The Hedgehog (HH) signaling pathway is one of the key pathways involved in embryogenesis, widely distributed and highly conserved throughout the evolutionary process from fruit flies to humans, playing a crucial role in the development of various organs. Studies have indicated that the HH signaling pathway plays an important regulatory role in the growth of ovarian follicles, proliferation of granulosa cells, maturation of oocytes, synthesis of steroid hormones, and process of ovulation. Based on the existing research findings, this article provides a detailed review of the regulatory functions of the HH signaling pathway in the growth of ovarian follicles, maturation of oocytes, ovulation, and synthesis of steroid hormones, as well as an overview of the latest research trends on several ovarian diseases that lead to a decline in fertility due to abnormalities in the HH signaling pathway, aiming to provide theoretical support for enhancing female reproductive capacity and the treatment of ovarian diseases.

Calf diarrhea is an important issue faced by the cattle industry. It has high incidence and mortality rates, high prevention and treatment costs, and even developmental delays and reduced production performance after recovery, threatening the industry's sustainable development. Functional oligosaccharides, as a type of water-soluble dietary fiber and prebiotics with bifidogenic properties, have been widely used for maintaining intestinal health. This article reviews the structure and function of functional oligosaccharides, as well as the factors that cause calf diarrhea. It also highlights the progress of research on the mechanism of functional oligosaccharides in alleviating calf diarrhea. These contents will provide an important reference for promoting calf intestinal health and the rational utilization of functional oligosaccharides.

The intestinal mucosal barrier is the first line of defense against the invasion of foreign pathogenic substances, and its integrity is essential to maintain intestinal health. β-defensins are small molecular peptides widely distributed in mammalian epithelial cells. They have biological properties such as antimicrobial, anti-tumor, anti-inflammatory and immunomodulatory properties, and have a broad spectrum of antimicrobial activity, small molecular weight, and are not prone to drug-resistant. β-defensins can regulate intestinal barrier function by regulating intestinal flora, the expression of tight junction of intestinal epithelial cells, the secretion of bacteriostatic substances in intestinal epithelial cells, and the immune function of intestinal mucosa, thus maintaining the homeostasis of the intestinal environment. In this paper, the biological characteristics of β-defensins and its function mechanism, the regulation of β-defensins on animal intestinal barrier function, regulatory effect of feed additive on β-defensins were reviewed, in order to provide reference for its application in animal intestines.

Bile acids are important products of cholesterol metabolites, and the bile acids secreted into the intestine can improve the structure and morphology of the intestine, maintain the integrity of the intestinal barrier, regulate the balance of the intestinal microflora, and enhance the functions of mucosal immunity through bactericidal, anti-inflammatory and signal transduction, which is conducive to the homeostasis of the intestinal environment, promotes the normal growth of animals and improves the production performance. This paper systematically elaborate the regulatory effects and mechanisms of bile acids on intestinal morphology, mucus barrier, microbial barrier and mucosal immunity in animals, in order to provide a theoretical basis for the use of bile acids to regulate intestinal health.

Intestinal stem cells (ISCs) are vital for maintaining intestinal homeostasis. Research indicates that intestinal commensal organisms, including microorganisms and parasites, along with their metabolites, play a crucial role in regulating host stem cells. These organisms can influence the renewal and differentiation of ISCs through direct or indirect mechanisms. This article delves into the impact of intestinal microbiota, worms, and their metabolites on ISCs, and provides an overview of current research on how related bacteria and metabolites affect the proliferation and differentiation of ISCs. The goal of this review is to offer innovative insights for potential future treatments of intestinal injuries.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV), which is mainly characterized by respiratory symptoms in pigs and reproductive failures in sows. PRRS has led to huge economic losses to the global swine industry. However, there is still no safe and effective vaccine or drug against PRRS. A comprehensive and in-depth understanding of the PRRSV life cycle can provide new ideas for PRRS prevention and control. Therefore, based on a brief description of the PRRSV life cycle, this paper focuses on the research progress of viral invasion, replication and transcription, translation and post-translational modification, assembly, with a view to providing a reference for the study of PRRSV pathogenic mechanism, prevention and control.

The porcine epidemic diarrhea (PED) is an infectious intestinal disease caused by porcine epidemic diarrhea virus (PEDV), which results in an outbreak of diarrhea in pigs and high mortality in newborn piglet. In 2010, a highly pathogenic mutant strain of PEDV emerged and spread globally, which has presently been one of the main pathogens of porcine diarrhea and caused large losses for the global swine industry. Vaccination is the primary measure for preventing and controlling PED. Currently, available commercial PED vaccines mainly consist of traditional inactivated and attenuated vaccines. With the development of genetic engineering technologies in recent years, significant advancements have been made in the study of subunit vaccines, virus-like particle vaccines, recombinant vector vaccines, transgenic plant vaccines, and nucleic acid vaccines. The frequent variations of PEDV continues to bring new challenges to the vaccine development. Therefore, efficient vaccine development platforms and engineering vaccines are crucial for future prevention and control of PED. This article provides a systematic review of the pathogenic characteristics of PEDV and the latest advancements in vaccine research, as well as prospects for future vaccine development, aiming to provide a reference for effective prevention and control of PED.

Cryptosporidiosis is a zoonotic disease caused by Cryptosporidium. Genomics plays an important role in the molecular biology of Cryptosporidium such as the complex life cycle process. Genome resequencing of Cryptosporidium and genome comparison of different Cryptosporidium species can reveal the genetic evolution of Cryptosporidium species and the formation mechanism of diversity. This paper reviews the progress of Cryptosporidium genomics, which is of great significance for the prevention and control of Cryptosporidium.

Parasitic diseases significantly hinder the development of animal husbandry. The limited range of therapeutic drugs has led to severe drug resistance and suboptimal treatment outcomes. Consequently, there has been an increased demand for new drugs effective in preventing and treating parasitic diseases. In response, numerous clinical trials demonstrate that combining two or more antiparasitic drugs can enhance efficacy, broaden the antiparasitic spectrum, and mitigate parasite resistance. The combination of levamisole hydrochloride, an imidazole thiazole drug, and oxyclozanide, a salicylamide drug, has proven effective in treating nematode and schistosomiasis, with its unique mechanism of action garnering extensive researcher interest. This article provides a comprehensive review of the mechanisms of action, design and synthesis, physicochemical properties, research status, and therapeutic efficacy of these dual-class drug combinations.

Mastitis is one of the more harmful diseases in dairy farming, which seriously affects the health of cows and dairy safety, and therefore requires timely preventive and control measures. Currently, there are problems such as inefficient diagnosis and unclear explanations of the pathogenesis of mastitis in dairy cows. With the rapid development of high-throughput sequencing technology, genomics, epigenomics, transcriptomics, proteomics, and other omics techniques have been gradually applied to dairy mastitis-related research, which promotes in-depth knowledge of the disease. This paper reviews the current status and research progress of the application of omics techniques in the pathogenesis and diagnosis of mastitis in dairy cows, aiming to provide a reference for scientific understanding and reducing the incidence of mastitis. By analyzing the multifaceted mechanism of mastitis in dairy cows, key regulatory pathways, and potential biomarkers, such as proteins like CHI3L1, LBP, GSN, GCLC, C4, and metabolites like lactic acid and tyrosine, were revealed, which can provide knowledge for the unveiling of mastitis and a new way for intervention and treatment. However, when applied in production, histology technologies still face challenges such as high cost and technical threshold. With the deeper integration and lower cost of multi-omics technology, it will help to discover more accurate biomarkers and gain insight into specific signaling pathways, providing a more scientific basis for the prevention, diagnosis, and treatment of mastitis in dairy cows.

Keratitis is an inflammation of the cornea caused by external pathogens or diseases, due to a reduction in the corneal defense ability. Pyroptosis is a programmed cell death related to inflammation. The pores formed on the cell membrane can allow the efflux of various inflammatory substances and mediate the inflammatory response. Studies have shown that keratitis is related to the occurrence of pyroptosis. Inhibiting the occurrence of pyroptosis can improve the clinical symptoms of the disease, which is a new direction for clinical treatment. This article reviews and summarizes the definition and morphological changes of pyroptosis, the mechanism of pyroptosis, the role of pyroptosis in keratitis, and the application of common pyroptosis inhibitors in ophthalmic diseases, to promote the research process of pyroptosis in keratitis.

The aim of this study was to explore candidate genes influencing age at 100 kg (AGE) and average daily weight gain at 100 kg (ADG) in pigs. Ear tissue samples were collected from 4 593 healthy adult pigs, including 2 563 boars and 2 030 sows, representing three breeds: Large White, Landrace, and Duroc. The age and body weight of the pigs were recorded, and the corrected AGE and ADG were calculated accordingly. The DNA from the samples was extracted using the phenol-chloroform method. Genotyping was performed using the "CC-1" 50K SNP chip, and SNP markers were imputed from 40 000 to 8 million after quality control. Subsequently, genome-wide association study was performed using the GEMMA mixed linear model on AGE and ADG traits. Candidate genes were identified by searching within a 1 Mb window upstream and downstream of significant SNP loci using BEDTools. Additionally, the study integrated expression quantitative trait locus data from 34 tissues in the PigGTEx, and colocalization analysis was conducted using R software to identify genes that shared causal variants with the GWAS signals. Through GWAS and colocalization analysis, candidate genes for AGE and ADG traits were identified. The significant SNP associated with both AGE and ADG traits is 1_270827213 on chromosome 1. Within a 1 Mb region upstream and downstream of this SNP, 32 candidate genes were identified. Colocalization analysis showed that, there were 10 genes had eQTL signals that colocalized with the GWAS signal for AGE, while 11 genes had eQTL signals colocalizing with the GWAS signal for ADG. Ultimately, the genes CRAT, GPR107 and USP20 were identified as candidate genes for AGE, and CRAT, GPR107, USP20, FNBP1, PTGES and HMCN2 were identified as candidate genes for ADG. This study provides molecular markers for pig breed improvement and lays the foundation for the functional gene discovery related to growth traits in pigs.

This study aimed to detect genome-wide copy number variations (CNVs) in Yunong-black pigs and identify candidate genes associated to growth-related traits in this breed. Growth-related traits were collected from 807 Yunong-black pigs (738 sows and 69 boars) across four generations (2rd-5th generation), with body weight range of 95 to 105 kg. The measured traits included body length (BL), body height (BH), chest circumference (CC), cannon bone circumference (CBC), legs buttocks circumference (LBC), backfat thickness (BF), and loin muscle depth (LMD). Ear tissue samples from the experimental population were genotyped using the "Zhongxin-1"50K SNP chip, and CNV detection was performed using PennCNV software. The CNV regions (CNVRs) were identified by merging overlapping CNVs and genome-wide association analysis (GWAS) was conducted using Plink software to examine associations between CNVs and growth-related traits. A total of 1 432 CNVs were identified on 18 autosomes and merged into 232 CNVRs. The CNVs ranged in size from 2.7 kb to 2.2 Mb, while CNVRs spanned from 4.5 kb to 2.2 Mb, covering 56.4 Mb or 2.50% of the autosomal genome. GWAS identified one CNV was associated with CC at the genome-wide significance level, and seven CNVs showed suggestive associated with CC, CBC and BF. The CNV most strongly associated with CC also showed a strong association with CBC. Additionally, the CLDN23 gene on SSC17 was significantly associated with BF, suggesting its potential role in regulating muscle development or fat deposition. This study provides new insights into the functional implications of CNVs in the Yunong-black pig, and offering valuable theoretical support for implement molecular marker-assisted selection in Yunong-black pigs breeding programs.

The study aimed to explore the regulatory effect of miR-375-3p on porcine adipocyte generation and its action mechanism targeting Fam229a. In this study, 10 healthy and disease-free boars of 30 days old were selected to be reared under the same conditions. Real-time fluorescence quantitative PCR (qPCR) was used to detect the expression distribution of miR-375-3p in the heart, liver, spleen and other tissues and the expression levels at different stages of adipogenic differentiation of adipocytes. Using miRNA mimics and miRNA inhibitors to overexpress and interfere with miR-375-3p, qPCR was employed to detect the effect of miR-375-3p on the expression of key adipogenic genes CEBPα, PPARγ, and FABP4. Oil red O staining and statistical analysis were used to assess the adipogenic capacity. Bioinformatics analysis and dual luciferase reporter assays were used to predict and identify Fam229a as a downstream target of miR-375-3p, and the function of Fam229a was subsequently validated. Finally, the downstream functions of Fam229a were predicted using PSIPRED software, Phyre2.0 software, and the STRING database, and qPCR was employed to detect the expression of early adipogenic key genes CEBPα, CEBPβ, ZFP423, and ZFP521 regulated by Fam229a. All of the above experimental methods were performed in triplicate to ensure biological reproducibility. The results showed that miR-375-3p was widely expressed in various tissues of pigs, with relatively high expression levels in lung and perirenal lipids. The temporal expression profile showed that the expression of miR-375-3p decreased first and then increased during the differentiation of porcine primary precursor adipocytes. After overexpression of miR-375-3p, the lipid droplets of porcine primary precursor adipocytes increased significantly, and the mRNA levels of adipocyte differentiation marker genes CEBPα, PPARγ and FABP4 increased significantly (P < 0.01). However, the results were reversed after interfering with miR-375-3p. In order to explore the action mechanism of miR-375-3p, a binding effect between miR-375-3p and Fam229a was found through qRT-RCR, binding site prediction and dual luciferase assay. Overexpression of miR-375-3p could significantly inhibit the expression of Fam229a (P < 0.05). After interference, the opposite result is indicated. Tissue expression profile showed that Fam229a was relatively high in liver and perirenal lipids. The temporal expression profile showed that the expression of Fam229a increased first, then decreased and then increased during the differentiation of porcine primary precursor adipocytes. After overexpression of Fam229a, the lipid drops were significantly decreased, and the mRNA levels of adipocyte differentiation marker genes CEBPα and PPARγ were significantly decreased (P < 0.05), while the results were opposite after interfering with Fam229a. The downstream mechanism of Fam229a regulating adipogenic differentiation was predicted by qRT-RCR technique and bioinformatics. This study results show that miR-375-3p can promote the adipogenic differentiation of porcine primary precursor adipocytes, and its target gene Fam229a inhibits adipogenic differentiation, revealing the regulation mechanism of adipogenic differentiation by miR-375-3p targeting Fam229a, and enriching the molecular regulatory network of miRNA.

This study aimed to identify key genes affecting drip loss in Laiwu pigs to improve pork quality and reduce economic loss caused by drip. Twenty-eight Laiwu pigs, including 19 boars and 9 sows with carcass weight of (72.30±1.26) kg, were selected as experimental animals. The meat quality traits, including 48 h drip loss, meat color, intramuscular fat content, and pH, were measured. RNA sequencing was conducted on the longissimus dorsi muscle tissue of these pigs. Then, differentially expressed genes (DEGs) were detected based on the criteria of log₂(fold change) ≥1 and an adjusted P≤0.01 between groups with high and low drip loss. Each group included three boars and one sow. By combining correlation analysis between DEGs and drip loss, as well as protein-protein interaction (PPI) analysis, the candidate genes influencing drip loss in Laiwu pigs were further identified. The results indicated that drip loss was significantly negatively correlated with the intramuscular fat content and the a^* value of meat color at both 1 h and 24 h post-mortem (P < 0.05) in Laiwu pigs. A total of 166 DEGs were identified between the high and low drip loss groups. These DEGs were significantly enriched in Gene Ontology (GO) terms and pathways related to cardiovascular system development and function, cytoskeleton and motility, and protein regulation and function. Correlation analysis using the 28 individuals revealed that 110 of the 166 DEGs were significantly correlated with drip loss (P < 0.05). Among the top ten of the most significantly correlated DEGs, two genes, KLF2 and IGF2, were identified, whose functions were reported to be related to drip loss. Furthermore, PPI analysis of DEGs identified two proteins, which were encoded by UBR1 and TTBK2, having the highest number of interactions with other proteins, and the mRNA expression levels of the two genes were significantly correlated with drip loss (P < 0.05). These gene functions were also reported closely related to drip loss in previous studies. Therefore, by combining the results of DEG analysis, correlation analysis, and PPI analysis, KLF2, IGF2, UBR1 and TTBK2 were identified as candidate genes affecting drip loss. This study lays the ground for elucidating the molecular mechanisms underlying the drip loss in pigs and offers valuable insights for the improvement of meat quality traits in pigs.

The aim of this study was to identify the core promoter activity of porcine CYP3A29 gene and the corresponding transcriptional regulatory factors, and to analyze the regulation of CYP3A29 promoter activity by transcription factors. Three healthy Yorkshire sows (30 kg) were used as experimental materials to detect the expression and distribution of CYP3A29 gene in pig tissues (heart, liver, spleen, lung, kidney, small intestine and muscle) by PCR and Western blot. The dual luciferase reporter vectors of CYP3A29 gene promoter with different fragment lengths were constructed by PCR amplification and were transfected into 293T and AML12 cell lines. The core promoter region of CYP3A29 gene was determined by detecting luciferase activity. The animal TFDB website was used to analyze the possible transcription factor binding sites in the core promoter region of CYP3A29 gene and segmented deletion dual-luciferase reporter vectors of the core promoter region were constructed. The binding site region of the transcription factor was further determined by detecting the luciferase activity. The dual-luciferase reporter vectors with transcription factor binding mutation sites and shRNA vector for transcription factor were constructed, which were used to explore the role of transcription factors in the regulation of the CYP3A29 core promoter. The results showed that the expression of CYP3A29 gene was the highest in pig liver. The -528- +62 bp region of CYP3A29 promoter had the highest activity among the 4 different regions (-2 026-+62 bp, -1 526-+62 bp, -1 026- +62 bp and -528- +62 bp), and was considered to be the core promoter region. The -528- -448 bp region of CYP3A29 promoter negatively regulated the core promoter activity and contained a potential transcription factor binding site of RUNX1. The mutant RUNX1 binding site significantly reduced the luciferase activity of the -528- +62 bp promoter, while the interference of the RUNX1 gene significantly increased the luciferase activity of the -528- +62 bp wild-type promoter, but had no significant effect on the luciferase activity of the -528- +62 bp mutant promoter, indicating that RUNX1 negatively regulated the core promoter activity of the CYP3A29 gene. The results of this study laid a foundation for further analysis of the transcriptional regulation mechanism of CYP3A29 gene.

This study aimed to investigate the effect of riboflavin (also known as vitamin B2, VB2) on the development of skeletal muscle in chicken embryos to promote its application in embryo feeding. The CCK-8, EdU, and immunofluorescence detection methods were used to identify the effects of adding different concentrations of VB2 (1.562 5, 3.125, 6.25, 12.5 and 25 μmol ·L^-1) on the proliferation and differentiation of chicken primary myoblasts. Different concentrations of VB2 solution (50, 100 and 200 μg) were injected into the yolk sac of 7 embryonic age chicken embryos, and a 0.9% saline control group was set up to detect the effect of VB2 on the development of skeletal muscle in chicken embryos. The experiment was divided into 4 groups, with 25 embryo eggs in each group. The results showed that adding different concentrations of VB2 at the cellular level promoted the proliferation activity of chicken primary myoblasts to varying degrees, but 6.25 μmol ·L^-1 VB2 was the optimal addition concentration. In addition, adding 6.25 μmol ·L^-1 VB2 significantly promoted the expression of proliferation marker genes CCND1 (P < 0.01), PCNA (P < 0.05), and CDK1 (P < 0.01), but had no significant effect on the expression of differentiation marker genes MYOD, MYOG, and MYHC (P>0.05); at the same time, EdU experiments also confirmed that adding 6.25 μmol ·L^-1 VB2 at the cellular level could significantly promote the proliferation of myoblasts (P < 0.05). In addition, the experiment of injecting VB2 solution into the yolk sac of chicken embryos showed that the injection of 50 μg VB2 significantly increased the expression levels of proliferation marker genes CDK1 (P < 0.01), PCNA (P < 0.001) and differentiation marker genes MYOD (P < 0.05), MYOG (P < 0.05) in the breast muscle tissue of 18 embryonic age chicken embryos; at the same time, the addition of 50 μg VB2 also significantly increased the fiber diameter (P < 0.05) and breast muscle weight (P < 0.05) of the breast muscle of 18 embryonic age chicken embryos. The above results show that VB2 can promote the proliferation of chicken primary myoblasts and the development of the breast muscle of chicken embryos. Therefore, the VB2 is an important nutrient that affects the development of the breast muscle of chicken embryos. This study provides an important theoretical basis and application value for the early embryonic nutritional regulation of high-quality broilers in China.

The aim of this study was to analyze the genetic diversity and population genetic structure of the Small-boned goat population, and to provide a theoretical basis for clarifying whether it is a new germplasm resource. In this study, the Small-boned goats, Hexi cashmere goats and Inner Mongolia cashmere goats (Albas) were used as subjects, 20 adult healthy ewes were selected from each group. Genome-wide nucleotide polymorphisms (SNPs) were detected using the SLAF-seq technique. Genetic diversity was calculated by using perl programming for minor allele frequency (MAF), observed heterozygosity (Ho), expected heterozygosity (He), Nei diversity index (Nei), polymorphic information content (PIC), and Shannon Wiener index (SHI); PopLDdecay was used for linkage disequilibrium analysis (LD); and VCFtools was used for population differentiation index (Fst). Population genetic structure was illustrated by principal component analysis (PCA) using EIGENSOFT, construction of NJ trees by MEGA X and population structure analysis by ADMIXTURE. Kinship was illustrated by calculating IBS distances using PLINK, calculating kinship using GCTA and constructing matrices. A total of 5 253 776 SNPs were detected, most of which were located in the intergenic region. Small-boned goats had the highest genetic diversity indexes except Ho (0.197). The mean MAF, He, Nei, PIC, and SHI of Small-boned goats were 0.216, 0.302, 0.312, 0.247, and 0.466, respectively. LD analysis showed that Small-boned goats had the highest r² and the slowest rate of decay. Fst showed that the Small-boned goat had a low level of differentiation from the Hexi cashmere goat (0.043) and a medium level of differentiation from the Inner Mongolian cashmere goat (Albas, 0.052). PCA analysis showed that Small-boned goats were more aggregated with Hexi cashmere goats and Inner Mongolian cashmere goats (Albas), and some of the Small-boned goats could be distinguished from the other groups from PC1>0. The results of the NJ-tree showed that the majority of the Small-boned goats converged into independent, and the rest had mixed groups. The population structure analysis showed that K=1 was the optimal number of groups. Small-boned goats had a unique genetic structure. The IBS distance matrix and the kinship G matrix showed the same results, with the 3 groups being more distantly related to each other and more closely related within the Small-boned goat groups. The results showed that the Small-boned goat was lowly differentiated from the Hexi cashmere goat and moderately differentiated from the Inner Mongolian cashmere goat (Albas). The genetic diversity of Small-boned goats was higher than that of the other two populations, but there were problems of selection pressure and small population size. Some of the individuals in the Small-boned goat population were closely related to each other, and inbreeding existed. This study provides a theoretical basis for the rational development and utilization of the Small-boned goat.

The aim of this study was to investigate the analysis of influencing factors and the estimation of genetic parameters of daily rumination time and daily milk yield of Holstein cows. Daily rumination time and daily milk yield were continuously collected from a pasture in Shandong Province from December 2022 to December 2023 by intelligent collar recording. Total 271 113 milk and daily rumination data were collected from 1 223 Chinese Holstein dairy cows; the effects of season, parity, lactation stage and temperature and humidity index (THI) on daily rumination time and daily milk yield were analyzed using a general linear model in SPSS Ver26.0 software; the maximum likelihood component estimates of each trait (covariate) were obtained based on the AI-REML module of DMU software, and the rumination time and daily milk genetic analysis of Holstein cows at different lactation stages were performed with the multi-trait repeatability animal model. It showed that the average daily rumination time was (523.4±82.66) min·d^-1 and the average daily milk production was (39.58±10.76) kg·d^-1. Different seasons, parity, lactation stages and THI had significant effects on rumination time and daily milk production. The heritability estimates of rumination time at different lactation stages ranged from 0.02 to 0.17, belonging to medium and low heritability traits; except for 101-200 days, the estimated heritability of daily milk production at different lactation stages ranged from 0.13-0.53, which belonged to medium to high heritability traits. In addition, the daily rumination time and daily milk yield had strong genetic correlation (0.45-0.99) at 5-40, 41-100 and 101-200 d period, and strong phenotypic correlation (0.59-0.99) at 5-40 and 41-100 d period. The rumination time collected by collar is a moderate heritability trait and has certain correlation with milk production traits. Strengthening the selection of rumination time can provide a new direction for the selection and breeding of milk production traits.

The aim was to analyze the genetic diversity and genetic structure of Simmental cattle populations to provide a theoretical basis for breeding programs and genetic improvement. In this study, 149 Simmental cattle were subjected to whole genome resequencing, and genetic structure, runs of homozygosity (ROH), and kinship and lineage construction were analyzed based on high-quality single-nucleotide polymorphisms (SNPs) sites obtained from all the genomic data. The results showed that the average sequencing depth of 149 Simmental cattle was 5×, and a total of 1 265 356 SNPs loci were identified after quality control, with an average minimum allele frequency of 0.067, an average polymorphic information content of 0.083, an average observed heterozygosity of 0.121, and an average expected heterozygosity of 0.157. The G matrix of kinship and the IBS distance matrix had similar results, and most individuals had medium level of kinship. A total of 70 genomic ROH were detected in 149 Simmental cattle with a total ROH length of 127 627.935 kb, of which 98.57% were between 1 to 5 Mb in length. The inbreeding coefficient based on ROH was 0.000 3, suggesting a low degree of inbreeding. In addition, evolutionary tree analysis classified these 149 Simmental cattle into 22 different family branches. In summary, the Simmental cattle herd showed relatively rich diversity and moderate relatedness. Inbreeding was observed in a few individuals, but the overall level of inbreeding in the population remained low.

The study aimed to understand the genetic basis of long non-coding RNA(lncRNA) regulating skeletal muscle development and fat deposition, the transcriptome analysis was performed on semitendinosus muscle of Lufeng and Leiqiong cattle to explore the lncRNAs that affect skeletal muscle development and fat deposition. In this study, 4 4-month-old female Leiqiong cattle and 4 4-month-old female Lufeng cattle with the same feeding condition were used to extract RNA from the semitendinosus muscle and then perform transcriptome sequencing to screen out differential genes with a threshold of Q < 0.05, and to perform GO and KEGG enrichment analysis and construct lncRNA-miRNA-mRNA and homeopathic targeting regulatory network. Finally, the expression levels of the differential genes were examined by RT-qPCR to verify the reliability of the sequencing results. The results showed that, using Leiqiong cattle as the control group, there were 146 differentially expressed lncRNAs, of which 71 were down-regulated and 75 were up-regulated, 355 differentially expressed mRNAs, of which 180 were down-regulated and 175 were up-regulated, and 34 differentially expressed miRNAs, of which 29 were down-regulated and 5 were up-regulated. GO enrichment analysis of lncRNAs showed that they were mainly enriched in calcium-activated potassium channel activity, actomyosin contractile ring, protein kinase A regulatory subunit binding, myosin Ⅱ complex, etc. KEGG enrichment analysis showed that they were mainly enriched in cGMP-PKG signaling pathway, RNA transport, Gap junction, insulin secretion and other pathways. The cis-targeted regulatory effects was predicted based on the positional relationship of lncRNAs and mRNAs, and 8 mRNAs were screened to be targeted and regulated by 7 lncRNAs, among which DKL1 was associated with muscle development. Then the competitive regulatory sub-networks affecting muscle development and fat deposition were screened by constructing ceRNA regulatory networks, in which ELN and FBOX32 were screened in relation to muscle development, and SLC27A6 and FABP5 were screened in relation to fat deposition. RT-qPCR results showed that the expression levels of the 9 differentially expressed genes were consistent with the expression trends of transcriptome sequencing, indicating that the sequencing results were reliable. In this study, differentially expressed lncRNAs, mRNAs and miRNAs were identified in the semitendinosus muscle of Leiqiong and Lufeng cattle, and the possible regulatory relationships of some differential lncRNAs, mRNAs and miRNAs were found, which are important for revealing the molecular regulatory mechanisms of muscle development and intramuscular fat deposition, and also provide a theoretical basis for improving meat quality of South China Yellow cattle.

This study was conducted to investigate the protective effect of the antioxidant gene peroxiredoxin I (PRDX1) of Tarim red deer in cells under oxidative stress. The Tarim red deer PRDX1 (CeyPRDX1) overexpression vector (pLJM1-CeyPRDX1-EGFP) was used to infect human immortalized keratinocyte HaCaT cells, and MTT method was used to establish the injury model of HaCaT cells under high temperature, salt (NaCl) and hydrogen peroxide (H₂O₂) stress conditions. Under this stress conditions, the effects of overexpression of CeyPRDX1 on cell apoptosis rate, mitochondrial membrane potential changes (JC-1), reactive oxygen species (ROS) content, malondialdehyde (MDA) content, and relative expression levels of SOD1, PTEN, and Caspase-3 genes were examined. The results showed that the 24 h of intervention with 200 mmol·L^-1 NaCl, 12 h of treatment with 0.50 mmol·L^-1 H₂O₂, and 4 h of cultivation at 42 ℃ were selected as the optimal stress conditions for cellular oxidative damage. Under stress conditions, overexpression of CeyPRDX1 gene could increase cell survival rate, and reduce apoptosis rate, mitochondrial membrane potential damage, ROS and MDA content induced by stress conditions such as NaCl, H₂O₂ and high temperature, effectively reducing oxidative stress-induced cell damage. The qRT-PCR results showed that under 3 stress conditions, compared to HaCaT cells that were not infected and HaCaT cells infected with empty lentivirus, HaCaT cells infected with CeyPRDX1 overexpressing lentivirus showed significantly increased mRNA expression level of SOD1 (P < 0.001), while significantly decreased mRNA expression levels of PTEN and apoptosis related gene Caspase-3 (P < 0.01). The results indicate that CeyPRDX1 gene may play an important role in protecting cells from oxidative stress damage in the adaptation of Tarim red deer to extreme drought environments and tolerance to high salt diets. Its role may be related to the expression regulation of genes such as SOD1, PTEN and Caspase-3.

The aim of this research was to identify candidate genes and genetic markers associated with stillbirth and mummified fetus traits in the first litter of Large White sows. A total of 1 805 Large White sows were genotyped using the "Zhongxin No. 1" 50K SNP chip. After filtering and selecting the data, a genome-wide association study (GWAS) was conducted, incorporating phenotypic data of stillbirths and mummified fetuses from the first litter. The results showed that 24 SNPs were significantly associated with the number of stillbirths, and 18 SNPs were significantly associated with mummified fetus traits, identifying a total of 34 candidate genes. Among these, 25 genes were related to stillbirths (such as SIL1, FSTL4, DHX38, PLK2, PDE4D, etc.), while 9 genes were associated with mummified fetus traits (including LRRTM4, ERC2, ARHGEF3, U6, etc.). A comparison with the Pig QTL database (Pig QTLdb) revealed that some of the candidate genes were located within previously reported genomic regions associated with stillbirth and mummified fetus traits, and significant SNPs exhibited linkage disequilibrium. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that the genes annotated upstream and downstream of the significant SNPs were closely associated with biological processes such as immune responses, cell signaling, apoptosis and cell division, as well as disease development. This study screened the candidate genes related to the traits of stillbirth and mummy in Large White pig population, and provided an important reference for molecular breeding.

The purpose of this study was to clarify the effect of FST gene on proliferation, apoptosis, and hormone secretion of porcine ovarian granulosa cells, and provide scientific reference for investigating the role of FST gene in the development of pig follicles. In this study, immortalized porcine ovarian granulosa cells were used as experimental materials, the granulosa cells were divided into 4 groups with 3 replications per group that included overexpression negative control group, overexpression group, inhibition negative control group and inhibition group. The overexpression plasmid and siRNA of FST gene were designed and constructed according to the mRNA sequence, and transfected into porcine ovarian granulosa cells to verify the overexpression and inhibitory effects. The CCK-8 and flow cytometry were used to analyze granulosa cell proliferation and apoptosis after transfection for 24, 48 and 72 h, ELISA was used to detect estradiol and progesterone content in granulosa cell. Furthermore, RT-PCR and Western blot were used to detect mRNA and protein expression level of cell proliferation, apoptosis, and hormone synthesis related genes. The results showed that, the mRNA and protein expression level of FST gene were significantly changed by overexpression plasmid and siRNA (P < 0.01); and FST gene could inhibit cell proliferation and induce cell apoptosis after transfecting overexpression plasmid and siRNA into porcine ovarian granulosa cells, and also significantly downregulated the mRNA (P < 0.05) and protein (P < 0.01) expression level of cell proliferation and apoptosis related genes FSHR, GDF9, BCL2 and CDKN1B. After interfering with FST expression, the estradiol and progesterone content in porcine ovarian granulosa cells were extremely significantly increased (P < 0.01), and the mRNA and protein expression level of hormone synthesis related genes STAR, CYP11A1, CYP19A1, 3β-HSD, and 17β-HSD were also extremely significantly increased (P < 0.01). In summary, in porcine ovarian granulosa cells, the decrease of FST gene expression level could accelerate cell proliferation, estradiol and progesterone secretion, and this function could be due to the upregulation of cell proliferation and apoptosis related genes and hormone synthesis rate limiting enzymes expression.

The study aimed to obtain and cultivate Rhode Island Red yolk sac mesenchymal stem cells (cYS-MSCs), then evaluate proliferative capacity and biological properties in vitro. The yolk sac of fertilized Rhode Island Red eggs, which had been incubated for 12 days, was selected for tissue adherent culture. The 5th generation of cYS-MSCs was chosen for immunofluorescence and RT-PCR analyses to detect surface marker genes. Additionally, the 3rd, 9th, and 15th generations of cYS-MSCs were selected, with 3 replicates established for each generation, to plot growth curves, calculate population doubling time, and assess colony formation rates. Genetic stability was evaluated through karyotyping. The 9th generation of cYS-MSCs was further selected for adipogenic and cartilage induction, with multidirectional differentiation capabilities assessed using specific staining and RT-PCR. The isolated cells were characterized by a long, spindle-shaped morphology, growing in either a swirling or parallel pattern. Immunofluorescence results indicated the expression of CD29, CD73, CD90, and CD166, while the hematopoietic stem cell marker gene CD34 and the panleukocyte marker gene CD45 were not expressed. Additionally, RT-PCR results confirmed the expression of CD29, CD44, CD73, CD90, and CD166, with CD34 and CD45 remaining unexpressed. The cYS-MSCs exhibited an ′S′ type growth pattern, with the population doubling time of the 3rd generation being extremely significantly lower than that of the 9th generation (P < 0.001). Furthermore, the population doubling time of the 9th generation was extremely significantly lower than the 15th generation (P < 0.001). The cloning rate of the 3rd generation was extremely significantly higher than the 9th generation (P < 0.001), and the cloning rate of 9th generation was also extremely significantly higher than the 15th generation (P < 0.001). Karyotype analysis revealed that the number and morphology of cYS-MSCs were normal (2n=78, zz). Specific staining and RT-PCR results demonstrated that cYS-MSCs could be successfully induced to differentiate into adipoblasts, chondroblasts, and osteoblasts. In conclusion, MSCs were successfully isolated from the yolk sac of chicken embryos, demonstrating the ability to be cultured and proliferated in vitro. The isolated cYS-MSCs exhibited the biological characteristics and multidirectional differentiation potential typical of MSCs. This differentiation potential suggests promising prospects for cell transplantation, providing a foundation and potential resources for the conservation of Rhode Island Red germplasm resources.

The aim of this study was to screen core genes and long non-coding RNAs related to seasonal estrus. Ovarian tissue were collected from 2 to 3 years old, non-pregnant female Chinese Merino and Hu sheep (3 sheep per group, each in anestrus, estrus and interestrus). RNA-seq technology was employed to identify differentially expressed genes and lncRNAs. GO and KEGG enrichment analyses, protein-protein interaction (PPI) network analysis and ceRNA network construction were performed to identify core genes and lncRNAs related to reproductive performance. RNA libraries for 6 distinct physiological states were constructed. There were 1 495 differentially expressed genes (DEGs) and 454 differentially expressed long non-coding RNAs (lncRNAs) be identified. Among these, 313 DEGs and 435 lncRNAs were common, while 1 182 DEGs and 19 lncRNAs were unique. GO and KEGG enrichment analyses revealed that common DEGs were significantly enriched in the processes related to cell division and the cell cycle, whereas the target genes of differentially expressed lncRNAs were enriched in intracellular signaling pathways and negative regulation of RNA polymerase Ⅱ promoter transcription. Unique DEGs were primarily associated with the positive regulation of cell migration. The target genes of lncRNAs were enriched in cell adhesion and integrin-mediated signaling pathways. There were 19 core genes identified by protein-protein interaction network analysis, which were predominantly enriched in oocyte meiosis and cell cycle pathways. These core genes exhibited higher expression level during the anestrus phase than those in interestrus. Further analysis of the ceRNA network highlighted the enrichment of genes in cell cycle-related signaling pathways. RT-qPCR results were consisted with the RNA-seq results. A total of 19 core genes and 28 lncRNAs were identified, along with 42 lncRNA-miRNA-mRNA interactions, providing potential regulatory targets for understanding the molecular mechanisms of seasonal reproduction in sheep.

The purpose of this study was to determine the optimal concentration of sodium butyrate in regulating the expression of cytokines and tight junction proteins in human intestinal epithelial cells (FH_S 74 int cells) by cell experiments, and to further explore the effects of sodium butyrate on ileum development, inflammatory factors and physical barrier function in young rats. In cell experiments, FH_S 74 int cells were selected to explore the effects of different concentrations of sodium butyrate on cell viability, expression levels of inflammatory factors and tight junction protein. In animal experiment, 32 newborn C57BL/6J mice were randomly divided into 4 groups, each with 8 mice, namely the control group (0.9% normal saline), SB20 group (20 mg·kg^-1 BW sodium butyrate), SB350 group (350 mg·kg^-1 BW sodium butyrate), and SB1000 group (1 000 mg·kg^-1 BW sodium butyrate). After continuous administration for 30 days, body weight, blood routine, ileal histological morphology, ileal cytokine and tight junction protein gene expression levels of mice were detected. In vitro, 12.5 and 25 mmol·L^-1 sodium butyrate increased the gene and protein expression of occludin in cells, and 25 mmol·L^-1 sodium butyrate significantly increased the gene expression of GPR109A and the gene and protein expression of ZO-1. In animal experiment, there were no significant differences in body weight and blood routine indexes among the groups of mice. Through ileal pathology, it was found that 350 mg·kg^-1 BW sodium butyrate significantly increased the ileal villus height but reduce the crypt depth of young mice (P < 0.05). At the same time, it increased the relative mRNA expression of ileal anti-inflammatory factors IL-4 and IL-10, while decreasing the relative mRNA expression of pro-inflammatory factor IL-18. 1 000 mg·kg^-1 BW sodium butyrate significantly increased the ratio of villus height to crypt depth of ileum (P < 0.05), decreased the crpyt depth and the relative mRNA expression of pro-inflammatory factor TNF-α. In conclusion, 25 mmol·L^-1 sodium butyrate can promote the gene expression of occludin, ZO-1, and GPR109A in FH_S 74 int cells, maintaining barrier function. Sodium butyrate at 350 mg·kg^-1 BW can promote the development of the ileum morphological structure of mice, increase villus height, reduce crypt depth, and simultaneously regulate the gene expression of intestinal inflammatory factors and tight junction proteins.

The objective of this experiment was to investigate the impact of tannic acid (TA) supplementation in low protein diets on blood biochemical parameters, muscle morphology and structure, flavor amino acids, and factors associated with myofibril development in weaned piglets experiencing immune stress induced by Escherichia coli (ETEC). Twenty-one healthy weaned piglets (Duroc×Landrace×Yorkshire), weighing approximately 6.75±0.07 kg, were selected and randomly allocated into three groups: basal diet group (CON group), basal diet+ETEC group (EK group) and basal diet+0.05% TA+ETEC group (MTA group). Each group consisted of seven replications, with one pig per replication. Weaned piglets in the EK and MTA groups were orally administered 20 mL of LB medium containing an ETEC concentration of 1×10¹² CFU·mL^-1 on days 29 and 31 of the experiment, while the CON group received the same volume of inactivated LB medium. On day 32, blood samples and longissimus dorsi were collected for assay analysis. The results were showed as follows: 1) The various treatments did not yield any statistically significant impact on the quantity and proportion of whole blood cells in weaned piglets (P>0.05). 2) Among the serum biochemical parameters, ETEC treatment significantly decreased the serum high-density lipoprotein (HDL) level and increased the total bile acid level, whereas TA treatment reversed these indices, and creatinine levels were significantly lower in the MTA group compared to both other groups. 3) ETEC treatment may induce alterations in the morphology and structure of the longissimus dorsi, particularly leading to a reduction in both average diameter and area of muscle fibers. However, supplementation of TA to a low protein diet effectively preserves the integrity of its morphology and structure. 4) The relative mRNA expression levels of MyoD and MSTN in the longissimus dorsi were significantly reduced in both the CON and MTA groups compared to the EK group (P < 0.05). Additionally, the relative mRNA expression level of MyoG was significantly higher in the EK group than in the CON group (P < 0.05), while there was a significant decrease in MyHC Ⅱ b mRNA abundance observed in the MTA group compared to both the CON and EK groups (P < 0.05). 5) The ETEC treatment significantly decreased the levels of lysine, leucine, methionine, essential amino acids, sweet amino acids, and total amino acids in longissimus dorsi (P < 0.05). However, feeding TA diets partially restored the content of the amino acids mentioned above. In conclusion, the inclusion of 0.05% TA in low-protein diets can enhance muscle development in immunostressed weaned piglets by preserving muscle morphology and structure integrity, as well as modulating the expression of myofiber-related genes and flavor amino acid content.

This paper aimed to investigate different energy supply rates simulated by varying the dietary NFC/NDF ratio to elucidate the role of energy supply in regulating urea nitrogen utilization efficiency and urea nitrogen metabolism fluxes of rumen microorganisms, and to provide a reference for the efficient urea utilization. In this study, by in vitro fermentation technique, forty fermentation flasks were randomly divided into 4 groups (n=10), and in order to deduct the ¹⁵N abundance background value, conventional urea (n=5) and ¹⁵N-urea (n=5, ¹⁵N¹⁵N-urea abundance: 99.08 atom%) were added to each group, and the NFC/NDF ratios of each group were 0.89, 1.10, 1.33, and 1.66, respectively. A 24 h in vitro fermentation was carried out and the fermentation broth was collected for the analysis of fermentation parameters, microbial ¹⁵N abundance and ¹⁵N metabolic flux. The results showed that microbial protein production and urea nitrogen utilization increased linearly (P < 0.05) with increasing dietary NFC/NDF ratio. Metabolomic analysis showed that dietary NFC/NDF ratios altered metabolic pathways such as the purine metabolism, ammonia cycle, and glutamate metabolism of rumen microorganisms.¹⁵N metabolic flux analysis of labelled metabolic fluxes showed that urea nitrogen fluxed to 34 nitrogen-containing compounds involved in amino acid, nucleotide, and energy metabolism. As the dietary NFC/NDF ratio increased, urea nitrogen flowed more to metabolites such as deoxy thymidylate, UDP-glucose and amino acids, which activated amino acid and nucleotide metabolic pathways. Under the in vitro conditions of this experiment, an increase in the proportion of dietary fast-fermenting carbohydrates promotes the conversion of urea nitrogen to microbial amino acids and nucleotides, improving urea nitrogen utilization and microbial protein production.

The aim of this experiment was to research and validate the prediction model of protein requirement of Guangming No.2 broiler. First, the protein growth requirement of broiler is measured (growth test), and then combined with the protein maintenance requirement measured in the previous stage of this project by the writer's team, a prediction model of protein requirement of broiler is established and verified (validation test). The growth test was designed with a completely randomized trial, and 132 1-day-old Guangming No.2 male and female broilers with similar body weight were selected, and they were divided into 2 treatment groups according to sex, with 6 replicates and 22 birds per replicate. They were fed a corn-soybean meal diet until 6 weeks of age. The body weight and body protein weight of broilers were measured weekly, and a linear regression equation between body weight and body protein weight was established, and the slope was the growth requirement of broiler protein. The validation test was carried out in three phases (0-10 days of age, 11-24 days of age, and 25-39 days of age). At 0, 11 and 25 days of age, 200, 150 and 100 healthy Guangming No.2 broilers were selected, and the broilers were randomly divided into control group and experimental group, with 5 replicates in each treatment group. The control and experimental diets were formulated according to Aviagen recommended standards and predicted protein requirements, respectively. The growth test showed that the production performance of 21 d and 42 d Guangming No.2 male broilers was significantly higher than that of female broilers (P < 0.01). The linear regression equation of broiler body weight and body protein weight was established in stages, and the regression model all reached significant level (P < 0.001), with R² of 0.99 in the early stage and 0.98 in the late stage. According to the linear regression equation, the growth requirement of body protein is calculated, and the growth requirement of diet protein is further calculated according to the deposition efficiency of dietary protein, which was every gram of ADG requires 0.251 and 0.228 g diet protein (the quantity unit was expressed in g·g^-1 ADG) for male and female broilers aged 1-21days, and 0.272 and 0.268 g·g^-1 ADG for male and female broilers aged 22-42days, respectively. Combined with the protein maintenance requirements measured in the previous stage of the writer's team, a prediction model of protein requirements was established: 0-21 days old: CPR=2.365×BW^0.75 +0.251×ADG (male), CPR=3.165×BW^0.75 +0.228×ADG (female); 22-42 days of age: CPR=2.955×BW^0.75 +0.272×ADG (male), CPR=2.560×BW^0.75 +0.268×ADG (female). The verification test showed that the body weight and ADG of broilers in experimental group were higher than those in control group at 0-10 days of age (P < 0.05), and lower than those in control group at 11-24 days of age and 25-39 days of age (P < 0.05). The protein content of the diet in the experimental group was predicted by the model based on the body weight, daily gain and feed intake of AA broilers. Due to the differences in the growth curves and feed intake changes between Guangming No.2 broilers and AA broilers, the prediction results may be inaccurate, which does not indicate that there are problems in the prediction model. Both the control group and the experimental group used Guangming No.2 broilers, so the protein requirement of the broiler diet could be calculated based on the actual body weight and daily gain, using a prediction model. After comparison, it was found that only the dietary protein supply of broilers in experimental group aged 1-21 days exceeded the requirement by 12.2%, the rest of the groups did not exceed 10%. These results indicate that the prediction model of dietary protein requirement of Guangming No.2 broiler proposed in this study is basically accurate.

Bovine respiratory disease complex (BRDC) is one of the diseases posing significant threats to the healthy development of the global cattle industry. The primary viruses causing BRDC include infectious bovine rhinotracheitis virus (IBRV), bovine viral diarrhea virus (BVDV), bovine parainfluenza virus 3 (BPIV3), and bovine respiratory syncytial virus (BRSV). Acquiring the distribution of the major viruses that cause BRDC is an important foundation for precision prevention and control of the disease, and for vaccine development. In this study, we investigated beef cattle, dairy cattle, yaks, and slaughterhouse cattle from various regions of China. Using PCR and RT-PCR techniques, we detected the main viruses causing BRDC in 750 clinical samples from 30 farms nationwide. We found that the individual detection rate of BRDC viruses in Chinese cattle herds was 9.47% (95% CI: 7.5, 11.8), and the herd detection rate was 33.33%(95%CI: 17.9, 54.3). BVDV was the predominant virus, with a positive detection rate of 5.07% (95% CI: 3.6, 6.9), but IBRV had the highest herd positivity rate at 20.00% (95% CI: 7.7, 38.6). The herd positivity rates of the other three viruses were all greater than 10%. In beef cattle, the main virus was BVDV with a positive detection rate of 5.62% (95% CI: 3.9, 7.8). In dairy cattle, only BVDV was detected, with a positive detection rate of 13.33% (95% CI: 3.8, 30.7). In yaks, only IBRV was detected, with a positive detection rate of 6.09% (95% CI: 2.5, 12.1). The results of correlation analysis showed that there was no statistically significant difference in the risk of BRDC infection among different breeds and regions. However, there were significant differences in the presence of season and age. Cattle were more susceptible in summer and autumn compared to spring. Cattle older than 14 weeks had a significantly higher risk of infection compared to those 14 weeks old or younger, particularly for BVDV infection. Further analysis of the presence of viruses in cattle with respiratory symptoms and apparently healthy cattle revealed that BVDV was the main virus in cattle with respiratory symptoms, with instances of mixed infections involving BVDV and BPIV3 or BRSV. In cattle without respiratory symptoms, IBRV had the highest positive detection rate. In conclusion, this study identified the distribution of the main BRDC viruses in Chinese cattle herds and determined the infection risks associated with different regions, seasons, and breeds. These findings provide a crucial foundation for the precise control of BRDC.

To study the effect of HE gene receptor binding domain variation on bovine coronavirus (BCoV) infection, recombinant plasmids pEGFP-HE420, pEGFP-HE424 and pEGFP-HE428 were constructed. Three recombinant plasmids were transfected into HCT-8 cells respectively and then infected with NX-2021-B2-HE424 to detect viral mRNA expression levels at adsorption, internalization and replication stages by RT-qPCR, and to detect viral intracellular and extracellular titers by TCID₅₀. The results showed that pEGFP-HE420, pEGFP-HE424 and pEGFP-HE428 could be expressed in HCT-8 cells. Compared with pEGFP-HE424, deletion or insertion of HE gene receptor binding domain did not affect the adsorption, internalization, replication and intracellular virus titer. Deletion of HE gene receptor binding domain decreased extracellular titers of BCoV. HE gene receptor binding domain insertion did not affect the BCoV extracellular virus titer. In this study, three recombinant plasmids of HE genes with only receptor binding domain variation was constructed to explore its effect on bovine coronavirus infection in HCT-8 cells, providing data support for the study of the infection mechanism of BCoV.

The aim of this study was to prepare the ASFV H108R protein, to evaluate the immunogenicity of the protein by immunization of mice and to provide a theoretical basis for the investigation of this protein as a candidate antigen for vaccines. The structure and basic physicochemical properties of the H108R protein were analyzed using bioinformatics tools, and the gene sequence encoding the protein 33-108 AA was selected for tandem fusion into a prokaryotic expression vector, transformed and induced to express by IPTG, and the target protein purified by nickel affinity chromatography. Immunized mice were examined by flow cytometry to detect spleen-activated CD4⁺ and CD8⁺T lymphocytes, mouse serum was isolated to determine specific antibody titres, and the specificity of polyclonal antibodies was determined by Western blot and IFA. The cytokine content of the serum was determined using the kit, and the serum was incubated with ASFV-GFP virus to assess whether the antibody could inhibit virus replication. Bioinformatics analysis showed that the H108R protein is a hydrophilic protein with a transmembrane region, no signal peptide, and more α-helices exist in the protein's ditertiary structure. SDS-PAGE and Western blot showed that the purified H108R protein was successfully expressed after IPTG induction. The protein was able to induce activation of mouse splenic T cells and stimulate the body to produce higher levels of cytokines after immunization of mice. The immunized polyclonal antibody with a potency of 1:128 000 was able to specifically bind to the ASFV-GFP virus and significantly inhibit viral replication after incubation with the virus. In this study, ASFV H108R protein was successfully expressed and produced, and immunization was able to induce activation of splenic T lymphocytes leading to an increase in cytokine levels, and the produced polyclonal antibody was able to inhibit virus replication, which had a good immunogenicity, and it lays a foundation for the in-depth study of the biological function of H108R protein and the research of vaccine.

Antibody detection is important for African swine fever virus (ASFV) infection diagnosis and vaccine research. Therefore, it is of great significance to establish a fully automatic chemiluminescent enzyme immunoassay (CLEIA) based on ASFV p72 protein antibody. For the detection, antibodies against p72 were captured by magnetic particles (MPs) coupled with purified p72 protein, then combined with alkaline phosphates (AP)-labelled p72, and ASFV p72 antibodies can be determined using a fully automated chemiluminescence analyzer. Under optimal working conditions, the cut-off value of the established CLEIA was 9.505 U·mL^-1 with a diagnostic specificity and sensitivity of 98.33% and 100%, respectively, and no cross-reactivity with other porcine pathogen-positive sera were found. The intra- and inter-batch coefficients of variation were less than 5% and 10%, respectively, and all components can be stored for more than 6 months at 4 ℃. The lowest detection limit for ASFV-positive standard serum was 1∶12 800. The total coincidence rate with the commercial kit was up to 95.90%, indicating a good applicability. In conclusion, the established CLIEA has the advantages of high sensitivity and reproducibility, combining with automated detection and simple operation steps, which can provide an important tool for the clinical diagnosis of ASF.

Long-stranded non-coding RNAs (lncRNAs) are a class of RNAs longer than 200 bp that lack protein-coding ability. lncRNAs have been shown to play important biological roles in viral replication. This study analyzed changes of lncRNAs in cells infected with porcine epidemic diarrhea virus (PEDV) and their effect on PEDV replication. Transcriptome sequencing was performed on Vero-E6 cells infected with PEDV at different time points. lncRNA 18850 was selected, and its overexpression plasmid was constructed. The effects of lncRNA 18850 overexpression on PEDV replication in Vero-E6 cells were analyzed by Western blot, qPCR and TCID₅₀ methods. Transcriptome sequencing was also used to detect gene changes in Vero-E6 cells overexpressing lncRNA 18850, as well as lncRNA 18850 target genes and differential protein interactions. The results showed that the expression of multiple lncRNAs in PEDV-infected Vero-E6 cells changed significantly, especially the expression of lncRNA 18850, which showed an upward trend at both 24 and 48 hours after viral infection. Overexpression of lncRNA 18850 significantly promoted the replication of PEDV compared with the control group. The genes LIF, IL11, EPHA2, CCND1, DUSP5, and CCN2 in Vero-E6 cells have target relationships with lncRNA 18850. The findings indicate that PEDV infection upregulates the expression of lncRNA 18850 in Vero-E6 cells, and lncRNA 18850 may promote viral replication by regulating multiple target genes, providing new perspectives and insights for a deeper understanding of the replication mechanism of PEDV as well as for exploring the potential therapeutic strategies against it.

This study aims to understand the latest infection and genetic evolution of porcine circovirus type 2 (PCV2) in various regions of Hunan Province. Using qPCR methods established in the laboratory, this study conducted viral nucleic acid detection on 1 244 samples of lymph nodes, swabs, brain tissue, and blood collected from 14 prefecture-level cities in Hunan Province, including Changsha, Yueyang, Huaihua, Changde, and Zhuzhou, from 2021 to 2023. PCV2-positive samples were then screened out, and the ORF2 gene sequences of PCV2 were analyzed through PCR amplification and sequencing. The results showed that 778 of the 1 244 samples were PCV2-positive, with an overall positive rate of 62.54%. Among them, Shaoyang City had the highest PCV2 positive rate of 92.55% (87/94), while Chenzhou City had the lowest positive rate of 18.28% (17/93). Sequencing analysis of the ORF2 gene of 57 PCV2 strains revealed that the proportions of PCV2a, PCV2b, and PCV2d were 12.28%, 7.02%, and 80.70%, respectively. The nucleotide and amino acid sequence homologies of the ORF2 gene among the sequenced strains ranged from 88.5% to 100.0% and 86.3% to 100.0%, respectively. There were 45 amino acid variant sites among 57 PCV2 strains, and 15 variant amino acid sites among PCV2d strains. Selection pressure analysis indicated that there were three positive selection sites in PCV2. This study suggests that PCV2 is currently mutating rapidly, with significant amino acid site differences among various strains. Consistent with previous reports, PCV2d has become the absolutely dominant epidemic strain in this study. These variations of PCV2 may weaken the protective effect of vaccines, which deserves attention.

Avian influenza (AI) and Newcastle disease (ND) are the two most threatening viral infectious diseases in the global poultry industry. Rapid and accurate pathogen detection and typing are of vital importance for the early control and transmission blocking of these two animal diseases. (Method) In this study, a high-throughput nucleic acid mass spectrometry method for detection of AIV and NDV was developed by combining micro multiplex RT-PCR, single base extension and MALDI-TOF mass spectrometry, and the sensitivity, specificity and repeatability of the method were evaluated. The results showed that the established method could detect AIV (type A), subtype H5, subtype H7, subtype H9, NDV and its velogenic/mesogenic strains synchronously. The analytical sensitivity of targets was 8.82-15.40 copies·μL^-1, and there was no cross-reaction with other avian pathogen nucleic acid. The reproducibility of 6 mixed targets at 12 copies·μL^-1 was above 75%. The detection results of 179 samples (including swab samples and tissue organs) collected in laboratory showed that the coincidence rate of universal target of AIV was 98.9%, and the other targets were 100.0% compared with real-time RT-PCR. So a new high-throughput alternative method is provided for differential detection of AIV, NDV, and their important subtypes/strains synchronously in this study.

In recent years, the infection rate of Mycoplasma synoviae (MS) in chicken flocks has been increasing. The inventory of laying hens in Hubei Province is relatively large, and all commodity breeds are raised. Currently, the situation of MS infection in laying hens in Hubei Province is not clear. Therefore, this study conducted an investigation into the infection status of MS in chicken flocks of large-scale laying hen farms in Hubei and explored the impact of MS infection on clinical eggshell apex abnormality (EAA). One thousand six hundred and thirty-seven serum samples and tracheal cotton swabs were collected from 82 chicken flocks in 36 large-scale laying hen farms in Hubei Province. The MS load was detected using the established real-time qPCR method, and the serum antibodies of vaccinated and non-vaccinated chicken flocks were detected using ELISA kits. Perform MS loading, pathological sectioning, and MS localization analysis on the oviduct of EAA laying hens. The results showed that in clinical samples, the MS positive rate of tracheal swabs was 9.41%; The positivity rate of serum antibodies was 92.24%, among which the positivity rate of antibodies in non-vaccinated chicken flocks was 89.04%, and the positivity rate of antibodies in 6-month-old chickens was 100%, while the positivity rate of antibodies in vaccinated chicken flocks was 95%; Out of 36 large-scale farms, 26 (72.22%) were infected with MS, and out of 82 chicken flocks, 61 (74.39%) were infected; All 10 breeds of laying hens were infected with MS. MS was detected in the oviduct of EAA laying hens, and the load in the magnum was significantly higher than that in the uterus, isthmus and infundibulum (P < 0.01), the uterus was higher than that in the isthmus and infundibulum (P < 0.05). In the oviduct of EAA laying hens, mucosal epithelial cell degeneration and glandular enlargement occur in the isthmus and uterus, as well as shedding and necrosis of glandular epithelial cells in the magnum. Moreover, MS positive signals were detected in the mucosal epithelium of the uterus and isthmus, as well as in the mucosal epithelium and surrounding tubular glands of the magnum, and among them, the magnum and uterus had the strongest positive signals. The infection of MS in laying hens in Hubei Province is severe, and different breeds are infected. MS mainly colonizes in the uterus and magnum of the laying hens' oviduct, causing pathological damage. This study provides data support for understanding the situation of MS infection in laying hens in Hubei Province and the study of MS induced EAA eggs.

Giardia duodenalis is one of the significant zoonotic enteric protozoan parasites instigating symptoms like abdominal pain, diarrhea, nausea, vomiting, and delayed development in the susceptible hosts. The Mongolian gerbil has long been employed as a stable and reliable animal model to establish infection and subsequently study pathogenic dynamics of various intestinal parasites. Although previous reports have established an infection model of G. duodenalis in Mongolian gerbils, a systematic assessment of its pathogenicity was lacking. With an aim to investigate the pathogenicity of G. duodenalis in Mongolian gerbils, this study meticulously used 30 three-week-old gerbils which were randomly divided into control and treatment (infected group). An oral dose of 5×10⁶ G. duodenalis trophozoites were administered to each gerbil to establish intestinal infection. Three gerbils from each group were randomly euthanized at the 3rd, 7th, 14th, 21st and 28th day post-infection to evaluate changes in their body weight, overall pathogenicity of G. duodenalis including its infection burden, gross pathological and ultrastructural alterations in the intestine, and fluctuation in cytokine levels. Morphological changes during the encystation process of G. duodenalis were also observed both internally and externally. The results revealed that artificially infected gerbils by G. duodenalis manifested retarded development and slow weight gain. After counting trophozoite burden and scanning electron microscopy of pathological sections of intestine it was evident that myriad of trophozoites were attached to the intestine. Following infection significant villous atrophy was witnessed with blunting morphology accompanied by deepening crypts resulting in decreased villous: crypt ratio. Serum levels of pro-inflammatory cytokines IL-1β, IL-6, IL-17, and TNF-α were elevated following G. duodenalis infection while IL-10 decreased. Encystation results showed that pH and bile content influence the morphological changes like rounding off of trophozoites coupled with flagellar and adhesive disc degeneration ultimately leading to cyst formation. This study successfully established Mongolian gerbil as a suitable animal infection model for G. duodenalis, and systematic evaluation of its pathogenicity laid a foundation for further research pertaining to G. duodenalis pathogenicity, host's immune responses and development of potential drug.

This study aimed to clone and express EnROP30 gene of Eimeria necatrix, and to evaluate the immune protection of recombinant protein EnROP30 in chicken. EnROP30 gene was cloned from total RNA of the second generation merozoites (MZ-2) of E. necatrix by RT-PCR, and inserted into the pGEM^Ⓡ-T-Easy vector by TA cloning. After sequencing analysis, EnROP30 cDNA was subcloned to the pET-28a(+) vector to obtain a recombinant prokaryotic plasmid. After transformed into E. coli BL21, the recombinant plasmid pET28a(+)-EnROP30 was induced to express by IPTG, and the recombinant protein rEnROP30 was identified and purified. BALB/c mice were immunized with the purified recombinant protein to prepare the anti-rEnROP30 polyclonal antibodies, which was used to detect the native protein EnROP30 and its localization in sporozoites (SZ) and MZ-2 of E. necatrix by Western blot and indirect immunofluorescence assay, respectively. Finally, the transcriptional level of EnROP30 in SZ and MZ-2 was analyzed by qRT-PCR. Three groups of chickens were immunized with rEnROP30 at three different doses (200 μg, 100 μg and 50 μg per chicken), respectively. At the same time, two groups of chickens, namely the unimmunized and challenged (UC) group, and the unimmunized and unchallenged (UU) group, were used as the controls. The immune protective effects were evaluated based on the survival rate, weight gain, relative weight gain, oocyst reduction, lesion score and anticoccidial index (ACI) of each group. The results showed that the target gene was 1 605 bp, coding 534 amino acids with a predicted molecular weight of 55.55 ku. The recombinant protein was about 65 ku and predominantly expressed as soluble form. Western blot analysis showed that the recombinant protein could be specifically recognized by mouse anti-His monoclonal antibodies and the convalescent serum of the chickens infected with E. necatrix. Native protein EnROP30 was detected in SZ and MZ-2 and had a molecular weight of 62 ku. EnROP30 protein was located in the top of SZ and MZ-2. The transcription level of EnROP30 in MZ-2 was significantly higher than that in SZ (P < 0.05). The survival rate of all the groups was 100%. The weight gain of the immunized groups was higher than that of the UC group, but their lesion scores were significantly lower than that of the UC group (P < 0.05). In the group immunized with 100 μg rEnROP30 per chicken, the relative weight gain (92.96%), the oocyst reduction (66.87%) and ACI (158.96) were the highest. It was showed that EnROP30 gene was cloned and expressed successfully; rEnROP30 had a good protective efficacy against E. necatrix.

The aim of this study was to construct a recombinant Lactobacillus reuteri expressing bovine lactoferrin peptides, and to enhance the secretion and expression of lactoferrin peptides by screening different signal peptides, thereby improving the application efficiency of the recombinant strains. A strain of Lactobacillus reuteri isolated in the laboratory was subjected to whole genome sequencing, the signal peptide gene for secreted proteins was screened, and recombinant strains expressing bovine lactoferrin peptides(LFCA)were constructed. The effect of LFCA secreted by the signal peptide was analyzed and compared by Western blot, ELISA, and laser confocal techniques, and the inhibition of proliferative activity of Staphylococcus aureus by LFCA secreted by the recombinant bacteria as well as the half-minimum inhibitory concentration were identified. The results showed that A2, A3, A4 and A8 signal peptides were able to express LFCA, of which A3 and A8 were able to successfully make them secreted into the culture supernatant, and A3 signal peptide increased the secretion and expression level of bovine lactoferrin peptides nearly 2-fold, which did not have significant effect on the growth performance of the strains, and the half-minimum inhibitory concentration of LFCA secreted by the strains was 97.50 μg·mL^-1 against Staphylococcus aureus. This study successfully screened the signal peptide which could secrete lactoferrin peptide efficiently, and the bacteriostatic and antiviral efficiency of the recombinant strain was significantly improved.

The aim of this study is to investigate the effect of adding yeast β-glucan (G70) to the diet on intestinal immune function in chickens immunized Newcastle disease (ND) vaccine. Sixteen 1-day-old healthy black-bone chickens were randomly divided into 2 groups, the Vaccine group and the G70+Vaccine group, and in the G70+Vaccine group, yeast β-glucan was added at 1 g·kg^-1 in the basal diet. At 14 and 28 days of age, ND vaccine was administered for the 1^st immunization and booster immunization. At 35 days of age, jejunum tissue was collected to determine the number of IgA⁺cells, the proportions of CD4⁺CD8⁺double-positive T cells, and the mRNA expression levels of immune-related genes in jejunum, then transcriptome sequencing was performed, and the differentially expressed genes were analyzed by GO function and KEGG pathway. The results showed that, compared with the Vaccine group, dietary G70 significantly increased the number of IgA⁺cells (P < 0.05) and the proportions of CD4⁺CD8⁺double-positive T cells in the jejunum (P < 0.05), and the mRNA expression levels of GATA-3, MHC-Ⅰ, MHC-Ⅱ, CCR7 and IFN-γ were significantly up-regulated (P < 0.05). RNA-seq showed that there were 559 differentially expressed genes in G70+Vaccine group, of which 54 were up-regulated and 505 were down-regulated, the GO analysis revealed that most of the differentially expressed genes were annotated to the GO entries related to amine metabolism and catabolism, protein synthesis, stimulation of TGF-β, and regulation of enzyme activities. KEGG signaling pathway analysis revealed that the differentially expressed genes were mainly enriched in signaling pathways related to cell synthesis and metabolism, such as ribosomes, MAPK, cell adhesion and local adhesion. In summary, the addition of yeast β-glucan (G70) to the diet increased the number of IgA⁺cells, promoted the proportions of CD4⁺CD8⁺double-positive T cells, and up-regulated the expression of immune-related genes in jejunum. The possible mechanism of improving chicken's intestinal immune function might be affecting the IL-17 receptor, TGF-β and MAPK signaling pathways by regulating the expression of immune-related genes such as IL-17RD, TGF-β and TMEM158.

Canine atopic dermatitis (CAD) is a complex inflammatory skin disease associated with alterations in the skin microbiota, immunity and skin barrier. Nodakenin is a coumarin glycoside with anti-inflammatory, antimicrobial and antioxidant properties. The aim of this study was to investigate the intervention effect of Nodakenin on 2, 4-dinitrotoluene (DNCB)-induced canine atopic dermatitis as well as the effect of skin microbiota. DNCB-induced canine atopic dermatitis was first established, and nodakenin was administered for 14 days as a topical application treatment. The effect of nodakenin treatment was assessed by body weight indexes, routine blood tests, serum biochemical tests, serum immunoglobulin E (IgE) levels, the degree of skin lesions and itching, and the results of 16S rRNA sequencing of skin microbiota. Compared with the DNCB group, the nodakenin group showed a significant increase in body weight, a significant increase in the total number of leukocytes and the percentage of neutrophils in the routine blood indexes (P < 0.05), while there was no significant difference in the serum biochemical related indexes (P>0.05), a significant decrease in the serum IgE content (P < 0.05), and a significant improvement in the degree of skin lesions and itching (P < 0.01). There were significant differences in both alpha diversity and beta diversity in the skin microbiota (P < 0.05), and at the phylum level, the relative abundance of the thick-walled phylum Firmicutes was significantly restored after nodakenin treatment (P < 0.01). At the genus level, nodakenin treatment significantly increased the relative abundance of Romboutsia, Clostridium_sensu_stricto_1, and Terrisporobacter (P < 0.01 and P < 0.05, respectively) and significantly decreased the relative abundance of Moraxella and Staphylococcus spp. relative abundance (P < 0.05). The application of nodakenin can effectively reduce the degree of skin lesions, alleviate itchy skin, reduce serum IgE levels, and regulate the imbalance of skin microbiota, thus improving the symptoms of canine atopic dermatitis and providing a new idea for drug development for CAD.

In recent years, strains of Brucella spp. resistant to trimethoprim-sulfamethoxazole have been isolated worldwide. Rapid detection of these resistant strains in samples can guide clinical medication. Currently, there are no rapid detection methods for Brucella trimethoprim-sulfamethoxazole-resistance strains. The aim of this study is to establish a rapid detection method for trimethoprim-sulfamethoxazole-resistance strains of B. melitensis. Based on previous studies, the isolates of B. melitensis with higher minimum inhibitory concentrations were selected. Whole genome sequencing was used to analyze SNPs and InDels, and variation sites were selected for developing a quantitative real-time PCR detection method using MGB modified probes. The results showed that B. melitensis bv.2 strain 63/9 could be the reference genome to analysis resistance strains, and 64 unique SNPs and InDels have been passed. Among them, twelve mutation sites were identified that could be used to establish a rapid detection method for resistant strains. A quantitative real-time PCR detection method developed using the A/G polymorphism at site 2 014 308 of chromosome 1 showed good specificity and could detect as low as 2×10¹ CFU, making it suitable for detecting trimethoprim-sulfamethoxazole-resistance strains in clinical samples. In conclusion, this study established a quantitative real-time PCR detection method for trimethoprim-sulfamethoxazole-resistance B. melitensis strains, providing a scientific basis for the prevention and control of drug resistant strains in China, and can also serve as a reference for clinical medication.