production, posing a threat to poultry farming. The pathogenicity of Apg is influenced by various factors, including cell wall structural components like capsule and lipopolysaccharide, as well as functional proteins secreted by the cell such as hemagglutinin and metalloproteinases. This paper provides a review of the research progress on the virulence factors of Apg, focusing on the biofilm system, secretory system, and bacterial iron capture system. The aim is to offer a theoretical reference for the scientific prevention and treatment of infectious coryza in chickens.

The purpose of this experiment was to explore methods to improve the accuracy of genomic selection (GS) for Duroc pigs’ carcass traits, and to provide a theoretical basis for improving the accuracy of GS for breeding pig carcass traits. In this study, corrected backfat thickness at 115 kg (BF115), corrected lion eye area at 115 kg (LEA115), corrected age at 115 kg (AGE115) were studied in 2 796 Duroc boars and 3 149 Duroc×Landrace×Yorkshire (DLY) commercial pigs. Additive animal model (AM) and additive add dominant animal model (ADM) were constructed to verify the impact of dominant effects on GS accuracy of Duroc pigs; exploring the impact of different combinations of DLY and Duroc pigs in the reference population on GS accuracy of Duroc pigs. The reference population were divided into the following groups: group 1, Duroc pigs as the reference group; group 2, with DLY pigs as the reference group; group 3, Duroc and DLY pigs were merged as reference group. The research results indicated that after adding dominant effects, the accuracy of GS for AGE115 increased by 2.84% to 4.87% in group 1, 2, and 3, while the accuracy of GS for Duroc BF115 and LEA115 increased by 1.37% to 16.18% in group 1 and 3; compared to group 1, the accuracy of GS for BF115 and LEA115 in the AM increased by 6.19% to 7.35% in group 3, and by 6.52% in the ADM. In summary, dominant effects can improve the accuracy of GS for pig carcass traits, and combining Duroc and DLY pigs as reference population can improve the accuracy of GS for some pig carcass traits, that it is recommended to include suitable hybrid pig population in the reference population for purebred pig breeding.

The study aimed to study the weekly changes of body weight and body composition of Wenchang chickens, and establish the empirical growth model to provide scientific basis for efficient breeding of Wenchang chickens. A total of 300 healthy 1-day-old female chicks with similar body weight were randomly divided into 6 replicates and fed conventionally until 119 days of age. The body weight and body composition of Wenchang chickens were measured weekly. The results showed that the body weight, carcass weight, feather weight, body protein weight and body fat weight of Wenchang chickens increased significantly with the increase of week age (P<0.001). Four nonlinear function formulas, Gompertz, Von Bertalanffy, Logistic and Richards, were used to fit the growth curves of body weight, body protein and body fat of Wenchang chicken, respectively. According to the determination coefficient (R²), mean square error, Akaike information criterion and residual sum of squares of regression models, it is found that Gompertz model has the highest degree of fit. The growth models of body weight, carcass, feather, body protein and body fat of Wenchang chickens were established using Gompertz model. Except for the feather moisture weight, the R² of the other component models was close to or exceeded 0.99. The mature weight of body weight, body protein and body fat were 2 382 g, 696.8 g and 1 037.1 g, respectively. The relative maturity rates were 0.023 2, 0.017 4 and 0.017 8, respectively. The Gompertz model can be used to predict the body weight and body composition weight of Wenchang chickens at different days of age. These models and their calculus formulas are helpful for genetic selection and production decision-making of Wenchang chickens, and also provide a basis for predicting the daily energy and amino acid requirements of Wenchang chickens in the future.

The study aimed to explore the genetic diversity, relationship and family structure of the New Pudong chickens population. In order to reveal the genetic relationship between individuals, "Jingxin No.1" SNP chip was used to detect the SNPs in the whole genome of 368 New Pudong chickens. The genetic structure, genetic relationship and inbreeding coefficient of New Pudong chickens were also analyzed. The result showed that a total of 55 023 SNPs were obtained. According to the quality control of Plink (V1.90) software, there were 42 267 remaining SNPs, among which 78% were polymorphic. The effective allele number was 1.552, the PIC was 0.237, the MAF was 0.226, the H_O was 0.355, and the H_E was 0.353. The average IBS genetic distance of New Pudong chickens was 0.280 9. The average IBS genetic distance of 110 roosters was 0.280 1. According to the cluster analysis (the standard is that the coefficient of genetic relationship was greater than or equal to 0.1), 110 roosters were divided into 32 families. The ROH length of each New Pudong chicken were 0-350 Mb, and the proportion of individuals with ROH length of 100-150 Mb was the largest. The average inbreeding coefficient calculated based on ROH value was 0.155, indicating a high degree of inbreeding. According to these results, this study reveals the population genetic structure and genetic diversity of New Pudong chickens at the genomic level, which can provide reference for future breeding and utilization.

The purpose of the study was to establishment of chicken skeletal satellite cell line by constructing SV40-LT gene with lentivirus and transforming primary chicken skeletal satellite cell line (PMSCs). In this study, chest tissues from 20-30 healthy AA chickens of 15 embryonic ages were collected, and a mixed enzyme digestion method was used to isolate and cultivate PMSCs. Randomly divided the cells into two groups, each with three replicates. The treatment group was transfected with SV40-LT lentivirus for 48 hours, and then replaced with medium containing 1 μg·mL^-1 puromycin for screening and cultivation based on the treatment group and the control group (virus not transfected). After all cells in the control group died, the cells in the treatment group were continuously subcultured to obtain a chicken skeletal muscle satellite cell line. Immunofluorescence assay was used to analyze the expression of the marker gene PAX7 in chicken skeletal muscle satellite cells; Cell proliferation characteristics were detected using CCK-8 and cell cycle analysis; Serum dependence and soft agar analysis were used to detect its malignant transformation status; Constructing an induced differentiation model to test its ability to induce differentiation. The 92% of cells in PMSCs were positive for the PAX7 gene, which could be used for subsequent research. Compared with primary chicken skeletal muscle satellite cells, SV40-LT was expressed 15-fold in chicken skeletal muscle satellite cell lines (IMSCs P12), and after continuous passage to 12th generation, both IMSCs P12 and PMSCs exhibited fibrous morphology. IMSCs P12 showed higher proliferative activity and did not undergo malignant transformation. Subsequently, IMSCs P12 was induced to differentiate. During the stage from 70% proliferation to 1 day of induced differentiation, the expression of PAX7 genes in IMSCs P12 and PMSCs was downregulated, while the expression of MyoD and MyHC genes was upregulated. This study successfully cultivated chicken skeletal muscle satellite cell line by transfecting the SV40-LT gene, which has similar characteristics to the primary chicken skeletal muscle satellite cells. The establishment of this cell line provided a new platform for studying functional genes related to poultry skeletal muscles, and also laid the foundation for the study of SV40-LT gene immortalization in poultry cells.

The purpose of this study was to explore the genetic loci and functional genes affecting the weight and meat quality traits of broilers by genome-wide association study (GWAS), and to accumulate theoretical basis for breeding of fast yellow-feathered broilers. The test subjects were 393 Lihua ephedra terminal paternal roosters, which were weighed and collected breast muscle to determine meat quality traits at 60 days of age. Individual genetic variants were obtained by whole genome resequencing, and GWAS analysis of body weight and meat quality traits was performed using PLINK, GEMMA and R software. A total of 2 SNP loci significantly associated with body weight and 8 SNP loci significantly associated with meat quality traits were screened (P<1.15×10^-7); The 52 loci associated with body weight and 296 loci associated with meat quality were identified at the potentially significant level (P<2.31×10^-6). By annotating these associated SNPs, a total of 99 candidate genes related to body weight, 27 to shear force, 28 to water binding capacity, 57 to pH, and 113 to meat color were selected. It was concluded that FSTL3, MBNL3, NPFF, PFKM, PRKG1, SLC13A5, MDFIC, FGD3, AQP1 may be key candidate genes for body weight and meat quality traits in broilers. The above research results provide important genetic variation sites and subsequent genes for important economic traits of fast type yellow-feathered broilers, and provide key basic data for improving molecular breeding methods for high-quality broilers and accelerating the progress of breed selection.

The purpose of this study was to clone the GPR35 gene sequence of goat, explore its expression characteristics and elucidate its effect on the differentiation of goat subcutaneous preadipocytes and its possible mechanism. In this study, the complete sequence coding for amino acids in protein (CDS) of GPR35 gene in one-year-old healthy male Jianzhou Big ear goats (n=4) were cloned by RT-PCR and analyzed by bioinformatics. Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of GPR35 gene in goat various tissues and goat subcutaneous adipocytes at different differentiation stages. Then GPR35 overexpression vector (pEGFP-N1-GPR35) and its interference sequence (Si-GPR35) were transfected into goat subcutaneous adipocytes and induced by oleic acid. The changes of lipid droplets were observed by Oil Red O staining and Bodipy staining, and the changes of triglyceride content were detected. Finally, qRT-PCR was used to detect the changes in the expression of marker genes related to adipocyte differentiation, triglyceride synthesis and decomposition (all samples have 3 biological sample replicates and 3 technical replicates). The results showed that the cloned goat GPR35 gene sequence was 1 167 bp and the open reading frame was 906 bp, encoding 301 amino acid residues. The expression of GPR35 gene in longissimus dorsi was significantly higher than that in other muscle tissues of goats (P<0.01); Its expression in subcutaneous fat content was significantly higher than that in other adipose tissues (P<0.01); It was significantly higher in spleen than that in other internal organs of goats (P<0.01). In addition to that, the expression of GPR35 gene was the highest in subcutaneous adipose cells at 120 h after induction (P<0.01). Overexpression and interference of GPR35 inhibited and promoted the content and aggregation of lipid droplets in subcutaneous adipocytes, respectively, and the relative content of triglyceride was significantly decreased and increased, respectively. The expression of C/EBPα, C/EBPβ, PPARγ, AP2 and SREBP1 genes was significantly down-regulated (P<0.01), and the expression of HSL was significantly up-regulated (P<0.01) after overexpressing GPR35 gene. However, the expression of C/EBPα, C/EBPβ, AP2, SREBP1 and DGAT1 genes was significantly up-regulated (P<0.01), and the expression of FASN was significantly down-regulated (P<0.01) after silencing GPR35 gene. These results suggest that GPR35 gene may be a negative regulator during the differentiation of goat subcutaneous preadipocytes, which may be achieved by regulating the expression of C/EBPα, C/EBPβ, AP2, SREBP1 or interacting with CXCL17 protein.

This study aimed to explore the effect of Snail1 gene on the proliferation and differentia-tion of bovine adipocytes and its potential molecular mechanism. In this study, an adenovirus was used to overexpress the Snail1 gene in Qinchuan cattle adipocytes. Experimental and control groups were established, each with 3 biological replicates. The effect of Snail1 on the proliferation and differentiation of bovine adipocytes was investigated by flow cytometry, CCK-8, EdU staining, Oil Red O staining, triglyceride determination, qRT-PCR and Western blot. The signaling pathways and target genes were further screened by RNA-Seq and dual-luciferase reporter experiments. The results showed that overexpression of Snail1 inhibited cell proliferation (CCK-8) and reduced the proportion of positive cells at the S-phase (EdU, P<0.05). Combined with flow cytometry tests, the results indicated that the up-regulation of Snail1 led to G1/S phase arrest of the cell cycle. Furthermore, qRT-PCR and Western blot results showed that Snail1 inhibited the expression of CCNB1 and CCND2 (P<0.05). The results of Oil Red O staining and triglyceride detection on bovine adipocytes on day 6 of induced differentiation showed that Snail1 inhibited the adipogenesis and triglyceride formation of bovine adipocytes. qRT-PCR analysis revealed that the overexpression of Snail1 inhibited the gene expression of PPARγ (P=0.06), FABP4 (P<0.05), and LPL(P<0.05), while the expression level of PIK3R3 was significantly upregulated (P<0.01);The Western blot results domonstrated that expression of Snail1 significantly inhibited FABP4 protein expression. Further analysis through RNA-Seq revealed that the differentially expressed genes induced by Snail1 overexpression were predominantly enriched in signaling pathway such as the PPAR, ECM-receptor interaction, PI3K-AKT, MAPK, nicotine addiction, and insulin secretion. Based on FIMO target gene screening, combined RNA-Seq data analysis, and luciferase assay, it was confirmed that the transcription factor Snail1 potentially affects the differentiation process of bovine adipocytes by targeting the antagonist SFRP2 of the Wnt signaling pathway. The results suggested that Snail1 gene inhibited the proliferation and differentiation process of bovine adipocytes, and may be involved in the regulation of bovine adipocyte differentiation through the positive feedback loop of "Snail1/SFRP2-Wnt/β-catenin-GSK3 β".

The purposes of this study were to evaluate the variations in gene expression between Mongolian horse and Thoroughbred, which underlying the coordinate changes in the immunophysiological responses to the specific environmental conditions and directional breeding selection pressure. In this study, jugular blood collected from healthy adult Mongolian horses (n=3) and Thoroughbred (n=3) was used to perform RNA-Seq and comparative analysis of the expression profiles between two horse breeds. Liver, spleen, lung, and jugular blood samples were collected from about 2-year-old healthy Mongolian horses (n=3) and Thoroughbred (n=3). qRT-PCR was used to analyze the relative expression of 7 genes of NF-κB signaling pathway in liver, spleen, lung and blood tissues between two horse breeds. Western blot was used to analyze the relative expression of RELA proto-oncogene NF-kB subunit (NF-κB p65) protein and interleukin 1 beta (IL-1β) protein in the lungs between Mongolian horse and Thoroughbred. The results showed that 19.69-23.95 Gb high-quality RNA-Seq data were obtained and 1 199 differentially expressed genes were identified. Functional analysis showed that many differentially expressed genes directly participated in the immune process. The qRT-PCR results showed that 7 genes of NF-κB signaling pathway were expressed in 4 tissues of Mongolian horse and Thoroughbred, but the difference in gene expression between breeds was manifested as tissue diversity. The expression of 7 genes in the lung of Mongolian horse was higher than that of Thoroughbred, excepting for tumor necrosis factor (TNF-α) (P≥0.05), while all other genes had biological statistical significance (P<0.05). The expression of NF-κB p65 protein and IL-1β protein in the lung of Mongolian horse was also higher than that of Thoroughbred (P<0.05). Many immune-related genes were differentially expressed in the immune tissues between Mongolian horses and Thoroughbred, which might be due to the adaptation of Mongolian horses to harsh environments and the sports performance directional breeding of Thoroughbred. The results of this study provided data support for further research on the molecular mechanism of the Mongolian horse immune function adapting to specific environment.

This research aimed to study the differences in structural composition, function of caecum and colon bacterial microbiota between Laiwu pigs and Duroc×Landrace×Duroc (DLY) crossbred pigs in order to enrich the understanding of the characteristics, including fat deposition, roughage tolerance and strong stress resistance of Laiwu pigs from the pespective of gut microbiota. In this study, 12 Laiwu pigs and 6 DLY pigs were used as experimental individuals. Bacterial V3-V4 region sequences of their caecum and colon were determined by 16S rDNA high-throughput sequencing. The composition, abundance, diversity and predicted biological function of the bacterial microbiota were analyzed. Bacteria which affected the characteristics of Laiwu pigs were identified through abundance comparison analysis between Laiwu and DLY pigs, discriminant analysis of species differences in LEfSe, and the correlation analysis between the bacterial genera and backfat thickness and serum biochemical parameters. The results showed that: 1) There were significant differences in backfat thickness, serum total protein, triglyceride, glucose and high-density lipoprotein cholesterol between Laiwu and DLY pigs (P<0.05). 2) Laiwu pigs have different bacterial diversity and relative abundance of cecal and colonic microbiota from DLY pigs. The cecal Simpson index of Laiwu pigs was significantly higher than that of DLY pigs (P<0.05). The relative abundance of Firmicutes of the caecum in Laiwu pigs was significantly lower than that of DLY pigs (69.16% vs. 89.59%, P<0.05). 3) The abundances of some bacterial genera of the caecum and colon were significantly correlated with backfat thickness and serum biochemical parameters (P<0.05). In addition, caecum had more bacterial genera related to these indicators and higher correlation coefficients than colon, indicating that bacterial microbiota of caecum may play a greater role in pig phenotype than those of colon. 4) Methanobrevibacter, Fusobacterium, Escherichia_Shigella, [Ruminococcus]_gnavus_group and Bacteroides were related to the fat deposition, roughage tolerance and strong stress resistance of Laiwu pigs, and may be the bacterial genera that affecting the characteristics of Laiwu pigs. This study systematically described the characteristics of caecum and colon bacterial microbiota of Laiwu and DLY pigs. The results not only extend understanding of the characteristics of Laiwu pigs from the perspective of intestinal microbiota, but also lay a foundation for the development and utilization of intestinal microbial resources of local pigs.

Based on previous data from comparative genomics, a structural variation (SV) with 189 bp in length was identified from the intron 15 of mitogen-activated protein kinase kinase kinase 4 (MAP3K4) gene in Xiang pig, which was defined as MAP3K4-I15-sv189. To explore the SV polymorphism in pig populations and its effects on gene expression, a PCR method was established using specific primers to discriminate SV genotypes and the gene expression level was determined via RT-qPCR technology. There found two genotypes, DD and ID, at the MAP3K4-I15-sv189 locus in Xiang pig population, in which the gene without SV region was indicated as D, while the reference gene containing the SV region was termed to be gene I. Of those, the frequency was 97.35% of allele D in Xiang pig population. In Yorkshire pig population, three genotypes, DD, ID and II, were measured with frequency of 80% in allele D. And frequency of allele D in Xiang pigs was significantly higher than that in Yorkshire pigs (P<0.05). The elements in the SV region were analyzed via Bioinformatics methods. The deletion of SV might lead to the loss of several repeat elements, which might change the expression of MAP3K4. A higher expression level of MAP3K4 mRNA was then tested from the lung of Xiang pigs compared with other tissues. And the highest level of MAP3K4 was found in the lung with genotype DD and the lowest from samples with genotype II. It indicated that the deletion of SV MAP3K4-I15-sv189 could accelerate the expression of MAP3K4 in pig lung. The results illustrated that the SV MAP3K4-I15-sv189 in MAP3K4 gene was dominated with genotype of deletion which resulted in the increase of gene expression in the lung, and would benefit to better undestanding the mechanism of susceptibility to diseases in Xiang pig lung.

The objective of this study was to investigate whether arachidonate lipoxygenase type 15B (ALOX15B) participates in the production of malondialdehyde (MDA) as well as reactive oxygen species (ROS), regulates the apoptosis of porcine sertoli cells and determines the role of JNK in the process. In this experiment, porcine sertoli cells from the testicular tissues of 20 3-week-old three-way hybrid piglets was isolated and randomly divided into 3 groups with 3 replicates in each group. Porcine sertoli cells were treated at 44℃ for 30 min as the heat stress model. After siRNA transfection of ALOX15B and addition of ALOX15B inhabitor (baicalein) or JNK inhibitor, the apoptosis rates of the cells were detected by flow cytometry, the expressions of phosphorylated JNK, ALOX15B and p53 were detected by Western blotting, and the contents of 8-HETE and 15-HETE were analyzed by ELISA. The levels of MDA and ROS were detected by the kit. The results showed that the combined treatment of baicalein and heat stress decreased the content of 8-HETE and 15-HETE (P<0.01), reduced the levels of MDA and ROS(P<0.01), and inhibited the apoptosis rate induced by heat stress (P<0.01). Under heat stress, bai-calein and ALOX15B siRNA inhibited JNK phosphorylation induced by heat stress (P<0.01). SP600125 inhibited the expression of p53 downstream by decreasing the phosphorylation of JNK (P<0.01), and reduced the apoptosis induced by heat stress (P<0.01). Meanwhile, compared to the heat stress group, SP600125 combined with heat stress also gave negative feedback on the protein level of ALOX15B (P<0.01), decreased the content of lipid peroxides 8-HETE (P<0.01) and 15-HETE (P<0.05), and reduced the level of MDA and ROS (P<0.01). In conclusion, ALOX15B regulates apoptosis of porcine sertoli cells under heat stress; JNK participated in the effect of ALOX15B, and regulated apoptosis of porcine sertoli cells through p53, and could have the feedback on the levels of ALOX15B, 8-HETE, 15-HETE, MDA and ROS.

The purpose of this study was to identify the oar-miR-200b promoter and investigate the effects of miR-200b on mitochondrial function in follicular granulosa cells. Sheep ovarian follicular granulosa cells were isolated and cultured in vitro, recombinant plasmids containing 3 different lengths of promoter region were constructed, and bioinformatics software and dual-luciferase reporting assays were used to predict and identify the oar-miR-200b core promoter region. Online tools were used to predict the binding site of transcription factor NR4A1, and the effect of overexpression of NR4A1 gene on miR-200b promoter activity and expression levels was detected by dual-luciferase reporter assay and qRT-PCR. Mitochondrial morphology and number, mitochondrial membrane potential, ROS levels, the concentration of ATP and mitochondrial respiratory chain complex Ⅰ were respectively detected by transfection miR-200b mimic and mimic NC, respectively, and the expression levels of mitochondrial oxidative phosphorylation-related genes were detected. The result indicated that the highest activity of miR-200b-P1 (-159/+36 nt) was regarded as the core promoter region of miR-200b. There was one binding site of NR4A1 in the miR-200b core promoter. The miR-200b promoter activity and miR-200b expression increased in NR4A1 overexpressed cells (P<0.01). Compared with mimic NC group, transfection of miR-200b mimic resulted in a significant decrease in the mean optical density (P<0.01) and a reduction in cell length (P<0.01) of granulosa cells, decreased mitochondrial membrane potential (P<0.05), concentrations of ATP (P<0.05) and mitochondrial respiratory chain complex I (P<0.01), and increased ROS levels (P<0.01), accompany with down-regulating the expression of ATP6V1G1 and NDUFV1 (P<0.01) and up-regulating the expression level of NOX4 (P<0.01). This study determined the core promoter region of oar-miR-200b (-159/+36 nt), NR4A1 positively regulated the transcription of miR-200b, and miR-200b might affect the process of mitochondrial OXPHOS and induce mitochondrial damage in granulosa cells. These results provide a theoretical basis for further revealing the mechanism of miR-200b during sheep ovarian follicular development.

The study aimed to explore molecular regulatory mechanism and genes regulatory network of goat reproductive traits. In this study, 3-year-old healthy ewes of 3rd parity with similar body size were selected. According to the reproductive performance records, 3 ewes with 1 lamb on average were collected as single-lamb group, and 3 ewes with more than 2 lambs on average were collected as multi-lamb group. Transcriptome sequencing technology was used to construct mRNA library, screen differentially expressed genes (DEGs) and conduct functional enrichment analysis between single-lamb group and multi-lamb group. Real-time fluorescence quantitative PCR was used to verify gene expression. Six mRNA libraries of ovarian tissues from Huai goats were constructed, with a total number of 41 163 239 raw reads and 37 908 182 clean reads on average. A total of 121 DEGs were found between single-lamb group and multi-lamb group (P<0.05), including 30 up-regulated genes and 91 down-regulated genes. The expressions of PTX3, MMP13, PAK1, ADAMTS1, COL1A2, CCN1, SLC4A10, FOS, NR4A1, NR4A2 and ADCY8 genes were significantly differentially expressed between single-lamb group and multi-lamb group (P<0.05). And these 11 DEGs were involved in the organs development and the secretion of aldosterone, cortisol, oxytocin and adrenocorticotropic hormone in the endocrine system. A total of 236 SNPs were screened in the above 11 DEGs. Seven SNPs were nonsynonymous mutations, among which, 4 SNPs were detected in ADAMTS1 gene, one was in PTX3 gene, and two were in CCN1 gene. The results showed that the expression of 11 genes were significantly different between single-lamb group and multi-lamb group. These DEGs were involved tissues and organs development and hormone secretion in endocrine system. These DEGs may be related to goat follicular development, ovulation and steroid hormone secretion. The results will provide scientific basis for elucidating the genetic mechanism of high fecundity of Huai goat.

The objective of the study was to investigate the effects of dietary supplementation with sodium humate on nutrient apparent digestibility, fecal microbial population and their metabolites in broilers. A total of 540 one-day-old 817 white-feathered broilers were selected for the trial and randomly divided into 3 groups (6 replicates per group, 30 birds per replicate). The control group was fed the basal diet and the test group was fed the test diets containing 0.3% and 0.5% sodium humate, respectively. The trial lasted for 42 days, and 3 days before the end of the trial, 2 broilers with similar body weight were selected from each replicate and were put into metabolic cages for metabolic tests. The results showed that: 1)Compared with the control group, dietary supplementation of 0.5% sodium humate significantly increased apparent digestibility of crude protein, phosphorus and ash (P<0.05) in broilers. 2)Compared with the control and 0.3% groups, dietary supplementation with 0.5% sodium humate significantly increased total bacteria and Lactobacillus concentrations (P<0.05) and decreased Escherichia coli concentrations (P<0.05) in broiler manure. Compared with the 0.3% group, dietary supplementation with 0.5% sodium humate significantly increased Prevotella concentration (P<0.05) in broiler manure. 3)Compared with the control group, dietary supplementation with 0.5% sodium humate significantly decreased ammoniacal nitrogen concentrations (P<0.05) and increased lactic acid concentrations (P<0.05) in broiler manure. Compared with the control and 0.3% groups, dietary supplementation with 0.5% sodium humate significantly decreased p-cresol concentrations (P<0.05) in broiler manure. 4) Compared with the control and 0.3% groups, dietary supplementation with 0.5% sodium humate significantly decreased tyramine, spermidine, spermidine and total amines concentrations (P<0.05) in broiler manure. Compared with the control group, dietary supplementation with 0.3% and 0.5% sodium humate significantly decreased putrescine concentrations (P<0.05) in broiler manure; Additionally, dietary supplementation with 0.3% sodium humate significantly decreased tyramine and spermine concentrations (P<0.05) in broiler manure. Therefore, dietary supplementation with sodium humate can improve nutrient apparent digestibility in broilers, promote beneficial bacteria growth and reproduction, increase lactic acid concentrations, inhibite harmful bacteria growth and reproduction, and then decrease ammonia nitrogen, p-cresol and biogenic amines and other odorous substances in manure, thus achieve the goal of pollution source reduction, and under the current condition, supplementation with 0.5% was more effective than 0.3%.

The purpose of this experiment was to study the effects of different treatments before and after transportation on the musle energy metabolism of lambs after slaughter, and to analyze the potential mechanism of lamb stress caused by transportation, so as to provide a reference for alleviating animal transportation stress and improving animal welfare. Sixty 4-month-old healthy Hu sheep male lambs with similar weight were randomly divided into three groups: control group, multivitamin electrolysis group and neomycin group, with 20 lambs in each group. All the experimental lambs were transported for 8 hours, and the day of transportation was recorded as the 0th day. The control group was fed with basal diet, the multivitamin electrolysis group was added with 375 mg·d^-1 in the basal diet for each lamb from 2 d before transportation to 7 d after transportation, and the neomycin group was treated with neomycin 200 mg·d^-1 per lamb after transportation from 0 to 7 d. Five lambs in each group were slaughtered on the 0th,7th and 14th day, and the longissimus dorsi muscle was taken to detect its glycolytic potential (GP)and the expression levels of related genes and proteins. The results showed that:1)The treatment significantly affected the muscle glycogen level (P<0.01), and the order of each group was as follows: neomycin group>multivitamin electrolysis group>control group. Time significantly affected the muscle glycogen, free glucose, hexokinase (HK)and lactate dehydrogenase (LDH)activities of the lamb (P<0.01). The muscle glycogen and free glucose levels of the lamb at 7th and 14th day were obviously higher than that of the 0th day after transportation. The HK activity of muscle in each group increased firstly and then decreased within 0-14 d after transportation, and reached the highest on the 7th day. The muscle LDH activity increased within 14 days after transportation, reaching the highest in the control group and neomycin group on the 7th day, and the highest in the multivitamin electrolysis group on the 14th day. 2)Treatment significantly affected the gene expression of LKB1 (P<0.05), and the order of each group was neomycin group>control group>multivitamin electrolysis group. Time extremely significantly affected the mRNA expression levels of AMPKα1, AMPKα2, LKB1 and CPT-1 (P<0.01), and significantly affected ACC gene(P<0.05) as well as the gene and protein expression levels of LKB1 in lambs (P<0.05). After transportation, the gene expression of CPT-1 and ACC in each group increased within 14 days, and the expression was the lowest on the 0th day. In summary, adding electrolytic multivitamin before and after transportation can improve muscle energy metabolism and inhibit the activation of AMPK pathway induced by transportation.

The aim of this experiment was to explore the effects of maternal betaine supplementation on the breast muscle development and mechanism of offspring goslings. A total of 540 healthy geese breeeders with similar body weight at 39 weeks old were randomly divided into 3 groups with 5 replicates per group and 36 geese per replicate, male and female ratio was 1∶5. Geese breeders in control were fed a basal diet, and the others in the experimental groups were fed the basal diet supplemented with 0.25% (LBT group) or 0.5% (HBT group) betaine. The trail lasted for 7 weeks, eggs were collected during the 7th week for consecutive day and hatched. After hatching, 160 healthy one-day-old goslings with similar body weight were selected from each group (half male and half famale), offspring goslings were fed the same basal diet, the trial last for 63 days. Ten goslings from each group were slaughtered and breast muscle samples were collected at 35 and 63 days of age, respectively. RT-qPCR and Western blot were used to detected the expression levels of muscle fiber types, growth-related genes and protein in breast muscle. The results showed that: maternal betaine supplementation significantly increased the body weight and breast muscle weight of offspring goslings in HBT group at the age of 35 days (P＜0.01), as well as in LBT group at the age of 63 days (P＜0.05 or P＜0.01), and significantly increased breast muscle index in HBT group at 63 day of age (P＜0.05). Compared with the CON group, the muscle fiber diameter and cross-sectional area from offspring goslings at 35 days of age in LBT and HBT groups and at 63 days of age in HBT group were significantly increased (P＜0.05), while the muscle fiber density of the 35-day-old HBT group was significantly reduced (P＜0.05), in addition, the RNA and protein content in HBT group at 35 and 63 days of age and RNA content in LBT group at 35 days of age were also significantly raised (P＜0.05); There were no significant change in the breast muscle fiber types, while the mRNA expression of Pax7 was significantly elevated in LBT and HBT group at 35 and 63 days of age. At 35 days of age, the IGF-2 content of the breast muscle was significantly increased in HBT group (P＜0.05) and the mRNA expression levels of IGF-2, IGF-1R, and mTOR were also significantly up-regulated in LBT and HBT groups (P＜0.05; except mTOR in LBT group, P=0.07). At 63 days of age, the contents of IGF-1 and IGF-2 was significantly enhanced in breast muscle from LBT and HBT groups (P＜0.05), which was line with higher mRNA expression of IGF-1, IGF-2, IGF-1R, and mTOR (except mTOR in LBT group) genes, compared with CON group (P＜0.05). In addition, maternal betaine supplementation significantly increased the protein expression of p-mTOR in breast muscle of 35-day-old offspring goslings in both experimental groups, as well as the protein expression of p-AKT and p-mTOR in breast muscle of 63-day-old offspring goslings in HBT group (P＜0.05). These results indicated that maternal betaine supplementation promotes the protein synthesis of breast muscle of goslings by activating IGFs-related signaling pathways, thus increase the breast muscle weight.

Bovine coronavirus (BCoV) is one of the major pathogens causing diarrhea in calves. With the rapid development of cattle production in Xinjiang and the increase number of introduced cattle breeds, BCoV infection is on the rise, but there is a lack of comprehensive understanding of its epidemiological status and patterns. In this study a two-year follow-up survey from 2020 to 2022 on the prevalence of BCoV infection was conducted in the main cattle production areas of Xinjiang. In total 652 fecal samples of diarrhea calves were collected and tested for BCoV by RT-PCR. Genetic evolution analysis was performed on the isolated and identified strains. The results indicated that, the total positive rate of BCoV was 23.93% (156/652), in which it varied seasonally, with the peak in the winter being as high as 50.85%. The incidence of BCoV was higher in the southern than in the northern Xinjiang. Compared with dairy calves, meat calves were more susceptible to infection. The phylogenetic analysis showed that the S gene of BCoV/China/XJ-CJ/2022 isolated in this experiment was most closely in relation to the 2018 strain MN982199.1, the N gene was in relation to the 2018 strain MK095169.1 BCOV-China/SWUN/LN4/2018, the M gene was in relation to the 2017 strain MK095148.1 China/SWUN/SC1/2017, and the HE gene was in relation to the 2017 strain MK095136.1 BCOV-China/SWUN/SC2/2017. There was no genetic recombination occurred between BCoV/China/XJ-CJ/2022 and the BCoV strains from other countries. After nucleotide and amino acid similarity analysis of five structural proteins, the results showed that the genes were still the most similar to the local strain FJ938064.1_E-AH187-TC in China, reaching the lowest 91.50% and 96.70%. The results suggested that the seasonal, geographical and interbreed variability of BCoV infection in the scaled cattle farms in Xinjiang could provide data support for the development of the accurate prevention strategies.

Porcine circovirus type 2 (PCV2) is a non-enveloped single-stranded negative-strand circular DNA virus that can cause immunosuppression in infected pigs. It is the main pathogen of porcine circovirus associated diseases and is widely spread around the world. The purpose of this paper is to explore the effect of decitabine (DAC) on the proliferation of PCV2 and its mechanism. In this study, a PK-15 cell model infected with PCV2 was constructed, and the cytotoxicity of DAC to PK-15 cells was detected by CCK-8 method, and then the effect of DAC on the proliferation of PCV2 was detected by fluorescent quantitative PCR, indirect immunofluorescence and Western blot. The effect of DAC on the global methylation of cellular genomic DNA was characterized by labeling 5-methylcytosine by indirect immunofluorescence method; Dnmt1(DNA methytransferase 1), as the target of DAC in PK-15 cells, was silenced by RNA interference technology, to further explore the inhibitory effect of DAC on PCV2 proliferation. The results showed that DAC had slight cytotoxicity at 20 μmol·L^-1, and showed obvious anti-PCV2 activity at a concentration of 10 μmol·L^-1, and could inhibit the proliferation of PCV2 at different times. DAC treatment of cells can significantly reduce the methylation level of genomic DNA in PK-15 cells and induce the expression of type Ⅰ interferon (Ⅰ-IFN) and interferon-stimulated genes (ISGs). Silencing Dnmt1, the target of DAC, significantly inhibited the proliferation of PCV2 and induced the expression of I-IFN and ISGs. This study confirmed that DAC can play an anti-PCV2 function by inhibiting DNA methylation and promoting the expression of I-IFN and ISGs. Furthermore, DAC has the potential to be developed into a clinical antiviral drug against PCV2, Dnmt1 is a potential target for activating the host's innate immune antiviral pathway, providing a theoretical basis for the development of new PCV2 antiviral drugs.

This study aimed to construct the BVDV-1(bovine viral diarrhea virus 1) E2 virus-like particles (VLP) by the insect cell-baculovirus expression system and to evaluate its immune efficacy in mice. The E2 gene sequence of BVDV-1 SWU-Z6 strain was optimized and synthesized according to the codon bias of insect cells, and used to generate the construct of the BVDV-1 E2 VLPs for insect cell-baculovirus expression system. The expression of E2 protein in recombinant baculovirus was validated by the indirect immunofluorescence (IFA) and Western blot, and the assembly of VLPs was evaluated through electron microscope. The VLPs were then purified using sucrose density centrifugation. Mice were immunized with different doses of VLPs plus MF59 adjuvant and CpG-ODN immune enhancer by intramuscular injection, with BVDV commercial inactivated vaccine group and PBS blank as control group. Four weeks after booster immunization, the mice were challenged using BVDV-1 SWU-Z6 strain via gavage. The immune protection effect of BVDV-1 E2 VLPs was evaluated based on weight changes and viral load in feces of mice. The recombinant plasmid pFast Dual BVDV-1 E2 was constructed by inserting the optimized E2 gene and verified by restriction endonuclease analysis and sequencing. After transformed into DH10.Bac competent cells, the recombinant baculovirus Baculo-BVDV-1 E2 were extracted and transfected into SF9 insect cells. The BVDV-1 E2 VLPs with diameter about 80-100 nm were observed by electron microscope. The E2 protein expressed by the recombinant baculovirus was further confirmed by IFA and Western blot analysis, exhibiting a good biological activity in vitro. The antibody titer in group injected with 50 μg of VLPs was about 1∶10 000 2 weeks after first immunization, and reached 1∶100 000 2 weeks after enhanced immunization by ELSIA. After the challenge with SWU-Z6 strain, the immunized mice showed weight loss 7 days after infection before beginning to recover. No virus was detected in feces collected on day 10 after infection. While the mice group injected 50 μg of BVDV-1 E2 VLPs twice showed no weight loss and virus replication in the digestive tract in comparison with the non-immunized mice. The BVDV-1 E2 VLPs expressed by the insect cell-baculovirus expression system induce an efficient BVDV-specific humoral immune responses in mice, and prevent the BVDV-1 SWU-Z6 strain infection.

To evaluate the protective efficacy of the inactivated vaccine against avian metapneumovirus (aMPV) subtype B in yellow-feathered broilers, the inactivated vaccine LN16-I strain prepared in this study was immunized by intramuscular injection at a dose of 0.5 mL per chicken to yellow-feathered broilers at the age of 3 weeks. Three weeks after immunization, the same injection method and dose were used once to reinforce the immunization. Serum was collected within 1-6 weeks and ELISA antibody and neutralizing antibody titers were measured. The results showed that the average ELISA antibody titer in the vaccinated group can reach 4.2×10⁴ post-priming immunization, the average neutralizing antibody titer reached 7.42 log2, and the positive rate was 100%. Three weeks after the booster immunization, the chickens in each group were challenged with virulent virus LN16 at a dose of 5 000 TCID₅₀ per chicken. The results showed that the chickens in the challenge control group had clinical symptoms such as cloudy or viscous nose, head shaking, and the incidence rate was 85% (11/13), while none of the chickens in the vaccinated group showed clinical symptoms. The number of virus copies in nasal swabs was further detected by RT-qPCR. The result showed that the control group chickens exhibited virus shedding, which reached a maximum at 3 days post-challenge, and the number of virus copies was 4.9×10⁶ copies·mL^-1, while the amount of virus shedding in the vaccinated group decreased by 99.43% compared with that in the control group. The pathological results showed that the turbinate and trachea of the chickens in the control group showed obvious pathological damage, while no obvious pathological changes were found in the vaccinated group. The results of this study show for the first time that the infection with aMPV subtype B can cause obvious disease in yellow-feathered broilers, and the inactivated vaccine LN16-I strain has a good immune protection effect on yellow-feathered broilers. This study provides theoretical support and technical guidance for the epidemiological investigation and effective prevention of aMPV subtype B in yellow-feathered broilers in China.

The purpose of this study was to express the full-length, truncated and bivalent fusion proteins of group Ⅰ fowl adenovirus serotype 4 (FAdV-4) fiber2 and serotype 8b(FAdV-8b) fiber by prokaryotic expression system, and evaluate its immunogenicity. Based on good antigenic truncated fragments (FP and BP) of FAdV-4 fiber2 and FAdV-8b fiber, a bivalent fusion fragment was constructed by SOE-PCR. The truncated protein, full-length and fusion protein were individually expressed in E. coli and identified by SDS-PAGE and Western blot. The SPF chickens were immunized using purified recombinant proteins and challenged with FAdV-4 and FAdV-8b. Survival rate of each group was calculated, and serum antibody levels of immunized chickens were detected by indirect ELISA. Viral loads of cloacal swab and the mRNA expression levels of cytokines IL-4, IFN-γ and TNF-α in liver were detected by qPCR post-challenge and histopathological observation. The constructed recombinant plasmids were successfully soluble expression in E. coli. The survival rate of BP-FP4 group was 58.3%, and that of other groups was 100%. The antibody level of full-length group (FL group and BL group) was higher than that of its corresponding fragment group (FP group and BP group), but the difference was not significant (P>0.05). At 3 days and 7 days post-challenge, the viral load in C8b group was significantly higher than that of immunized group (P<0.05). At 7 days post-challenge, the viral load in BP-FP8b group was significantly higher than that in BP and BL groups (P<0.05), but there was no significant difference between BL and BP group (P>0.05). The mRNA expression of three cytokines (IL-4,IFN-γ and TNF-α) in immunized groups were significantly higher compared to the negative control group (P<0.05). The mRNA expression of IFN-γ and TNF-α in FL group were significantly lower than those in BP-FP4 group (P<0.05), and there was no significant difference between FL group and FP group (P>0.05). The mRNA expression of IL-4 and IFN-γ in BL group were significantly lower than those in BP-FP8b group (P<0.05). Histopathological observation revealed that the liver lesions in the challenge groups were the most severe, followed by the FP and BP groups, and no significant pathological changes were observed in the FL and BL groups. This study showed that the immunoprotection of the truncated protein and the full-length protein of FAdV-4 fiber2 and FAdV-8b was better than that of the fusion protein. Therefore, the truncated protein could be used to replace the full-length protein in the preparation of fowl adenovirus subunit vaccine.

The aim of this study was to design a safer and more effective multi-epitope vaccine candidate using the adhesion proteins of Mycoplasma gallisepticum (MG). In this study, bioinformatics methods were used to predict B and T cell epitopes of several adhesin proteins (CrmA, GapA, Mgc2, PvpA) of MG. The screened antigenic epitopes were synthesized into a new epitope gene peptide (named mEA). The secondary structure, hydrophilicity, antigenicity, and tertiary structure of mEA were analyzed by Biology Online software. The recombinant plasmid pET32a-MG-mEA was constructed, identified by restriction endonucleases (BamH Ⅰ and Hind Ⅲ) and introduced into E. coli, BL21 (DE3) for induction of expression and purification by DNA sequencing.Western blot was used to verify the reactivity of the recombinant protein with chicken negative/positive sera. The purified MG-mEA recombinant protein was mixed 1∶1 with adjuvant and prepared as a multi-epitope vaccine and the immunogenicity of the fusion protein was analysed by SPF chicken immunoassay. The results showed that the secondary structure of the mEA sequence was relatively stable and the antigenicity was good. The recombinant plasmid was amplified by PCR to obtain the target band with a size of 1 680 bp, which was consistent with the prediction. SDS-PAGE detected that the relative molecular mass of the expressed protein was about 48 ku, mainly in soluble form. The multi-epitope vaccine immunised SPF chickens showed the highest antibody levels in all immunised groups at 10 d after the second vaccination, with statistically significant differences between the three recombinant protein immunised groups compared to the PBS group (P<0.05). The results showed that MG infection significantly damaged the tracheal mucosa structure and that the recombinant protein+adjuvant vaccine was effective in providing effective protection. In summary, the results of this study provide a new avenue for the development of a safe and effective MG vaccine candidate.

In this study, major bacteria in Enterobacteriaceae were isolated from 52 samples collected from two dairy farms in Hainan Province in 2021. The virulence genes detection, plasmid type, biofilm-forming ability and enterobacterial repetitive intergenic consensus (ERIC) molecular typing of isolates were studied in order to clarify the distribution, prevalence and biological characteristics of the isolates in Enterobacteriaceae in milk and dairy farm environment. First, the isolates in Enterobacteriaceae were identified by 16S rDNA PCR. The results showed that 49 strains of Enterobacteriaceae were isolated from 52 samples, including 32 strains of Escherichia coli, 13 strains of Klebsiella pneumoniae, 3 strains of Enterobacter cloacae and 1 strain of Enterobacter aerogenes. The results of virulence gene testing showed that ompA had the highest detection rate. Plasid typing is performed by PCR-based replicon typing created by CARATTOLI. The results showed that the detection rate of K plasmid was the highest among 49 isolated bacteria. The biofilm-formating ability of isolates was qualitatively detected using the Congo Red Agar method. Thirty-seven isolates were positive. The genetic relationship and diversity of Enterobacteriaceae were determined by ERIC-PCR. The results showed that 49 strains of Enterobacteriaceae were divided into 17 types (I-XVI), and the dominant type was type VI (all Escherichia coli), with a total of 20 strains. The overall study showed that Enterobacteriaceae widely existed in the milk and environment, causing a high risk of human and animal diseases. It is suggested that hygiene and standardized operation in breeding and transportation are crucial.

Klebsiella pneumoniae infection in rabbits often causes multiple respiratory diseases and digestive system diseases, which seriously harms the healthy breeding of rabbits. In this study, the suspected dead rabbits in a rabbit farm in Henan Province were dissected and the tissues were collected. The pathogenic bacteria were isolated and identified by morphological observation, PCR identification and sequencing analysis, and the pathogenic bacteria were identified and analyzed by virulence gene detection, histopathological observation, drug sensitivity test and pathogenicity test. The results showed that gram-negative and oval suspicious pathogens were obtained by bacterial isolation and culture. The length of amplified fragment of 16S rRNA gene was 1 494 bp, and the homology with ON925022.1 was 99.79%, which indicated that the diseased rabbits were infected by Klebsiella pneumoniae. Histopathological observation showed that most organs of the diseased rabbits were swollen and bleeding, and a large number of inflammatory cells infiltrated into the alveolar wall, and the intestinal villi mucosa epithelium fell off. Virulence gene amplification showed that allS, kfu and uge genes were positive. The results of drug sensitivity test showed that the pathogen was sensitive to amikacin, polymyxin B, cefoperazone and ceftriaxone, and was resistant to other drugs at varying degrees. To sum up, Klebsiella pneumoniae is the pathogen causing the disease in this rabbit farm, and the current experiment provides reference for better prevention and control of Klebsiella pneumoniae in rabbits.

The aim was to study the regulation of the c-di-GMP pathway metabolic gene STM1827 on the biofilm of Salmonella Typhimurium and its related biological functions. Firstly, we analyzed the effect of STM1827 on the content of c-di-GMP by measuring the level of c-di-GMP; Further analyzed the effect of STM1827 on biofilm formation through experiments such as biofilm and motility; Finally, the effects of STM1827 on the adaptability of Salmonella Typhimurium to environmental stress were analyzed through experiments such as oxygen stress and disinfectant stress. The results showed that the intracellular c-di-GMP level of the mutant strain 269ΔSTM1827 was 33.16% higher than that of wild strain. In terms of biofilm formation, compared with the wild strain, the mutant strain 269ΔSTM1827 had a 2.19-fold increase in the ability of biofilm formation, a 1.3-fold increase in the content of biofilm related extracellular polysaccharide, and a significant increase in the expression of extracellular polysaccharide synthesis genes (P<0.000 1). In terms of motility, the motility of 269ΔSTM1827 was decreased by 13%, and the expression of flagella-related genes was significantly decreased (P<0.05). And STM1827 gene mutant enhanced the adaptive ability of Salmonella Typhimurium under oxygen stress and disinfectant stress. The study showed that STM1827 can degrade c-di-GMP, inhibit the formation of biofilm by inhibiting the synthesis of extracellular polysaccharide and promoting bacterial motility, and ultimately lead to the decrease of bacterial tolerance under oxygen and disinfectant stress. This study provides a theoretical basis for the screening of salmonellosis targets and the development of prevention and control measures.

To study the mechanism of pyroptosis induced by highly pathogenic virulence island (HPI) of E. coli from Saba pig in IPEC-J2 cells, The E. coli HPI screened in the laboratory and the E. coli ΔHPI constructed by CRISPR/Cas9 gene knockout technology were used to infect IPEC-J2 cells to observe cell damage, LPS+ATP was used as a positive control. RT-qPCR was used to determine the mRNA transcription levels of key factors in the pyroptosis pathway, including NOD-like receptor thermal protein domain associated protein 3 (NLRP3), apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, Gasdermin D (GSDMD), and inflammatory factors interleukin-18 (IL-18) and interleukin-1β (IL-1β) at 0.5, 3, 6, 9, and 12 h after treatment in each group. Laser confocal microscopy was used to observe the assembly and expression of NLRP3/Caspase-1 inflammasome complex. Western blot was used to determine the expression levels of GSDMD and its activated form GSDMD-N protein. PI staining was used to detect membrane integrity. The results showed that LPS+ATP treatment significantly promoted the occurrence of pyroptosis. The cells in the negative control group grew normally with clear contours. The cells in the E. coli infection group and LPS+ATP group showed obvious cytopathic effects (CPE) such as deformation, detachment, and blurred boundaries. HE staining showed that E. coli HPI infection caused more severe cell damage than E. coli ΔHPI infection. The mRNA levels of key factors and inflammatory factors in the pyroptosis pathway in the E.coli HPI group were significantly (P<0.05) or extremely significantly (P<0.01) higher than those in the E. coli ΔHPI group at 3-9 h. NLRP3/Caspase-1 inflammasome complex was assembled in the cytoplasm, and the protein fluorescence intensity of NLRP3/Caspase-1, the expression levels of GSDMD and GSDMD-N protein, and the positive rate of PI staining in the E. coli HPI group were extremely significantly (P<0.01) higher than those in the E. coli ΔHPI group. The CPE, HE staining, mRNA levels of key factors and inflammatory factors in the pyroptosis pathway, expression levels of GSDMD and GSDMD-N protein, and co-localization of NLRP3/Caspase-1 in the E.coliHPI group were similar to those in the LPS+ATP group. The results suggest that E. coli HPI from Saba pig induces pyroptosis in IPEC-J2 cells by upregulating the mRNA levels of key factors in the NLRP3/ASC/Caspase-1 signaling pathway, inducing the assembly of NLRP3/Caspase-1 inflammasome complex, promoting the expression of pyroptosis marker proteins GSDMD and GSDMD-N, and punching holes in the cell membrane of IPEC-J2 cells.

This study aimed to isolate virulent Salmonella phages and provide a new biological agent for the prevention and control of bacterial diseases. The phage with Salmonella as host was isolated, and then we investigated the biological characteristics and genome features. The result showed that a virulent phage named SP3 was isolated, which charactered with a regular polyhedron head with approximately 70 nm in diameter and a long contractile tail with 130 nm in length. SP3 was a broad-spectrum phage that can lysate a total of 8 Salmonella strains in 3 serotypes. In addition, the optimal MOI of SP3 was 0.000 1, the incubation period was 40 min, and the outbreak period was 40-160 min. We also found that SP3 is stable under the pH ranges of 4 to 11 and temperatures ≤50 ℃, respectively. The full length of phage SP3 genome was 88 039 bp, the GC content was 38.85%, and it contained 22 tRNAs and 130 ORFs, 32 of which encoded proteins with known functions, but did not carry genes related to drug resistance, virulence and lysogen. SP3 was closely related to E. coli phage and Salmonella phage, both of which belonged to Ounavirinae subfamily and Felixounavirus genus, and genomic rearrangements were also detected. The phage SP3 has strong adaptability to the environment and clear genetic background, which is expected to become an antibiotic substitute for Salmonella prevention and control.

To explore the mechanism of apoptosis and necroptosis induced by di(2-ethylhexyl)phthalate (DEHP) exposure through ROS/PTEN/PI3K/AKT pathway. HD11 cells were used in this study, and control group(C group), 30 μmol·L^-1 DEHP group(L group), 60 μmol·L^-1 DEHP group(M group), 90 μmol·L^-1 DEHP group(H group)were set. After cells were treated with different concentrations of DEHP for 24 h, cell survival was detected by CCK-8, oxidative stress was detected by biochemical kit, included reactive oxygen species (ROS)levels, malondialdehyde (MDA)content, total antioxidant capacity (T-AOC)levels, and total superoxide dismutase (T-SOD)and glutathione peroxidase (GSH-PX)activities. AO/EB staining and flow cytometry were used to detect the rate of apoptosis and necrosis, mRNA and protein expressions of PTEN/PI3K/AKT pathway genes and apoptosis and necroptosis related genes were detected by qRT-PCR and Western blot. The results showed that, compared with the control group, DEHP considerably reduced the viability of HD11 cells, with the median inhibitory concentration (IC₅₀)being 180.644 μmol·L^-1. Besides, compared with the control group, AO/EB staining and flow cytometry validated the L, M and H DEHP group with typical characteristics of apoptosis and necroptosis. Additionally, compared with the control group, ROS expression levels in groups L, M and H showed an increasing trend (P<0.01), and the content of MDA were considerably increased (P<0.01), the T-AOC level was significantly decreased (P<0.01), and the activities of T-SOD and GSH-PX were significantly decreased (P<0.01). It was suggested that DEHP exposure induced oxidative stress injury of HD11 cells. In addition, compared with the control group, DEHP exposure up-regulated the mRNA and protein expression levels of PTEN/Bax/Caspase-9/Caspase-3/RIPK1/RIPK3/MLKL and down-regulated the mRNA and protein expression levels of PI3K/AKT/BCL-2(P<0.01), these results suggested that DEHP exposure induced apoptosis and necroptosis of HD11 cells through the PTEN/PI3K/AKT signaling pathway. Overall, DEHP can induce apoptosis and necroptosis in HD11 cells by modulating the ROS/PTEN/PI3K/AKT axis, and there is a dose-effect manner.

The purpose of this study was to investigate the effect of Danshensu, the monomer component of Salvia miltiorrhiza, on the repair of skeletal muscle injury, in order to provide the theoretical basis for clinical treatment. The skeletal muscle injury models were established and treated with Danshensu. The process of skeletal muscle repair was observed via H.E. and Masson staining. Western blot and Q-PCR were used to detect the expression of muscle regeneration related factors (α-Actinin, eMyHC, MyoD, MyoG, Pax7 and MSTN), inflammation related factors (IL-6 and IL-10), macrophage type related factors (F4/80), scar tissue formation related factors (Collagen Ⅰ and α-SMA) and intermuscular adipose-related factors (PPAR-γ). The results showed that Danshensu reduced Evans Blue uptake of injure gastrocnemius muscle and significantly increased the expression levels of α-Actinin and eMyHC. Meanwhile, the expression levels of muscle regeneration related factors, MyoD, MyoG and Pax7, were also significantly increased and MSTN was obviously decreased after adding Danshensu. Danshansu significantly reduced the expression of F4/80 and accelerated the transformation of M1 macrophages into M2 macrophages, reducing the IL-6 mRNA level and increasing the IL-10 mRNA level. Masson staining showed that Danshensu reduced the distribution of collagen fibers. In addition, it also reduced the expression of Collagen I and α-SMA. Meanwhile, Danshensu significantly increased the expression of PPAR-γ in muscle. Therefore, Danshensu can promote muscle fiber regeneration and the formation of intermuscular adipose, reduce the formation of scar tissue, thereby accelerating the process of skeletal muscle repair and improving the quality of skeletal muscle repair.

The study was conducted to verify the therapeutic effect of Maxing Shigan Oral Liquid on artificial infection of Mycoplasma gallisepticum. Eighty-seven 1-day-old healthy chickens were routinely raised to 20 days of age and randomly divided into infection control group, blank control group and treatment group. The blank control group was fed normally without infection. The chickens in the infection control group and the treatment group were injected with 1.5 mL (10⁹ CCU·mL^-1) of Mycoplasma gallisepticum M17 bacterial solution by nasal drip, eye drop and air sac injection, once a day for 4 days. On the 5th day (D1) of infection, the treatment group was fed with 1.5 mL Maxing Shigan Oral Liquid per liter of water for 5 days, and observed for 6 days after drug withdrawal. The clinical symptoms such as respiration, cough, runny nose, nasal throwing, feeding and mental state of chickens in each group were observed and scored from 1 day (D0) to D11 days before administration. The total score of clinical symptoms, cure rate, effective rate and inefficiency were calculated. The body weight of the chickens was weighed on D0 and D12, and the average weight gain was calculated. On D2, D6 and D12 days, 6 chickens in each group were sacrificed to observe the pathological changes of lung and air sac, and the lungs were collected for histopathological observation. The total score of clinical symptoms in the treatment group decreased significantly on D3 (P<0.05). On D2, D6 and D12, the scores of lung and air sac lesions in the treatment group were significantly lower than those in the infection control group (P<0.05), and the histopathological lesion score of the lung was also significantly lower than that in the infection control group (P<0.05). There was no abnormality in the blank control group. The cure rate of the treatment group was 76.5%, the effective rate was 94.1%, and the ineffective rate was 5.9%. The average weight gain of the infection control group and the treatment group was significantly lower than that of the blank control group (P<0.05). Maxing Shigan oral liquid can significantly improve the pathological damage of air sac and lung tissue of broilers, and has a good therapeutic effect on broilers artificially infected with Mycoplasma gallisepticum.

To study the chemical composition of Areca catechu L. seed in deep processing, UHPLC-QE-Orbitrap-MS technology was used to analyze and identify the composition of Areca catechu L. seed. Gradient elution was performed on ACQUITY UPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm) with 0.1% formic acid aqueous solution (A) to 0.1% formic acid acetonitrile solution (B) as mobile phases. The flow rate was 0.3 mL·min^-1, the column temperature was 35 ℃, and the sample volume was 10 μL. An electric spray ion source (ESI) was used with a scanning range of 100-1 200 m/z and positive and negative ions were collected simultaneously, and the detection mode was Full MS-ddMS². The original data were analyzed using Compound Discoverer 3.3 software, and the structures of the compounds were identified using the local database and mzCloud online database. Eighty-five compounds of Areca catechu L. seed in deep processing were detected, including 3 alkaloids, 24 flavonoids, 15 organic acids, 7 phenols, 7 terpenoids, 6 amino acids, 4 carbohydrates, 4 esters, 2 amides, 2 sphingolipids, 1 steroid, 1 phenylpropanoid, 1 coumarin and 8 other species. The UHPLC-QE-Orbitrap-MS detection technology quickly identified the compound of Areca catechu L. seed in deep processing, and the research results provided scientific basis for exploring the application prospects and developing high value-added products of Areca catechu L.

In this study, metagenomics was used to explore the fecal microbiota differences between the young pigeons (RP) and older pigeons (LP). The results showed that Firmicutes, Proteobacteria and Actinobacteria were the dominant bacterias. The contents of Bifidobacterium, Corynebacteriun, Psychrobacter and Streptococcus in RP group were significantly higher than those in LP group, while the relative abundance of Staphyloccus, Brachybacterium, Kurthia and Escherichia in LP group were significantly higher than those in RP group. Further LEfSe analysis showed that the differentially expressed genes in the two groups were enriched in 5 KEGG pathways. Antibiotic resistance genes (ARGs) analyzation showed that a total of 198 ARG,were identified. And the prevalence rate of multi-drug ARGs was more than 40%, which indicated that the antibiotic resistance problem in pigeon is seriously. The current study provided scientific datas for further researches on gut microflora and healthy breeding, and mechanism of antibiotic resistance mechanism.

This study aimed to conduct multiple stepwise regression and growth curve fitting analysis on the body measurement traits of Saanen dairy goats, explore the correlation and regression relationship between different body measurements, construct a growth curve for body measurement traits, so as to provide a theoretical basis for revealing the development patterns of Saanen dairy goats on body measurement, and provide practical reference for the selection and management of standardized farm of dairy goats. A total of 874 healthy Saanen dairy goats from different age of months and genders were used as the research subjects, and 11 body measurement traits were measured, including 4 general body measurement traits: body height, body length, chest circumference, and tube circumference; Three traits reflecting the reproductive performance of bucks: rump length, rump width, and testicular circumference; Four udder traits including udder circumference, udder depth, teat spacing, and udder height from the ground. Principal component analysis was applied to reduce the dimensions of the above three types of traits, and multiple stepwise regression was used to construct a regression equation for general body measurement traits reflecting reproductive performance traits of bucks and udder traits of does. The Bertalanffy model was used to fit the growth curve of general body measurement traits. The results indicated that: 1) There was a highly significant correlation (P<0.01) within the three types of traits, and the first principal component after dimensionality reduction represented the overall appearance factor among the three types of traits. The second principal component in does udder traits can be explained as a factor related to hindquarters height. 2) The regression fit of general body measurement traits to traits reflected buck reproductive performance was relatively high, ranging from 0.76 to 0.87, while the regression fit of udder traits of does was only 0.15 to 0.40. 3) Bertalanffy had a good fitting effect on general body measurement traits, except for a fitting degree of 0.60 for does tube circumference, all other traits were above 0.75, and the growth curves of bucks’ body measurement rose faster than that of does. In conclusion, there is a correlation between the body measurement traits of Saanen dairy goats, and the general body measurement traits of bucks can accurately predict the traits that reflected buck reproductive performance. The Bertalanffy model is good to fit the general body measurement traits in dairy goats.

The purpose of this study is to investigate the effect of 1-methylhydantoin on Nav Channels 1.8, which can provide a theoretical basis for pain intervened and different ways to achieve the ultimate goal of "pain minimization". Sixty SPF SD rats were randomly divided into six groups.The group was treated with 1-methylhyne (group MH), lidocaine (group L) and ambroxol (group A), respectively. On the 21st and 28th day after modeling, Western blot was used to detect the protein expressions of Nav1.8 in the L4-L6 dorsal root ganglia of rats, and patch-clamp technique was used to detect the peak currents of Nav1.8 channels under the effect of MH. Comparing with the group of model,the results showed that MH could decrease the expression of Nav1.8 significantly (P<0.05). MH inhibited the channel peak currents of Nav1.8 to a certain extent. The current inhibition rates of 10 μmol·L^-1 and 100 μmol·L^-1 MH to Nav1.8 were 6.34%±3.13% and 14.6%±1.52% respectively, which is showing a dose-response relationship. It is suggested that MH can decrease the expression of Nav1.8 in DRG of rats with chronic pain, and inhibit Nav1.8 current at the same time, thus producing analgesic.