Sex determination in the sexually reproducing animals refers to the phenomenon in which the animals develop into female or male individuals and exhibit morphological and physiological differences after gender differentiation. Sex determination in animals is divided into environmental and genetic types. Currently, it has been identified that some genes play important roles in animal sex determination. The sex determination of animals is also closely related to DNA methylation, such as the global DNA methylation of mammalian X chromosome. However, the role of DNA methylation in sex determination varies significantly among different animal species. This article reviews the influencing factors and key genes of animal sex determination, as well as the association between DNA methylation of genes and sex determination in different species, which provides useful references for further research on the mechanism of animal sex determination.

Oocyte cryopreservation is of significant importance for human assisted reproductive technology and livestock production.This technology aids in the conservation of genetic resources from superior breeds and the maintenance of biodiversity.However, due to their small surface area-to-volume ratio and low membrane permeability, oocytes are highly susceptible to damage during freezing.This includes mechanical injury caused by ice crystal formation, oxidative stress, and the toxicity of cryoprotectants, all of which severely reduce their developmental competence after thawing. Compared to other animals, the high lipid content within equine oocytes reduces their cryotolerance, making cryopreservation considerably more challenging. Although offspring can be obtained from vitrified immature oocytes, the developmental competence of vitrified-thawed oocytes remains to be enhanced compared to their fresh oocytes.This article primarily reviews the types of damage caused by vitrification to equine oocytes and the factors influencing the cryopreservation efficiency of equine oocytes.It aims to provide a reference for further establishing and optimizing vitrification protocols for equine oocyte cryopreservation.

Chinese local livestock breeds, through continuous evolution and adaptation, have become well-suited to their local agricultural ecological environments. Although they may exhibit lower performance in certain production traits, they possess numerous advantages such as high environmental adaptability, tolerance to coarse feeds, and strong disease resistance. These qualities make them indispensable for crossbreeding and the development of new breeds. In recent years, driven solely by the pursuit of economic efficiency and productivity, superior foreign breeds have been extensively introduced. This has led to the gradual phasing out of many domestic local breeds, replaced by their foreign counterparts, posing severe threats to the conservation of domestic local breed resources. With the advancement of cryobiology, the use of biobanks for cryopreservation has become a widely adopted method for conserving germplasm resources. However, during the cryopreservation of animal germplasm, oxidative stress significantly constrains their long-term preservation. Studies have shown that adding certain antioxidant compounds can effectively mitigate oxidative stress and prevent cryodamage to cells. However, traditional antioxidants suffer from low bioavailability and poor clinical efficacy. Nanoparticles (NPs), as an emerging material, possess unique physical structures that enhance bioavailability. This has led to their widespread application in fields such as assisted reproductive technologies, biomedicine, biopharmaceutics, and cosmetics. Therefore, this review focuses on the emerging applications and efficacy of nanoparticles in cryopreservation. It summarizes some of the most promising research progress and comprehensively discusses both the limitations and advantages of using nanoparticles for the cryopreservation of animal germplasm resources. The aim is to provide novel solutions for the cryopreservation of animal genetic resources.

Single nucleotide polymorphism (SNP) chips, as highly efficient genotyping tools, offer advantages such as high genotyping accuracy, fast detection speed, simple analysis workflows, and low detection costs. These chips have been widely applied in molecular genetics research and genomic breeding. Particularly in avian genetic breeding, the application of SNP chip technology has significantly enhanced breeding efficiency and accuracy. This article aims to provide a comprehensive analysis of SNP chip technology, focusing on the two major categories: solid-phase chips and liquid-phase chips. The technical principles and unique advantages of each category are thoroughly explored. Additionally, the article summarizes the latest research advancements of SNP chip technology in chicken genetic breeding, detailing its applications and contributions in commercial breeding, identification of important economic trait loci, disease resistance breeding, breed identification, and germplasm resource protection. The findings indicate that SNP chip technology has played a crucial role in promoting genetic improvement in chickens and has provided a powerful tool for the protection of breed diversity. Looking ahead, with further technological advancements, the potential applications of SNP chips in chicken genetic breeding are expected to be even more extensive.

Enhancing the milk yield of dairy cows is crucial for strengthening the core competitiveness of our country's dairy industry and fostering innovation and development within the sector. The detection of mammary development across various stages in dairy cows is instrumental in preemptively identifying reserve cows with high lactation potential, promptly monitoring the mammary condition of both lactating and dry dairy cows, and providing a critical reference for the feeding and management practices tailored to different stages of dairy cows. Currently, conducting a comprehensive assessment of mammary development in dairy cows poses challenges due to cost and maneuverability factors. In this paper, we review the characteristics of mammary development of cows in different stages and summarizes both domestic and international methods for evaluating mammary development. It provides a detailed summary of sample collection and developmental evaluation indicators from invasive and non-invasive perspectives. The application advantages and development prospects of histological, molecular biological, and imaging techniques in assessing mammary development in dairy cows are highlighted. The content of this review aims to enrich mammary evaluation technologies of dairy cows in China, and provide technical guidance for improving the yield of dairy cows and perfecting the breeding system of high-yielding dairy cows.

The Big Food Perspective is an innovative concept formed in the new stage of national development to adapt to China's modernization strategy and improve people's healthy diet. The range of food involved in livestock and poultry mainly includes meat, eggs and dairy products, which play an indispensable role in human diet and are of great significance to human health and life-sustaining activities. The consumption structure of livestock and poultry food in our country is gradually optimized, and the output and consumption keep a good momentum of increasing year by year. Livestock and poultry food production capacity will gradually increase, and production and consumption will increase steadily. But the growth rate will be slowing down, and the import quantity is expected to decline. In this process, science and technology support is increasingly prominent. However, problems such as low yield of varieties, tight forage resources, and insufficient popularization of intelligent equipment still need to be overcome. In the future, the development of livestock and poultry food industry should focus on scale, intelligence and green. Increaseing invesment should be allocated in biological breeding, forage resource development, and alternative protein resource research to improve the supply and safety of livestock and poultry food. This paper reviewed the dynamic characteristics of per capita consumption ratio, supply and consumption of livestock and poultry food. Based on this, the future trend was forecasted scientifically. An in-depth analysis of livestock and poultry food supply and demand structure of the changing process and development trends in China would be conducted. And to comprehensively assess the status quo of technological innovation and the bottlenecks encountered in this field, then explore the potential growth points of livestock and poultry food supply. Thus proposed a series of strategic suggestions and countermeasures based on technological support, to contribute to the sustainable development of the livestock and poultry food industry under the Big Food Perspective.

In recent years, as the global greenhouse effect has become more serious, frequent extreme weather has caused huge economic losses to animal husbandry. Especially in the summer when heat waves are frequent, continuous high temperatures can cause heat stress reactions in ruminants, resulting in stunted growth and development, increased mortality, and serious restrictions on the high-quality development of animal husbandry. Studies have found that heat stress has a particularly significant impact on the intestinal health of young ruminants, causing disordered intestinal microbial flora structure, oxidative stress damage, and increased barrier permeability. However, most existing studies only explore intestinal damage caused by heat stress at a single time point, but the latest research reveals that intestinal barrier function is not static but shows circadian rhythmic changes. To this end, this article reviews the effects of heat stress on the circadian rhythm of the digestive tract biological clock and barrier function of ruminants on a diurnal time scale, in order to find potential pathways and mechanisms for heat stress to regulate digestive tract barrier function through the biological clock, and provide new ideas for precise production and environmental management of ruminants.

N6-methyladenosine (m⁶A) methylation represents a dynamic and reversible post-transcriptional modification regulated by m⁶A methyltransferases, m⁶A demethylases, and m⁶A reader proteins. It is recognized as the most prevalent RNA modification in eukaryotic organisms. Recent studies have progressively elucidated the biological roles of m⁶A methylation in livestock and poultry production, highlighting its involvement in regulating critical life processes, including growth and development, reproductive physiology, glucose and lipid metabolism, oxidative stress, and cellular damage. Alterations in factors such as nutrition, environment, and disease within livestock and poultry production can disrupt m⁶A methylation modifications, thereby impacting the physiological metabolic processes of these animals. Nonetheless, nutritional regulation strategies have the potential to restore methylation homeostasis, thereby facilitating the regulation of growth, development, and diseases in livestock and poultry. This article provides a comprehensive review of these topics, offering novel insights and strategies for enhancing growth performance and ensuring the health of livestock and poultry.

Highly pathogenic avian influenza (HPAI) H5 viruses have circulated in wild birds, causing widespread avian influenza outbreaks across various regions globally and significant economic losses to the poultry industry. Simultaneously, this subtype is persistently crossing species barriers and causing infections in a wide range of mammalian species, as well as in humans. In 2024, an unprecedented outbreak of HPAI H5N1 in dairy herds occurred in the USA, with the virus spreading within and between herds, and spilling over into humans, cats and poultry, which together indicated an increased public health risk. Here we present an overview of the evolution and epidemiology of H5 influenza viruses, examining the current dynamics of infection in both mammals and humans, and providing reference for the prevention and control of its public health and safety risks.

The coronavirus (CoV) is a single-stranded positive-sense RNA virus with a lipid envelope, which is an important pathogenic agent of humans and animals. CoV infections mainly cause acute or chronic symptoms of respiratory system, liver, stomach and intestines, and nervous system of host. CoV mainly infects humans and a variety of animals, seriously endangering human health and the development of animal husbandry. Most of the spike (S) protein of CoV is composed of the S1 and S2 subunits, which are responsible for receptor binding and membrane fusion during the virus's infection of cells, respectively. It plays an important role in the virus's tissue or host tropism and virulence, and is a key protein for CoV to infect cells. This review focuses on the structure and function of the S protein of CoV, with the aim of providing a reference for exploring the invasion mechanism of CoV.

Brucellosis is a zoonosis caused by Brucella infection, which is prevalent worldwide and seriously threatens the health of humans and animals. Brucella has a wide range of hosts. In addition to humans and livestock, it can also cause disease in a variety of wild animals. Some studies have shown that there is a certain correlation between the prevalence of brucellosis in wild animals and that of brucellosis in livestock. Even in some areas where livestock brucellosis is purified, the case reports of wild animals leading to brucellosis in livestock have occurred currently, but there are few reports of brucellosis infection in wild animals. To know global cases of wildlife brucellosis, this study analyzed cases on wildlife brucellosis reported by the World Organization for Animal Health (WOAH) from 2012 to 2022. The analysis considered multiple factors, including year, region and country, host species, and Brucella strains. The results showed that a total of 6 307 cases of brucellosis in wildlife were reported in WOAH from 2012 to 2022. According to the year analysis, the largest number of cases was reported in 2015, with 1 248 cases, followed by 2014, with 1 224 cases. By region and country analysis, the highest number of cases was reported in Europe (6 094 cases), followed by Africa (163 cases) and the Americas (49 cases). The largest number cases of countries reported were Italy (2 477 cases), followed by Spain (2 365 cases), Estonia (928 cases), South Africa (163 cases), Hungary (121 cases). According to the analysis of host species, wild boar (5 862 cases) was the wildlife with the highest number of reported cases of brucellosis, followed by African buffalo (140 cases), igoat (55 cases), roe deer (47 cases). According to the analysis of Brucella strains, the infection rate of Brucella suis was the highest in the reported cases, accounting for 92%. Followed by the Brucella abortus, accounting for more than 6%. The results of this study showed that brucellosis was prevalent in a variety of wildlife in many regions and countries in the past ten years, with Brucella suis as the main epidemic strain. The data of this study provided a basis for the prevention and control of brucellosis in wildlife in the world.

Skin wound healing is a complex physiological process involving inflammation, tissue regeneration, and extracellular matrix remodeling. Dysregulation of this process can lead to excessive scarring or chronic ulcers, impairing skin function. This review summarizes the molecular mechanisms underlying wound healing in animal skin and explores various repair strategies, including novel drug development, enhanced drug delivery, regulation of hormones and external signals, gene therapy, and tissue engineering approaches. These methods improve wound healing outcomes by optimizing drug delivery, modulating inflammatory responses, and promoting vascularization and tissue regeneration. This study aims to provide new insights into animal skin wound healing, highlight potential research directions, and contribute to the development of safer and more effective therapeutic strategies to enhance animal health and welfare.

The use of antibiotics in livestock and poultry farming accounts for more than 50% of the global antibiotic use, and the antibiotics resistance genes (ARGs) from livestock and poultry farms are one of the important sources of environmental antibiotic resistance. The primary reason is that ARGs could enter the environment carried by livestock and poultry manure. Their threat is manifested through the transmission of horizontal gene transfer (HGT), which impacts other environmental microorganisms. This paper provides a comprehensive review of the research advancements related to the HGT of ARGs derived from livestock manure. Firstly, this review elaborates that fecal matter is a significant source of antibiotis and ARGs in the environment, then summarizes the four pathways of HGT which leads to the environmental spread of ARGs, including conjugative transfer, transduction, transformation, and transport via outer membrane vesicles (OMVs). The important factors (antibiotics, heavy metals, organic pollutants and micro/nanoplasties, etc.) affecting the HGT of ARGs in the environment is also discussed. Finally, some current recent research techniques for HGT is listed as well. This paper summarizes the current status of ARGs, emerging environmental pollutants from livestock and poultry manure sources, which can provide solid theoretical basis for the research on pollution control of ARGs in livestock and poultry manure sources.

Host defense peptides (HDPs), as antimicrobial peptides (AMPs), are an important component of innate immunity and can protect the host against pathogens invasion. HDPs have broad-spectrum antimicrobial activity and are less susceptible to the production to drug resistance, which are potential candidates for the development of new anti-infectives. The antibacterial mechanism of action of HDPs is complicated, which not only acts on cell wall and membrane, but also affects the synthesis of nucleic acid, protein and metabolism. This article reviews the mechanism of action of HDPs on pathogens including bacteria, fungi, viruses, and parasites, which provides reference for the application of HDPs.

The study aimed to investigate the population polymorphism of short interspersed nuclear element (SINE) insertions/deletions in the 3'UTR of the ENTPD1 gene and their effect on gene expression in the Xiang pig. Xiang pigs were used as research subjects and Large White pigs were used as controls. The detection of population polymorphism of SINE insertions/deletions in the 3'UTR region of the ENTPD1 gene in Xiang pig and Large White pig populations was performed by PCR. The functional elements in SINE sequences was analyzed using databases and softwares such as UCSC, SINE Base, miRNA Base, PITA and RBP suite; The mRNA levels of ENTPD1 in different tissues (heart, liver, spleen, lung and kidney) of individuals of 6-month-old and mRNA levels of ENTPD1 in lung tissues of individuals with different genotypes of healthy adult Xiang pig was detected using Real-time fluorescence quantitative PCR (RT-qPCR) technology. The protein levels of ENTPD1 in lung tissues of individuals with different genotypes was detected using Western blotting technique. The results showed that there was a 297 bp SINE polymorphism site in the 3'UTR of ENTPD1 in Xiang pig, it was located at 466 bp downstream of the terminator codon. Bioinformatic analysis revealed that the SINE belonged to the Pre0_SS tRNA family and contained the RNA polymerase Ⅲ promoter, multiple RNA-binding proteins, and binding sites for miRNAs; Three genotypes, insertion (SINE+/+), deletion (SINE-/-) and heterozygote (SINE+/-), were detected in Xiang pigs and Large White pigs. The frequency of deletion (SINE-/-) genotype in Xiang pigs was significantly higher than that in Large White pigs (P < 0.01), and the frequency of SINE- allele was significantly higher than that in Large White pigs (P < 0.05). The variation showed moderate polymorphism in both populations, but only in Xiang pig population conformed to Hardy-Weinberg equilibrium. Detection of ENTPD1 mRNA in different tissues of Xiang pig showed higher mRNA levels in spleen and lung tissues. The mRNA and protein assays of ENTPD1 in lung tissues of individuals with different genotypes of Xiang pigs showed that the mRNA levels of SINE-/- and SINE+/- genotypes were highly significantly higher than those of SINE+/+, and the protein levels of SINE-/- genotypes were highly significantly higher than those of SINE+/- and SINE+/+. The results suggest that SINE insertion negatively regulates the expression of ENTPD1 gene.

This study aimed to identify candidate genes and molecular markers associated with body weight and body size traits in Turpan black sheep using genome-wide association study (GWAS), thereby providing a scientific basis for genetic breeding and resource development of Turpan black sheep. In this study, 129 healthy male Turpan black sheep aged 1 to 3 years, raised under the uniform conditions, were selected. Phenotypic data for 9 traits were measured: body weight, body height, body length, chest circumference, tube circumference, chest width, chest depth, tail width, and tail length. Peripheral blood samples (5 mL) were collected from the jugular vein using EDTA-K₂ anticoagulant vacuum tubes for DNA extraction. Qualified DNA samples underwent genotyping-by-sequencing (GBS). Raw sequencing data were processed, filtered, and annotated using software such as samtools and bcftools.Subsequently, population genetic structure analysis, population stratification assessment, and GWAS were performed using GCTA, TreeBeST, and GEMMA softwares, respectively. Sequencing yielded 160.88 G of raw data. After quality control, 157.87 G of clean data were obtained, identifying 460 766 SNP loci. GWAS detected 74 significant SNPs (P < 10^-5) associated with the 9 traits. Annotation revealed 39 candidate genes. Haplotype analysis showed that multiple significant SNPs were located within haplotype blocks. This study identified important genetic loci and candidate genes associated with body weight and body size traits of Turpan black sheep.The findings provide crucial insights for elucidating the genetic mechanisms underlying these traits and lay the foundation for molecular marker-assisted breeding in Turpan black sheep.

This study aimed to prepared goats that overexpress the BLOC1S1 gene and investigate their biosafety, thereby providing the research model and platform for establishing brucellosis resistance models and for the genetic breeding of transgenic animals. In this study, 5 multiparous and healthy female Shaanbei White Cashmere goats (aged 2 to 3 years) were used as donors for superovulation, and after natural mating with rams, primordial embryos were obtained via fallopian tube flushing, using a microinjection system, BLOC1S1 lentiviral concentrate was injected into the zona pellucida gap of the primordial embryos, which were then cultured until they reached the 2 cell stage before being transferred into the fallopian tubes of 15 multiparous female Shaanbei White Cashmere goat recipients aged 2 to 3 years. After lambing, techniques such as PCR, quantitative real-time reverse tranion PCR, and Western blotting were employed to identify the overexpression of the BLOC1S1 gene in F0 generation lambs, and the physiological and biochemical parameters of blood, along with growth and development indicators, were monitored and compared between BLOC1S1 gene overexpressing goats and wild type goats. The results demonstrated that a total of 13 lambs were obtained following pronuclear microinjection and embryo transfer, and PCR analysis identified 7 positive BLOC1S1 gene overexpressing goats, resulting in a positive rate of 53.8%, furthermore, qRT-PCR results revealed a significant upregulation of BLOC1S1 gene mRNA expression in brain (P < 0.01), spleen (P < 0.001), and lung (P < 0.05) tissues, and additionally, BLOC1S1 protein expression levels were markedly increased in spleen tissue (P < 0.001). Compared to wild type goats, goats overexpressing the BLOC1S1 gene exhibited no significant differences in blood physiological parameters, including white blood cell (WBC) count and neutrophil (Neu) levels, nor in biochemical parameters such as total bilirubin (TBIL) and glucose (GLU) (P>0.05). Additionally, the results of growth and development monitoring analyses indicated that there were no significant differences in birth weight, body weight at 1 to 6 months age, or growth traits from 3 to 6 months age between BLOC1S1 gene overexpressing goats and wild type goats (P>0.05). This study successfully prepared goats that overexpress the BLOC1S1 gene, and the growth and development, along with blood physiological and biochemical indicators of these gene overexpressing goats, exhibited no significant differences when compared to wild type goats, which finding provides a theoretical basis for establishing a Brucella disease resistance model and a biosafety evaluation system.

This study aimed to construct a methodological framework for the 9-point linear type evaluation system of dairy goats and provide technical support for dairy goat breeding practices in China. The study based on the type evaluation systems of dairy goats in Europe and the United States. Scoring traits were selected according to their heritability (h²≥0.15) and economic importance. Using 36 720 phenotypic records from 1 530 Saanen dairy goats, the biological extreme range of scoring traits were determined through normal distribution analysis. A phenotypic-linear score-functional score conversion model was developed. According to China's production systems and breeding objectives, the weight allocation for body regions and scoring traits was quantified, the deduction rules for defective traits, and a scoring model with a standardized grading method, as well as the fundamental conditions for linear type evaluation of Chinese dairy goats were established and clarified, and the map of each scoring trait was drawn by Adobe Illustrator 2022 software. Twenty scoring traits, covering four body regions—udder system (42%), legs and feet (28%), dairy strength (20%), and rump (10%)—were selected, defined, and quantified with their respective weights, meanwhile, 20 defective traits and their corresponding deduction values were proposed. By determining the biological extreme ranges of the 20 scoring traits, a 9-point linear scoring system was established based on their normal distribution frequency, accompanied by 60 illustrative diagrams. This study will provide a scientific technical framework for the linear evaluation of conformation traits of Chinese dairy goats. It will also hold significant theoretical and practical importance for establishing a genetic evaluation system for dairy goats and promoting genetic improvement in the seed industry in China.

This experiment aimed to estimate the genetic parameters of body weight and daily weight gain at different growth stages of a new beef cattle breed "Huaxi cattle" and assess its genetic progress from 2011 to 2024. The Huaxi Cattle Breeding Alliance collected data from 13 212 Huaxi cattle (3 794 bulls and 9 418 cows), comprising 64 406 records of body weight and daily weight gain at various stages from 2009 to 2024. Fixed effects such as sex, birth year, birth season, birth place, and current province were considered, with age in days as a covariate. The REML algorithm was used to estimate the heritability of body weight and daily weight gain at each stage, and ASREML software was employed to calculate the breeding values of all individuals, followed by an estimation of genetic progress for each trait. The heritability estimates for birth weight, 6-month weight, 12-month weight, 18-month weight, 24-month weight and 36-month weight were 0.17, 0.21, 0.24, 0.51, 0.54 and 0.49, respectively. The heritability estimates for daily weight gain from birth to 6 months, 6-12 months, 12-18 months, 18-24 months and 24-36 months were 0.23, 0.17, 0.28, 0.42 and 0.14, respectively. The annual genetic progress for birth weight, 6-month weight, 12-month weight, 18-month weight, 24-month weight and 36-month weight was 0.06, 0.49, 0.71, 2.18, 3.03 and 1.90 kg, respectively. The annual genetic progress for daily weight gain from birth to 6 months, 6-12 months, 12-18 months, 18-24 months and 24-36 months was 0.123, 0.087, 0.165, 0.256 and 0.025 kg·d^-1, respectively. This study represents the systematic evaluation and analysis of the genetic parameters of growth traits in the Huaxi cattle population, as well as an assessment of its genetic progress, providing a theoretical foundation for the continued breeding of Huaxi cattle and beef cattle breeding in China.

The aim of this study was to investigate the monoallelic expressions and DNA methylation status of the forkhead box P2 (FOXP2) gene in bovine tissues and placentas. The bovine tissues of two growth periods (calves of 0 d old and adult cows of 4-5 years old) and full-term placentas naturally delivered were collected. The expression levels of the FOXP2 gene were analyzed in bovine 6 tissues (heart, liver, spleen, lung, kidney and brain) and placentas by qRT-PCR. The monoallelic expressions of the FOXP2 gene were analyzed using the method of direct sequencing of RT-PCR products based on the single nucleotide polymorphism (SNP). The analysis of DNA methylation status in bovine tissues, placentas and sperms was conducted using sulphite sequencing method. The results showed that the expression of FOXP2 gene in all 6 tissues (heart, liver, spleen, lung, kidney and brain) of adult bovine was higher than that of calf tissues. Based on the SNP site of g.53771685G > A situated in the 3' non-coding region of the FOXP2 gene, monoallelic expression of FOXP2 was observed in 6 tissues of calves. However, in adult cattle, FOXP2 showed monoallelic expression only in brain tissue and biallelic expression in other 5 tissues (heart, liver, spleen, lung and kidney). The FOXP2 gene was mathernally expressed in the placenta. Hypomethylation was showed at the promoter and the 5' end of FOXP2 X1 splice in bovine tissues, placentas and sperms. These results indicated that the FOXP2 gene is an imprinted gene and the maternal allele is expressed in bovine placenta, and the monoallelic expression in bovine tissues presents growth period- and tissue-specific. Hypomethylation is in the promoter and 5' regions of FOXP2 gene, indicating that DNA methylation is not involved in the regulating monoallelic expression of bovine FOXP2 gene.

This study aimed to screen for key genes influencing coat color formation in silver foxes. Under the same feeding conditions, 4 healthy male silver foxes and 4 healthy male red foxes of similar weight at 9 months of age were selected and divided into the silver fox group (YS) and the red fox group (CS). The mid-dorsal skin samples of the two groups were collected for transcriptome sequencing (RNA-Seq) and quantitative proteome sequencing with tandem mass tagging(TMT), and differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were screened and analysed for functional enrichment, to identify candidate genes related to the formation of coat colour in silver foxes. The results showed that a total of 2 403 differentially expressed genes (DEGs) and 203 differentially expressed proteins (DEPs) were identified in the skin of the silver fox compared with the red fox. Functional enrichment analysis revealed that 15 DEGs and 6 DEPs were involved in melanin biosynthesis and metabolic processes through melanin synthesis, PI3K-Akt, cAMP, Wnt, and MAPK signaling pathways. Correlation analysis between transcriptomic and proteomic data indicated that 12 genes, including TYRP1 and NRAS, exhibited consistent significant changes at both mRNA and protein expression levels. Protein-protein interaction (PPI) network analysis further confirmed that TYRP1, NRAS, MLANA, LEF1, TYR, KITLG, EDNRB, GSK3β, AKT2 and ERBB3 were key genes in melanin synthesis. Validation of the sequencing results was performed using RT-qPCR and Western blot analyses. In this study, TYRP1 and NRAS were identified as possible key candidate genes affecting the coat color phenotype of the silver fox, providing a theoretical basis for the molecular regulatory mechanism of coat color formation in the silver fox and for molecular breeding of foxes.

The purpose of this study was to explore the differences in components and peptide sequences among sika deer velvet antlers, red deer velvet antlers and New Zealand red deer velvet antlers from the perspective of comparative proteomics, and to provide accurate scientific basis for the identification and quality evaluation of deer velvet antler. In this study, the tissues of sika deer velvet antlers, red deer velvet antlers and New Zealand red deer velvet antlers collected from adult healthy male deer were used as experimental materials and were divided into three groups, including 10 two-branched sika deer velvet antlers, 10 three-branched red deer velvet antlers and 7 three-branched New Zealand red deer velvet antlers. After sample processing, 4D-DIA proteomics technology was used in combination with bioinformatics methods to analyze differentially expressed proteins and differential peptides. A total of 7 377 proteins and 56 739 peptides were identified in this study. There were 6 486 proteins and 41 878 peptides common to sika deer velvet antler, red deer velvet antler and New Zealand red deer velvet antler. In total, 640 differentially expressed proteins were identified. The 461 differentially expressed proteins were identified in sika deer velvet antler vs. red deer velvet antler comparison group, which 270 were up-regulated and 191 were down-regulated. The 391 differentially expressed proteins were identified in sika deer velvet antler vs. New Zealand red deer velvet antler comparison group, which 246 were up-regulated and 145 were down-regulated. The 96 differentially expressed proteins were identified in red deer velvet antler vs. New Zealand red deer velvet antler comparison group, which 50 were up-regulated and 46 were down-regulated. The differentially expressed proteins were mainly involved in biological processes such as the negative regulation of peptidase activity, the regulation of acute inflammatory responses, and the regulation of protein activation cascades. The differentially expressed proteins were mainly involved in pathways such as the intestinal immune network for IgA production and the complement and coagulation cascades. The 5, 1 and 3 candidate characteristic peptides were screened for sika deer velvet antlers, red deer velvet antlers and New Zealand red deer velvet antlers, respectively. There are certain differences in protein expression levels and peptide sequences among sika deer velvet antlers, red deer velvet antlers and New Zealand red deer velvet antlers. This study can provide a basis for the identification of sika deer velvet antlers, red deer velvet antlers and New Zealand red deer velvet antlers, and also offer certain theoretical support for clarifying the differences in biological among sika deer velvet antlers, red deer velvet antlers and New Zealand red deer velvet antlers.

The study aimed to further address the limitations of traditional linear regression models in capturing the complex relationships between genotype and phenotype, and improve the accuracy of genomic prediction by integrating omics data using machine learning. This study was based on two datasets containing both genotype and transcriptome information: 1) The Huaxi cattle dataset involved 3 economically important traits: live weight, carcass weight, and net meat weight; 2) The rice dataset included 3 agronomic traits: yield, grain, and kilo-grain weight (KGW). Five-fold cross-validation was employed, and Pearson correlation coefficients were used to evaluate the accuracy of estimated breeding values. We first compared the prediction performance using single-omics data as input, and then applied an autoencoder to perform dimensionality reduction and construct latent matrices as new relationship matrix for model training. The results showed that using transcriptomic data instead of genomic data as model input improved prediction performance, with accuracy increases of 44.2% and 27.4% in the rice and Huaxi cattle datasets, respectively. Furthermore, incorporating latent matrices extracted via autoencoders further enhanced prediction accuracy by 4.10% in rice and 6.81% in Huaxi cattle compared to traditional genomic relationship matrix. Correlation analysis revealed that the latent matrix exhibited strong nonlinear relationships with the original omics data. Using transcriptomic data as model input and incorporating relationship matrices constructed via autoencoders can improve the accuracy of selection, provide valuable insights for sustained genetic improvement in breeding programs.

This study aimed to explore the relationship between changes in plasma steroid hormones and superovulation effects in Tan sheep after superovulation treatment. Thirty-three healthy multiparous Tan sheep aged 4.5±0.1 years were subjected to superovulation using the CIDR+FSH+PG method. Blood samples were collected on the day of artificial insemination (AI, Day 0) and embryo collection (Day 6), and plasma was separated. Plasma samples were divided into 4 groups based on the number of ovarian corpora lutea observed on the embryo collection day and the sampling time: high-response group on AI day (HCA), low-response group on AI day (LCA), high-response group on embryo collection day (HC6), and low-response group on embryo collection day (LC6), with 8 samples in each group. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to target and detect changes in plasma steroid hormones. The results showed that progesterone (P4), pregnanediol (PdG), deoxycorticosterone (DOC) and 2-methoxyestrone) (2-ME) in plasma of Tan sheep on embryo collection day were significantly higher than those on the day of AI, while 4-methoxyestrone (4-ME) in plasma on the day of AI (LCA and HCA groups) was significantly higher than that on the day of egg collection; The concentration of 17α-hydroxyprogesterone in the HCA group (FC=1.50) was significantly higher than that in the LCA group (P < 0.05). The concentrations of progesterone (FC=0.52) and deoxycorticosterone (FC=0.50) in the HC6 group were significantly higher than those in the LC6 group (P < 0.05). Additionally, the concentrations of progesterone and deoxycorticosterone on the embryo collection day (6 d) showed a strong correlation with the number of corpora lutea and the total number of embryos. The results indicate that after superovulation treatment, plasma steroid hormones in Tan sheep change, and the concentrations of progesterone and deoxycorticosterone affect the superovulation effect on the embryo collection day.

This study aimed to investigate the molecular mechanisms underlying the differences in developmental competence of oocytes from different grades of bovine cumulus-oocyte complexes (COCs) after in vitro maturation. Bovine COCs derived from ovaries of bovine from slaughterhouse were classified into two groups (grades Ⅰ-Ⅱ (better)and grades Ⅲ-Ⅳ (poor)) based on cumulus cell layers, compactness, and cytoplasm appearance according to the International Embryo Technology Society manual. The experiment was divided into 3 replicates, each replicate group contained more than 100 COCs. After in vitro maturation, transcriptome analysis of oocytes was performed using Smart-seq2 technology and the differentially expressed genes (DEGs) were validated. Non-targeted metabolomics was employed to analyze differential metabolites in the culture medium of different COC grades. The results showed that: 1) Grade Ⅲ-Ⅳ COCs exhibited significantly lower cleavage rate (60.59%±6.54%) and blastocyst rate (22.11%±2.79%) compared to grade Ⅰ-Ⅱ COCs (78.09%±3.01%, 32.56%±4.33%, P < 0.05); 2) Smart-seq2 analysis identified 541 significant DEGs between grade Ⅰ-Ⅱ and grade Ⅲ-Ⅳ oocytes before maturation, and 860 DEGs after 22-24 h of maturation (after in vitro maturation). GO and KEGG enrichment analyses revealed that these DEGs were primarily associated with glucose metabolism and oxidative phosphorylation pathways; 3) Digital PCR demonstrated significantly lower copy numbers of glycolysis-related gene PKM and electron transport chain complex Ⅰ inhibitor gene NDUFA4L2 in grade Ⅲ-Ⅳ oocytes compared to grade Ⅰ-Ⅱ (P < 0.01). After maturation, grade Ⅲ-Ⅳ oocytes showed significantly lower copy numbers of electron donor generation-related gene IDH3α compared to grade Ⅰ-Ⅱ (P < 0.05). Interestingly, grade Ⅲ-Ⅳ cumulus cells exhibited significantly lower NDUFA4L2 mRNA expression (P < 0.05) but higher PKM and IDH3α mRNA expression than grade Ⅰ-Ⅱ (P < 0.05, P < 0.01); 4) Metabolite analysis identified 69 significantly upregulated and 71 downregulated metabolites in different grades of bovine cumulus-oocyte complexes culture medium. Further functional enrichment analysis showed these metabolites were primarily involved in tricarboxylic acid cycle (TCA), and glycolysis/gluconeogenesis pathways. Relative abundance analysis of key metabolites (α-D-glucose, pyruvate, lactate) revealed significantly higher α-D-glucose content in grade Ⅲ-Ⅳ maturation medium (P < 0.01); 5) Supplementation of sodium pyruvate, a glycolysis product, significantly improved cleavage rate (72.54%±4.79% vs. 61.71%±4.75%) and blastocyst rate (30.63%±4.15% vs. 22.72%±2.39%, P < 0.05) in grade Ⅲ-Ⅳ COCs. This study suggests that the poor developmental competence of grade Ⅲ-Ⅳ COCs after in vitro maturation may result from insufficient glycolytic activity in oocytes coupled with insufficient energy substrate supply from surrounding cumulus cells. The addition of sodium pyruvate significantly improved the developmental competence of grade Ⅲ-Ⅳ COCs, which provide a theoretical basis for enhancing COC utilization and embryo production efficiency.

The purpose of this study was to investigate the effects of chronic oxidative stress on trace elements in organs and tissues of weaned piglets. Twelve weaned Duroc×Landrace×Yorkshire crossbred piglets were randomly divided into control group and oxidative stress group, with 6 piglets in each group. The control group was fed a basal diet, and the oxidative stress group was fed the basal diet supplemented with 10 g·kg^-1 body weight dose of D-galactose.The experiment detected the trace elements in the organ tissues of weaned piglets and the apparent digestibility of the trace elements. The results showed that the contents of Mn in liver, Fe and Mn in kidney, Zn in jejunum, Fe, Cu and Mn in colon of weaned piglets in oxidative stress group were significantly lower than those in the control group (P < 0.05). The contents of Fe and Zn in liver, Fe and Cu in jejunum, Fe and Cu in ileum of weaned piglets in oxidative stress group were significantly higher than those in the control group (P < 0.05). Compared with the control group, the apparent digestibility of Cu, Fe and Mn were significantly decreased in oxidative stress group (P < 0.05).In summary, the chronic oxidative stress model induced by D-galactose can change the content of trace elements in various organs and tissues of weaned piglets and reduce the metabolic efficiency of apparent digestibility in piglets.

The aim of this experiment was to investigate the effects of dietary Bacillus subtilis (BS) on growth performance, antioxidant and immune function, intestinal morphology and intestinal flora of piglets challenged with enterotoxigenic Escherichia coli (ETEC). A total of 24 Duroc × Landrace × Landrace piglets with a body weight of 8.30±0.15 kg at the age of 35 days were randomly divided into 4 groups with 6 replicates per group and 1 pig per replicate. Piglets in the NC group were fed a basal diet and 10 mL·kg^-1 BW sterile culture medium, while piglets in the PC group were fed a basal diet and 10 mL·kg^-1 BW ETCT (1×10⁹ CFU·mL^-1) bacterial solution. The piglets in the LBS and HBS groups were fed a basal diet supplemented with 500 and 800 mg·kg^-1 Bacillus subtilis, respectively, and both fed 10 mL·kg^-1 BW ETCT (1×10⁹ CFU·mL^-1) bacterial solution. The pre-trial period lasted for 7 days, and the experimental period lasted for 14 days. The results were showed as follows:1) During the trial period from 1 to 7 days and from 1 to 14 days, diarrhea rate was significantly increased in PC group compared with NC group (DR), while DR in LBS group and HBS group were decreased compared with PC group; From 8 to 14 days, the average daily feed intake of PC group was significantly lower than that of NC group (P < 0.05), and the average daily gain of LBS group was significantly higher than those of PC group and HBS group (P < 0.05), the average daily feed intake of LBS group was significantly higher than that of HBS group (P < 0.05). 2) The activities of SOD and CAT in serum of piglets in PC group were significantly lower than those in NC group (P < 0.05), the content of PCO was significantly higher than that in the NC group, and the SOD in LBS group was significantly higher than that in PC group and HBS group (P < 0.05). 3) The serum IgA content of piglets in PC group was significantly lower than that in NC group (P < 0.05), and the serum IgG content in LBS group was significantly higher than that in PC group and HBS group (P < 0.05). 4) The villus height and crypt depth of jejunum in LBS group were significantly higher than those in PC and HBS groups (P < 0.05). Compared with PC group, crypt depth of colon of piglets in HBS group was significantly decreased (P < 0.05).5) Lefse analysis showed that compared with the PC group, the abundance of Streptococcus in colon and Lactobacilales, Peptostreptococcus and Alkoprevotella in rectum were significantly increased in LBS group (P < 0.05). In conclusion, dietary supplementation of 500 mg·kg^-1 Bacillus subtilis can affect intestinal morphology and microflora structure of piglets, improve their antioxidant and immune capabilities, and then improve the growth performance and diarrhea of ETEC challenged piglets.

The aim of this study was to investigate the effects of different dietary metabolizable energy and standard ileal digestible lysine levels on the lactation performance, milk components, apparent digestibility of nutrients, energy and nitrogen metabolism markers in serum, fecal microbiota, and growth performance of piglets in high-producing Shenxian sows. Sixty multiparous Shenxian sows with a total litter size of at least 11 piglets were randomly divided into six groups of ten replicates each, with one pig per replicate. A 2×3 factorial design was used, with two metabolizable energy (ME) levels (13.46 and 13.72 MJ·kg^-1) and three standard ileal digestible lysine (SID Lys) levels (0.65%, 0.85%, and 1.05%), resulting in six treatments. The experiment lasted for 35 days from parturition to weaning. The results showed that the body weight loss and backfat loss of lactating sows were significantly reduced when the dietary energy was 13.72 MJ·kg^-1 ME (P < 0.05), and the average daily gain (ADG) and weaning survival rate of piglets were significantly increased (P < 0.05), with the above indicators being optimal at 0.85% SID Lys. The apparent digestibilities of dry matter (DM), crude protein (CP), ether extract (EE) and gross energy (GE) of lactating sows were significantly improved in the 13.72 MJ·kg^-1 ME group (P < 0.05). The digestibility of crude protein (CP) in sows fed 0.65% SID Lys was significantly lower than that of the other two groups (P < 0.05). The levels of lactose and fat in milk were significantly increased with rising dietary ME (P < 0.05), and the highest level of milk protein was observed in the 0.85% SID Lys group (P < 0.05). The serum triglyceride (TG) concentration of lactating sows was significantly increased (P < 0.05), while the creatinine (CRE) concentration was significantly reduced in the 13.72 MJ·kg^-1 ME group (P < 0.05). The blood urea nitrogen (BUN) level showed a parabolic change with lysine level increased (P < 0.05). The energy level of 13.72 MJ·kg^-1 ME resulted in a significant increase in Streptococcus abundance (P < 0.05), while Turicibacter and Romnoitsia decreased significantly (P < 0.05), and Clostridium_T showed a linear decrease with increasing lysine concentration (P < 0.05). In conclusion, the dietary energy level of 13.72 MJ·kg^-1 ME and the lysine level of 0.85% SID Lys in high-producing Shenxian lactating sows ensures excellent body condition, accelerates energy metabolism and nitrogen metabolism of the body, ensures high-quality milk components, improves the intestinal micro-ecological environment, and promotes growth of the piglets.

The purpose of this study was to explore the effect of valine (Val) on the proliferation of bovine myoblasts and its underlying molecular regulatory mechanisms. A single variate experimental design was used to detect the effects of different concentrations of Val on cell proliferation by CCK-8 and EdU in order to determine its optimal concentration. Molecular docking methods were used to predict the interactions between Val and AMPK and mTOR proteins, respectively. Cells are divided into 6 groups: Control group (0 mmol·L^-1 Val), 0.5 mmol·L^-1 Val group (0.5 mmol·L^-1 Val), Val+AMPK activator AICAR group (0.5 mmol·L^-1 Val+1 mmol·L^-1 AICAR), Val+AMPK inhibitor Compound C group (0.5 mmol·L^-1 Val+5 μmol·L^-1 Compound C), AMPK activator AICAR group (1 mmol·L^-1 AICAR) and AMPK inhibitor Compound C group (5 μmol·L^-1 Compound C). In this study, qRT-PCR and Western blot were used to detect the effects of Val on the mRNA and protein expressions of proliferation-related factors PAX7, PCNA and CDK1, as well as AMPK/mTOR signaling pathway key factors AMPK and mTOR in bovine myoblasts. The results showed that compared with the control group, after 24 h of 0.5 mmol·L^-1 effected on bovine myoblasts, cell viability was extremely significantly increased (P < 0.001), and the rate of EdU-positive cells was extremely significantly increased (P < 0.01), the mRNA expression levels of PAX7 and PCNA (P < 0.05)and the protein expression levels of PAX7 were significantly increased (P < 0.05).The molecular docking prediction results showed that Val and AMPK were connected by two hydrogen bonds, and three hydrogen bonds could be formed between Val and mTOR, and the combination between Val and AMPK and mTOR was more stable. Compared with the control group, the protein expression level of mTOR was significantly increased after 0.5 mmol·L^-1 Val was applied to bovine myoblasts alone (P < 0.05), and the protein expression level of AMPK was significantly decreased (P < 0.05). Compared with the 0.5 mmol·L^-1 Val group, the mRNA expression level of mTOR and the protein expression levels of mTOR and PAX7 in the 0.5 mmol·L^-1 Val+1 mmol·L^-1 AICAR group were extremely significantly decreased (P < 0.01), and the mRNA and protein expression levels of AMPK were significantly increased (P < 0.05); The mRNA expression level of mTOR in 0.5 mmol·L^-1 Val+5 μmol·L^-1 compound C group was significantly increased (P < 0.05).This study showed that Val could promote the proliferation of bovine myoblasts by negatively regulating the AMPK/mTOR signaling pathway, revealing the molecular mechanism by which Val affect the proliferation of bovine myoblasts through the AMPK/mTOR signaling pathway.

The aim of this study was to investigate the effects of milk replacer with different protein levels on the diarrhea rate, serum immunity indices, intestinal organ indices and morphology, and small intestinal microbiota in lambs. Forty-eight healthy second-generation 3-day-old male lambs of East Friensian Sheep×Hu sheep crossbreeds with same genetic background and birth weight (4.34±0.15 kg) were selected and randomly divided into four groups by a one-way experimental design, namely, low protein level milk replacer group (L-Group, 22% protein group), the medium-low protein level milk replacer group (ML-Group, 24% protein group), the medium-high protein level milk replacer group (MH-Group, 26% protein group), and the high-protein level milk replacer group (H-Group, 28% protein group). The formal test was carried out after 5 d of pre-test feeding period, and slaughter performance measurement and test sample collection were conducted after 54 d of feeding. The results showed that: 1) The rate of diarrhea in the MH and H groups were extremely significantly higher than that in the ML and L group (P < 0.01). 2) The concentration of IL-2 in MH and H groups were significant higher than that in L group (P < 0.05), the concentration of IgA, IgG, IgM, TNF-α, IL-6, IL-1β didn't present significant differences (P>0.05). 3) There was no significant difference in intestinal organ index (P>0.05). 4) The depth of duodenal crypt in MH group was significantly deeper than that in L and ML groups (P < 0.01).The ratio of villi height to crypt depth in L and ML groups was dramatically higher than that in MH and H groups (P < 0.01). 5) Sequencing of jejunal 16S rRNA showed that there was no significant difference in alpha diversity among groups (P>0.05). At the phylum level, the Actinobacteriota relative abundance of H group was significantly lower than that of L and ML groups (P < 0.05). At the genus level, the Lachnospiraceae_NK3A20_group relative abundance of H group was significantly higher than that of L and ML groups (P < 0.05). In summary: milk replacer protein levels exceeding 24% increased the rate of diarrhea and serum IL-2 levels in lambs, and decreased the ratio of duodenal villi height to crypts depth, and the relative abundance of the beneficial intestinal bacterium Actinobacteria phylum was reduced when the protein level reached 28%. Therefore, under the experimental conditions of this study, it was concluded that milk replacer protein levels not exceeding 24% were more favorable to the intestinal health of lambs.

The aim of this study was to investigate whether oral gavage of theabrownin (TB) could alleviate the negative effects of oxidative stress on antioxidant capacity in the liver of rats, in which the oxidative stress model was established via feeding the diet containing oxidized fish oil (OFO), and tea polyphenols (TP) were used as a positive control. Thirty-two male Wistar rats weaned at 21 days of age (average weight was 55.58 g) were randomly allotted into 4 groups, including control group, OFO group, OFO+TP group and OFO+TB group. Both the daily oral doses of TB and TP were 200 mg·kg^-1 body weight. From day 9 to day 23, the challenge of oxidized fish oil was executed. The trial duration was 23 days. The results showed that feeding the diet containing OFO decreased body weight, induced the dysfunction of liver histomorphology, significantly enhanced liver organ index, serum alanine aminotransferase activity, and the levels of lipid peroxide, malondialdehyde, protein carbonyl and 8-hydroxydeoxyguanosine of liver (P < 0.05), significantly reduced total antioxidant capacity, catalase and glutathione peroxidase activities of liver (P < 0.05), significantly up-regulated the mRNA relative expression of Bax, Caspase3 and Keap-1 of livers (P < 0.05), and significantly down-regulated the mRNA relative expression of Bcl-2, Nrf2, HO-1 and NQO-1 of livers (P < 0.05) in rats. Oral infusion TB or TP might, to some extent, alleviate the negative effects of the diet containing OFO on body weight, liver morphology and function of rats, improve the antioxidant capacity and the mRNA relative expression of apoptosis-related and Nrf2-pathway-related genes in the liver of rats fed the diet containing OFO (P < 0.05). In summary, oral administration of TB could ameliorate the liver damage induced by OFO challenge in rats, which was associated with the improvement of antioxidant capacity and apoptosis. These effects were similar with TP.

Felis domesticus allergen 1 (Fel d 1), the major allergen responsible for cat-induced allergic diseases, can trigger symptoms ranging from mild allergic rhinitis to life-threatening asthmatic reactions. This study developed a novel immune intervention strategy based on an antigen delivery system: utilizing recombinant Escherichia coli Nissle 1917 (EcN) to achieve in vivo expression of rFel d 1, aiming to prevent allergic reactions induced by rFel d 1. The gene sequence of the major Fel d 1 was obtained from the NCBI GenBank database. The coding sequences (CDS) of Chain 1 and Chain 2 were selected, with a covalent linkage formed between the C-terminal cysteine residue (Cys) of Chain 1 and the N-terminal valine residue (Val) of Chain 2 to generate recombinant Fel d 1 (rFel d 1). A triple GS repeat sequence was introduced at the N-terminus of rFel d 1, while a 6×His tag was added to the C-terminus. After codon optimization, the construct was cloned into the pBad vector to generate the prokaryotic expression vector pBad-rFel d 1. The pBad-rFel d 1 plasmid was then transformed into EcN competent cells to construct the recombinant E. coli strain EcN^{rFel d 1}. In a murine allergy model, the preventive efficacy of EcN^{rFel d 1} against rFel d 1-induced allergic reactions was comprehensively evaluated. We found that pre-administration with the recombinant E. coli strain EcN^{rFel d 1} capable of expressing rFel d 1 in vivo could effectively prevent airway hyperresponsiveness and the elevation of IgE levels, protecte lung tissues from pathological changes and avoid local or systemic allergic reactions specifically activated by rFel d 1, and prevent the imbalance of Th1/Th2 and Th17/Treg in the body. Based on the inherent colonization advantages and sustained-release properties of probiotic EcN, this study developed a preventive recombinant E. coli strain EcN^{rFel d 1}, Which effectively prevents rFel d 1-induced allergic reactions by modulating Th1/Th2 and Treg/Th17 immune balance while inducing the production of blocking antibodies.

This study was conducted to develop a primer set for Nanopore targeting sequencing (NTS) to simultaneously detect ten porcine pathogens and explore the potential for detecting all porcine pathogens on this platform. Primers for ten pathogens were designed using Primer Premier 6 software. The specificity of the ten primers was verified by PCR and agarose gel electrophoresis. The optimal primer concentration in the multiplex PCR system was explored using nanopore sequencing technology by comparing the detection numbers of pathogens at different concentration gradients. The sensitivity of multiplex PCR was verified using the optimal primer concentration obtained in the previous step. The detection sensitivity was determined by amplifying pathogen templates at different concentration gradients and analyzing the corresponding results. Finally, a conformity test between fluorescence quantitative PCR and NTS was performed using clinical samples (n=201). The agarose gel electrophoresis images for verifying primer specificity all showed a single, bright, and clear band, and no bands were amplified for the other ten porcine pathogens not selected in this study. All ten pathogens could be detected in the reaction system when the final concentration of primers was 3 and 5 μmol·L^-1, while all other tested concentrations failed to detect all ten pathogens. All positive samples in the four replicates could be detected when 5 μL of the nucleic acid template with a concentration of 9×10² copies·mL^-1 was added. In the detection of clinical samples, more pathogen-positive samples were detected by NTS. After the chi-square test, there were statistically significant differences in the detection results of five pathogens (P < 0.05), while no statistically significant differences were found in the detection results of the other five pathogens (P>0.05). After kappa value analysis, excellent consistency was observed in the detection of six pathogens, and good consistency was observed in the detection of four pathogens. The results indicate that the NTS detection method can simultaneously detect ten different porcine pathogens. For samples with inconsistent detection results compared to the control method, further verification by a third-party detection method is required.

The goal of this study was to develop a sensitive, cost-effective, portable, rapid visual detection method for porcine deltacoronavirus (PDCoV) and transmissible gastroenteritis virus (TGEV) IgG antibodies. This study conjugated the N proteins of PDCoV and TGEV to carboxylated magnetic bead surfaces to form a trimeric complex detection system of "PDCoV-N/TGEV-N protein-specific IgG-enzyme-labeled antibody". We established an immunomagnetic bead-based indirect ELISA method combined with a smartphone-assisted colorimetric sensing platform for rapid visual detection of PDCoV and TGEV IgG antibodies in porcine serum. Optimized the experimental conditions of IMB-based indirect ELISA for the detection of PDCoV IgG antibody, with the optimal reaction conditions being as follows: magnetic beads dosage 25 μg; antigen coating concentration 5 μg·mL^-1 (30 min); blocking solution containing 2.5% BSA combined with skim milk powder (37℃ for 2 h); serum incubation for 60 min; enzyme-labeled secondary antibody at 1∶8 000 dilution (30 min); TMB substrate development for 10 min. Under these optimized conditions, this method demonstrated antibody detection titers of 1∶32 768 for PDCoV and 1∶8 192 for TGEV, with intra-/inter-assay coefficients of variation both < 8%, and showed no cross-reactivity with positive sera against seven common porcine pathogens. Clinical validation revealed overall agreement rates of 95% (PDCoV) and 96.25% (TGEV) compared with neutralization assays in 80 clinical samples. The detection method established in this study, based on an immunomagnetic bead-integrated smartphone-assisted colorimetric sensing platform, exhibits high specificity, sensitivity, and reproducibility. This field-deployable approach enables rapid visual on-site detection of PDCoV and TGEV in clinical serum samples, offering a robust solution for epidemiological monitoring and vaccine effectiveness assessment in PDCoV/TGEV control programs.

The aim of this study is to establish a rapid, sensitive, and quantitative detection method for porcine epidemic diarrhea virus (PEDV) based on fluorescent microsphere immunochromatographic detection, providing technical support for on-site rapid detection of porcine epidemic diarrhea. By adopting a double-antibody sandwich method in conjunction with fluorescent microsphere immunochromatographic technology, the PEDV murine polyclonal antibody labeled with time-resolved fluorescent nanoparticles (TRFNPs) was prepared and utilized as a fluorescent probe. Subsequently, the anti-PEDV N protein monoclonal antibody and the goat anti-mouse IgG antibody were employed as the test line (T line) and quality control line (C line), respectively, to assemble a fluorescent microsphere immunochromatographic antigen test strip. The sensitivity of the method was evaluated by detecting a gradient dilution of PEDV. The specificity of the method was verified by testing several other common swine disease viruses. The clinical application value of the method was assessed by testing simulated fecal samples and clinical samples. The results showed that the method had high sensitivity. There was a good linear relationship between the viral titter (ranging from 10^3.3 to 10^6.0 TCID₅₀·mL^-1) and the T/C value of the test strip. The limit of detection was as low as 10^3.1 TCID₅₀·mL^-1, indicating that the method can be used for quantitative detection of PEDV. The sensitivity of the test is approximately 10 times higher than that of commercial colloidal gold test strips. The operation is simple and timesaving, with a detection time of 15 min. The method has good specificity and shows no cross-reactivity with TGEV, PoRV, PDCoV, PRRSV, CSFV, and inactivated ASFV. The results of repeatability and intermediate precision experiments showed that the intra- and inter-batch coefficients of variation of the method were both less than 10%. The method is suitable for the detection of PEDV in simulated fecal samples and clinical samples. A total of 50 clinical samples were tested simultaneously using this method and RT-PCR, the consistency rate of the test results was 96%. Here, we developed a TRFNPs-based fluorescent microsphere immunochromatographic assay (FMICA) for the detection of PEDV antigen, which might provide a tool for rapid clinical detection of PEDV.

The aim of this study was to express the protective antigen S1 of porcine epidemic diarrhea virus (PEDV) using porcine Limosilactobacillus reuteri as a live bacterial vector, and to evaluate the immune effect after oral immunization of newborn piglets, in order to develop a safe and efficient oral mucosal vaccine preparation. In this study, the recombinant lactic acid strain pPG-T7g10-S1/L. ruteri J 31 expressing PEDV S1 was constructed. The growth characteristics, genetic stability of the recombinant plasmid, and adhesion characteristics of IPEC-J2 cells in vitro were evaluated. The results showed that the growth characteristics of the recombinant strain pPG-T7g10-S1/L. ruteri J 31 did not change significantly compared with the parental strain. The plasmid could be inherited stably and had a strong ability to colonize in the jejunum of piglets. After oral immunization of newborn piglets with the recombinant PEDV, the level of mucosa-specific SIgA antibody and serum specific IgG antibody were produced, and both of them had the activity of neutralizing PEDV, and could induce Th1, Th2 and Th17 types of cellular immune responses in piglets. In conclusion, in this study, the recombinant Limosilactobacillus reuteri pPG-T7g10-S1/L. ruteri J 31 was successfully constructed. Oral immunization with the pPG-T7G10-S1/L. ruteri J 31 could induce significant humoral immunity, mucosal immunity and Th1, Th2 and Th17 cellular immune responses in piglets. These results laid a theoretical and experimental foundation for the development of oral PEDV vaccine candidates.

The study aims to investigate the impact of prolyl oligopeptidase (POP) on the replication of foot-and-mouth disease virus (FMDV) in PK15 cells. Firstly, the effect of FMDV infection on POP expression in PK15 cells was analyzed using Western blot and real-time quantitative PCR (RT-qPCR). Secondly, a POP eukaryotic expression plasmid was constructed, and the effect of overexpression of POP on FMDV replication was assessed through Western blot, RT-qPCR, and virus titer assays (TCID₅₀); Thirdly, specific siRNA targeting the POP gene was synthesized, and effective siRNAs sequences for interfering with endogenous POP expression were identified using RT-qPCR. The impact of downregulating endogenous POP expression on FMDV replication was further evaluated by Western blot, RT-qPCR, and virus titer assays (TCID₅₀). The results indicated that FMDV infection did not significantly affect POP expression in PK15 cells. Overexpression of POP significantly promoted FMDV replication in PK15 cells, with viral replication level increasing in a dose-dependent manner as the dosage of POP expression increased. Downregulation of endogenous POP expression using siRNA significantly inhibited FMDV replication. This study revealed that the promoting effect of POP protein on FMDV replication from the host cell level at the first time. This finding provides a foundation for further exploration of the mechanism by which POP promotes FMDV replication.

Coronavirus is one of the most important pathogen causing respiratory and digestive diseases in humans and animals. In order to enrich the epidemiological data of coronaviruses in China, we conducted this study on the isolation of caprine coronavirus (cpCoV) from diarrheal goat samples in Xinjiang, and further identified the biological characteristics systemly. Rectal swab samples of goats suffering diarrhea from Xinjiang were tested positive by RT-PCR for epidemiological investigation, and the positive samples were treated and inoculated with HRT-18G cells. A novel cpCoV, named as cpCoV/XJCJ2301G, was isolated and identified with obvious cytopathogenic effect. The virus was detected positively by IFA, the purified virus was observed as the diameter of 100 nm with a crown-like surrounding by transmission electron microscopy. According to the genetic evolution analysis, the XJCJ2301G strain was belong to Embecovirus of β-CoVs, which formed an independent evolutionary cluster together with the present cpCoVs. The NS4a and NS4b gene sequences of XJCJ2301G strain have a 75 nt deletion, resulting in truncation or elongation of non-structural proteins. Through sequence alignment analysis of the S gene, it was found that the differences between cpCoV and BCoV mainly present in the CTD region. CpCoV was isolated from Xinjiang for the first time, and this strain enriched the epidemiological data of animal coronavirus. Notably, there was a stepwise pattern deletion between NS4a and NS4b among bovine coronavirus, caprine coronavirus, cpCoV/XJCJ2301G and rabbit coronavirus. Strain XJCJ2301G should be a key node and play a vital role in the evolution of herbivore coronaviruses.

This study aimed to construct a recombinant adenovirus expressing the structural protein VP1 of bovine enterovirus (BEV), and to perform preliminary verification of its immunogenicity and safety. The BEV VP1 gene was amplified by PCR and cloned into the adenoviral vector pDC316-CMV-copGFP. The recombinant plasmid pDC316-VP1-copGFP obtained was co-transfected with pBHGlox-△E1, 3cre into 293 cells, and then virus titer assay and RT-PCR were carried out to identify the virus; the construction of pET-32a-BEV-VP1 prokaryotic expression plasmid was constructed and BEV VP1 protein was purified after optimising the induction time and temperature; negative control adenovirus rAdv-copGFP, recombinant adenovirus rAdv-VP1, and blank control PBS were used to immunise BALB/c mice by myocardial injection to observe the clinical symptoms of mice; mice were infected with BEV after 21 days of immunisation and liver, spleen, lung, spleen and blood samples were collected from mice on day 0, 5, 10, and 15. The samples were collected from mice on day 0, 5, 10 and 15. After 21 days of BEV infection, the liver, lung, spleen, kidney and small intestine were collected on day 0, 5, 10 and 15. Tissue RNA was extracted and pathological sections were made, and the viral load in each tissue was detected and the histopathological changes were observed; the serum of adenovirus-immunised mice was tested for the response to the prokaryotic expression of the VP1 protein by Western blot; and the levels of total IgG antibody, specific antibody, and cytokine IFN-γ, IL-4 in the immunised serum were detected by ELISA. The adenoviral vector pDC316-VP1-copGFP was successfully constructed; the optimal conditions for VP1 prokaryotic expression were induced at 37 ℃ for 8 h, and pET-32a-BEV-VP1 protein was successfully purified; the serum of adenoviral-immunised mice was able to recognise the purified VP1 protein; the recombinant adenoviral immunisation was able to make the mice produce specific antibodies, which increased the levels of humoral immunity and cellular immunity. The recombinant adenovirus immunisation can make mice produce specific antibodies, thus improving humoral and cellular immunity. The recombinant adenovirus expressing BEV VP1 protein was successfully constructed in this study, and it could induce humoral and cellular immune responses in mice after immunisation, thus exerting a certain degree of protection against BEV infection, and laying a foundation for the further development of a new BEV vaccine.

This study aimed to prepare Clostridium perfringens α-ferritin nanoparticle antigens and evaluate its immunogenicity in mice. The full-length α gene containing D56G and H68G point mutations and the Spytag sequence, as well as the gene fragment ferritin containing the ferritin gene and SpyCatcher sequence were cloned into the prokaryotic expression vector pET 28a vector respectively. Recombinant proteins were expressed followed by identification and purification. The cytotoxicity, lecithinase activity and haemolytic activity of purified α_m2ST were analysed. The purified α_m2ST and FeSC were docked in vitro. The docking product of α-ferritin (α_m2-Fe) and α_m2ST were mixed with adjuvant, respectively, and used for mice immunization. Mice serum was collected every week post immunization to measure IgG, IgG subtype antibody levels and the serum neutralization titers; on day 28 post the second immunization, splenocytes were isolated to analyze T-cell subsets and IFN-γ secretion levels. The results showed that at 28 ℃, α_m2ST and FeSC were predominantly expressed in a soluble manner. The α_m2ST was free of cytotoxicity, lecithinase activity, or hemolytic activity after detection, indicating that the α_m2ST is non-toxic. After in vitro docking of α_m2ST and FeSC, it was confirmed that the α_m2-Fe formed nanoparticles with a diameter of 32.7-50.7 nm, with a main peak at 37.8 nm, after analyses with SDS-PAGE, TEM, and DLS. Following immunization in mice, the α_m2-Fe group exhibited high levels of IgG2a antibodies (P < 0.001). On day 21 after the second immunization, the serum neutralization titer in the α_m2-Fe group was 64-fold, significantly surpassing the 32-fold titer in the α_m2ST group (P < 0.05). By day 28 after the second immunization, the IFN-γ levels in the supernatant of stimulated splenocytes were also higher in the α_m2-Fe group compared to the α_m2ST group (P < 0.05). Flow cytometry analysis revealed that the α_m2-Fe group produced a higher proportion of effector CD8⁺ T cells (P < 0.000 1). Mice experiments confirmed that the α_m2-Fe nanoparticle antigens effectively induce both humoral and cellular immunity in mice.

The aim of this study was to investigate the use of liposomes as a carrier to deliver phage into the cell and the antibacterial effect of phage liposomes on Staphylococcus aureus in bovine mammary epithelial cells. In this study, liposome-encapsulated phage SDQ was prepared using the thin-film hydration method. The acid-base stability and thermal stability of the phage liposomes were determined using the double-layer plate method. The particle size, polydispersity coefficient, and Zeta potential of phage liposomes were measured using a laser particle size analyzer, while the structural characteristics of the phage liposomes were observed through transmission electron microscopy. The effect of phage liposomes on the viability of MAC-T cells was determined using the CCK-8 assay. The efficiency of cell entry and the intracellular and bactericidal activity of phage liposomes were measured using the plate counting method. The results showed that liposomes made from soybean lecithin, cholesterol, Tween 80, and octadecylamine were prepared in a ratio of 8:2:1:0.5, and phage liposomes (Lip-p-7) were created by encapsulating phage SDQ. The encapsulation rate of Lip-p-7 was greater than 50%, and its activity is stable in the range of temperature (4-60 ℃) and pH value (2-11). The average particle size of Lip-p-7 was 1 262.5 nm, and the shift of Zeta potential from neutral to negatively charged signified the successful encapsulation of phage SDQ by the liposome. The polydispersity index (PDI) was less than 0.7, suggesting a uniform dispersion. When stored at 4 ℃, the prepared Lip-p-7 could maintain its intact activity for up to 90 days, demonstrating good stability. Liposomes can effectively increase the number of phage SDQ entering cells and decrease the number of Staphylococcus aureus in MAC-T cells. In conclusion, this study provides a reference for the treatment of intracellular bacterial infections by using liposomes as a carrier to deliver bacteriophages into the cells.

The infection status of Rickettsia, Coxiella, and Bartonella in cat fleas Ctenocephalides felis collected from selected areas of China was investigated in this study. From January, 2019 to July, 2023, 294 fleas were collected from the body surface of pet dogs and cats across multiple Chinese regions. After preliminary morphological identification, the ITS1 sequences of fleas, gltA and ompA gene sequences of Rickettsia, IS1111 sequences of Coxiella, and gltA and ropB genes sequences of Bartonella were amplified with PCR. The positive products were sequenced and then subjected to sequence alignment and phylogenetic analysis. The results showed that all of 294 collected specimens were identified as cat flea C. felis. Results of molecular detection and genetic evolutionary analysis demonstrated that R. felis, C. burnetii and Bartonella were found in cat flea C. felis, with prevalence of 13.3% (39/294), 1.7% (5/294) and 25.9% (76/294), respectively. Among the infections of Bartonella, B. henselae was detected with a prevalence of 0.3% (1/294), B. koehlerae was 0.3% (1/294), B. clarridgeiae was 0.7% (2/294), and Bartonella spp. were 24.5% (72/294). In addition, co-infections involving R. felis and Bartonella spp. were identified. The cat flea C. felis was identified as the predominant flea species infesting pet cats and dogs in China, indicating potential of C. felis as a vector for multiple zoonotic agents. It was proven for the first time that cat flea C. felis carried C. burnetii. Rickettsia and Bartonella carried by C. felis in China showed significant geographical divergence and genetic diversity.

Previous studies have found that Mycobacterium smegmatis, which heterogeneically expresses the Rv3435c gene, can significantly inhibit the inflammatory expression of macrophages. In order to explore the function of Rv3435c gene in this process, second-generation sequencing was used to analyze the transcriptome differences of RAW264.7 cells infected by Rv3435c recombinant Mycobacterium smegmatis. Results were as follows: The mRNA expression levels of IL-1β, TNF-α and IL-6 at 6, 12, 24 and 48 h were detected by RT-qPCR, and 12 h with the highest expression levels was selected as the transcriptomic sequencing time point. The original data obtained by sequencing were analyzed for data quality and compared with the result statistics, and the data quality was confirmed to be good. The sample relationship analysis suggested that Rv3435c significantly changed the gene expression of mouse macrophages. A total of 14 077 differential genes were obtained by filtering and comparing the data, of which 278 were significantly up-regulated and 118 were significantly down-regulated. The differential genes obtained by GO annotation are mainly concentrated in immune system process, response to external stimulus, defense response and so on. KEGG pathway enrichment analysis was carried out for differential genes, mainly concentrated in environmental information processing, cellular processes, human diseases, and organic systems. Ten core genes were identified by protein interaction analysis, which were IL-6, IL-1β, CCL2, Mmp9, Spp1, Itgam, Cdc20, Ccna2, Kif11 and Ccnb2. Fluorescent quantitative PCR experiments were performed on the differential genes to verify the reliability of the transcriptome results. According to the selected DEGs and RT-qPCR results, we speculated that SPP1 was a potential target for the function of Rv3435c, and the results were verified by RT-qPCR, ELISA and Western blot. This study provides important data support for the function exploration of Rv3435c gene of Mycobacterium tuberculosis.

Metformin (MET) is a commonly used hypoglycemic drug in clinical practice, but its abuse or improper use has adverse effects on animal health. This article aims to explore the effects of high-dose MET on chicken, the growth metabolism of healthy chicks and its potential regulatory mechanisms. Eighty 1-day-old chicks were randomly divided into a control group (40 chicks, 20 males and 20 females) and an experimental group (40 chicks, 20 males and 20 females). Starting from 7 days old, the experimental group chicks were orally administered 0.6 g·kg^-1 MET-HCl (the dosage was calculated based on chicken weight, dissolved in 1 mL of physiological saline) daily for 23 consecutive days, the control group was orally administered an equal amount of physiological saline daily. The daily food intake of each group of chicks was recorded. On day 0 (the first day after hatching) and day 30, the weight of each group of chicks after fasting for 12 h was measured, and the average daily food intake (ADFI), average daily weight gain (ADG), and feed/gain ratio (F/G) were evaluated. We analyzed the levels of adenosine triphosphate (ATP) and growth related hormones in the serum of 30-day-old chicks; measured ATP levels and mitochondrial respiratory enzyme activities in the liver, kidneys, and muscles; detected the mRNA levels (in the liver, kidney, and muscle) and protein expressions (in muscle) of ATP synthase, liver kinase B1 (LKB1)/AMP-activated protein kinase α2 (AMPKα2) pathway and its downstream factors, as well as the mRNA and protein expressions of insulin (INS) and its receptor, insulin-like growth factor 1 (IGF1) and its receptor and their downstream factors using RT-PCR and Western blot. The results showed that high-dose MET can significantly reduce the chicken weight, decrease average daily weight gain, and improve the feed to weight ratio. MET reduced ATP, INS, growth hormone (GH), and IGF1 levels in the serum of chicks. Meanwhile, MET reduced ATP levels in the liver, kidneys, and muscles, as well as the activities of mitochondrial respiratory enzymes (NADH, cytochrome C oxidase, and ATP synthase). In addition, MET reduced the expression of ATP5A, activated the LKB1/AMPKα2 signaling pathway, inhibited the expressions of mammalian target of rapamycin (mTOR), INS and its receptor, IGF1 and its receptor, phosphatidylinositol-3-kinase (PI3K), protein kinase B (AKT), cyclin-dependent kinase 2 (CDK2), and cyclin E1, and increased the levels of p21 and p27. Our research findings suggest that high-dose MET may have a negative impact on the chicken growth metabolism by activating the LKB1/AMPKα2 signaling pathway, which provides some supporting evidence for avoiding the abuse or improper use of MET in clinical practice.

The aim of this study was to investigate the effect of oleic acid (OA) treatment on the secretion of ghrelin and its related mechanisms. MGN3-1 cells (gastric starvation tumor cells) were treated with OA and GPR120 inhibitor (AH-7614), respectively, and the following experiments were performed: 1) Determine the processing conditions for OA and group them together; 2) the amount of acylated starvation hormone (AG) was detected in the supernatant of the cells by ELISA; RT-qPCR was used to detect the gene expression of ghrelin, GOAT (growth hormone-releasing peptide O-acyltransferase), GPR120 (long-chain fatty acid receptor), and ACCα (acetyl-coenzyme A carboxylase) in the cells of each group; Western blot was used to detect the protein expression of GPR120, PKC, and IP3R; and flow cytometry was used to detect the intracellular Ca²⁺ concentration. The results showed that: 1) The low, medium and high concentrations of OA were determined to be 20, 40 and 60 μmol·L^-1, respectively, and the treatment time was 8 h. 2) The treatment of high concentration of OA highly significantly down-regulated cellular ghrelin and GOAT gene expression and decreased AG secretion (P < 0.01), significantly increased Ca²⁺ concentration (P < 0.01), significantly or highly significantly up-regulated gene expression of GPR120 and ACCα and protein expression of GPR120, IP3R and PKC (P < 0.01 or P < 0.05); AH-7614 treatment significantly or highly up-regulated the gene expression of GOAT and ghrelin and protein expression of IP3R, elevated AG secretion levels, and down-regulated GPR120 and ACCα gene expression and protein expression of GPR120 and PKC(P < 0.01 or P < 0.05); in contrast to the AH-7614 group, treatment with AH-7614 followed by OA resulted in significant or highly significant reduction in AG secretion levels and ghrelin and GOAT gene expression levels(P < 0.01 or P < 0.05), and a certain degree of up-regulation of the expression of GPR120 and ACCα genes as well as of the protein expression of GPR120, IP3R and PKC. In conclusion, the results suggest that OA treatment reduced ghrelin secretion in MGN3-1 cells. The possible mechanism is that OA treatment inhibition of GOAT activity through activation of GPR120 and its downstream PKC/IP3R/Ca²⁺signaling pathway on the one hand, and activating fatty acid β-oxidation through up-regulation of ACCα expression on the other hand, the two pathways synergistically inhibit GOAT activity, resulting in decreased cellular ghrelin secretion and reduced AG production.

The study aimed to investigate the mechanism of action by which the addition of dandelion to diets can enhance immune function in geese, using a network pharmacology approach. The Symmap database was used to collect the active ingredients and targets of dandelion. The Gene Cards database was used to search for immunosuppressive targets. The intersecting targets were subjected to protein-protein interaction (PPI) network analysis, Gene Ontology (GO) functional enrichment analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and molecular docking was performed between the core targets and the active components of dandelion. Subsequently, the outcomes of these analyses were used to conduct animal experiments to test the growth performance of the geese, immune-related indices, and relative expression of the core target mRNAs. The results showed that 190 key targets of dandelion to improve the immune function of geese were screened out through the database, and core targets, such as CASP3, IL-6, ACTB, and STAT3, were obtained by protein interaction analysis. GO and KEGG enrichment analyses showed that dandelion might be involved in the positive regulation of gene expression, response to hypoxia, negative regulation of apoptotic process, and carbohydrate metabolic process, and then exert immune regulatory effects through multiple signaling pathways such as FoxO signaling pathway, apoptosis, Toll-like receptor signaling pathway, MAPK signaling pathway, p53 signaling pathway, and NOD-like receptor signaling pathway. The results of molecular docking showed that the main active ingredients of dandelion had good docking activities with IL-6 and STAT3. The results of animal experiments showed that the addition of dandelion to the diet significantly increased growth performance and serum IgA and IgG levels, reduced IL-6 content, reduced the mRNA expression of IL-6 and STAT3, and increased the expression of FoxO1 and FoxO3 in the jejunal tissues of geese. This study demonstrated that adding dandelion to the diet could enhance the immune function of the organisms through the FoxO pathway, which provides a theoretical reference for the further development of dandelion as a feed supplement for geese.

Changes in the expression of apoptotic factors in endometrial epithelial cells of dairy cows in the state of high concentration of non-esterified fatty acids (NEFA) were explored to be influenced by forkhead box protein O1 (FoxO1). The effect of silent information regulator 1 (SIRT1) expression was evaluated by real-time fluorescence quantitative PCR and Western blot after the addition of the activator resveratrol (Resveratrol), the expression profile of FoxO1 was clarified in the endometrial epithelial cells of dairy cows. Changes in the expression of key factors of the FoxO1 pathway and apoptosis-associated factor proteins were examined after the addition of Resveratrol and high concentrations of NEFA. The protein and mRNA expression levels of SIRT1 were up-regulated (P < 0.01) and Ac-FoxO1 protein expression levels were down-regulated (P < 0.01) by the addition of Resveratrol compared with the control group; Caspase-3 protein expression levels were not significantly affected, showing a down-regulation trend but not statistically different (P>0.05); the protein expression levels of Bcl-2 and Bcl-xl were up-regulated (P < 0.01); the protein expression levels of Bax and Bax/Bcl-2 were down-regulated (P < 0.05). Compared with the NEFA group, in the NEFA+Resveratrol group, IGF-1, SIRT1 and p-AKT protein expression levels were up-regulated (P < 0.01); FoxO1, p-FoxO1, Ac-FoxO1, FoxO1/p-FoxO1, AKT and AKT/p-AKT protein expression levels were down-regulated (P < 0.01). Bcl-xl and Bcl-2 protein expression levels were upregulated (P < 0.01), Caspase-3, Bax and Bax/Bcl-2 protein expression levels were downregulated (P < 0.01). The results of laser confocal assay showed that the expression level of FoxO1 was suppressed in the NEFA+Resveratrol group compared with the NEFA group (P < 0.01). SIRT1 expression can be effectively activated by Resveratrol, FoxO1 expression was suppressed, and the expression level of apoptotic factors in the endometrial epithelial cells of dairy cows was reduced in the state of high concentration of NEFA.

This study aims to utilize the CRISPR/Cas9 gene editing technology to investigate the role of p5cr (pyrroline-5-carboxylate reductase) in the synthesis of swainsonine by Metarhizium anisopliae, providing a theoretical basis for the biosynthesis and industrial production of swainsonine. In this study, Metarhizium anisopliae was used as the object of study, qRT-PCR method was used to detect the gene expression levels of p5cr, sdh, pks, and p450 in the swainsonine biosynthetic pathway. The correlation analysis was conducted on the production of swainsonine and the expression levels of related genes of Metarhizium anisopliae after 1 to 7 days of fermentation. The p5cr gene was selected as the target gene. CRISPR/Cas9 gene editing technology was applied to knock out and overexpress the catalytic gene p5cr, in order to validate the correlation between the p5cr gene and swainsonine synthesis in Metarhizium anisopliae. The results demonstrated a strong positive correlation between swainsonine production and the expression level of the pks gene, while extremely strong correlations (P < 0.001) were observed with the expression levels of the p5cr, sdh, and p450 genes. Knockout and overexpression of the p5cr gene in Metarhizium anisopliae led to a significant decrease (P < 0.01) and increase (P < 0.001) in swainsonine yield, respectively. This study reveals that the p5cr gene exhibits an extremely strong association with swainsonine biosynthesis in Metarhizium anisopliae and plays a crucial regulatory role in its biosynthetic pathway. These findings provide a foundational framework for further investigations into the function and regulatory role of the p5cr gene.

This study aimed to explore the effects of curcumin (Curcumin, Cur) on the inflammatory response of lipopolysaccharide (LPS)-induced bovine mammary epithelial cells (MAC-T) from the ferroptosis pathway. MAC-T cells in the logarithmic growth phase were randomly grouped according to the experimental requirements and underwent corresponding treatments, with three replicates in each group. The MTT method was used to determine the effects of different concentrations of LPS/Cur on the survival rate of MAC-T cells; RT-PCR was employed to detect the transcription levels of inflammation-related genes (TNF-α, IL-6, IL-1β, iNOS, CXCL2, CCL2) and ferroptosis-related genes (PTGS2, SLC7A11, GPX4, FTH1, ACSL4); BODIPY 581/591 C11 staining was utilized to detect the lipid peroxidation level of cells; kits were used to measure the contents of MDA, LPO, GSH, and the GSH/GSSG ratio in cells; Ferro-Orange staining and the Fe²⁺ detection kit were used to determine the Fe²⁺ content in cells; ELISA kits were used to detect the concentrations of TNF-α, IL-6, and IL-1β in cells; Western blot was used to detect the relative expression levels of TNF-α, IL-6, and IL-1β proteins. The results revealed that compared with the CON group, treatments with 10 μg·mL^-1 LPS or 20 μmol·L^-1 Cur had no significant effect on the survival rate of MAC-T cells. After LPS treatment, the expression levels of pro-inflammatory related genes (TNF-α, IL-6, IL-1β, iNOS, CXCL2, CCL2), ferroptosis-related genes (PTGS2, ACSL4), and the related protein (ACSL4) in cells were significantly elevated (P < 0.01); the expression levels of anti-ferroptosis genes (SLC7A11, GPX4, FTH1) and protein (GPX4) were significantly decreased (P < 0.01); the MDA content and LPO concentration were all significantly increased (P < 0.01); the GSH content and GSH/GSSG ratio were both extremely decreased (P < 0.01); the Fe²⁺ content was extremely significantly increased (P < 0.01); the concentrations and protein expression levels of TNF-α, IL-6, and IL-1β were all extremely increased (P < 0.01). Compared with the LPS alone treatment group, the expression levels of pro-inflammatory related genes, ferroptosis-related genes and related proteins in the LPS and Cur-H co-treatment group were extremelydecreased (P < 0.01); the expression levels of anti-ferroptosis genes and proteins were extremely increased (P < 0.01); the lipid peroxidation level, MDA content, and LPO concentration were all extremely decreased (P < 0.01); the GSH content and GSH/GSSG ratio were both significantly increased (P < 0.01); the Fe²⁺ content was significantly decreased (P < 0.01); the concentrations and protein expression levels of TNF-α, IL-6, and IL-1β were all extremely decreased (P < 0.01). Additionally, the ferroptosis activator Erastin significantly inhibited the alleviating effect of Cur on LPS-induced inflammation. In conclusion, Cur can alleviate LPS-induced inflammatory responses in MAC-T cells by inhibiting ferroptosis.

The aim of this study was to investigate the regulatory effect of betaine on oxidative stress in chicken embryo liver cells (CEL). The study was carried out to explore the potential mechanism of betaine on oxidative stress and lipid metabolism in CEL with oxidative stress model induced by oleic acid. Cells were divided into control (Con) group, oleic acid (OA) group and OA+ betaine (OA+BT) groups (the betaine concentrations were 4, 6, 8 and 10 mmol·L^-1, respectively) according to different treatments, with 6 replicates in each group. After 24 h treatment, cell proliferation activity was detected by CCK-8 method. Oil red O staining was used to analyze the histopathological changes of the cells, and the intracellular reactive oxygen species (ROS) level was measured, and then cell pellets were collected to determine the antioxidant related indicators. The results showed that 4-10 mmol·L^-1 betaine had no significant effect on the proliferation activity of CEL cells (P>0.05). Oil red O results showed that a large number of red lipid droplets appeared in CEL cells in the OA group, and the contents of total cholesterol (TC) and triglyceride (TG) were higher than those in the Con group (P < 0.05). However, compared with the OA group, red lipid droplets in CEL cells were decreased in the 4 mmol·L^-1 OA+BT group, and the TG content was significantly decreased (P < 0.05). In addition, when compared with the Con group, the levels of malondialdehyde (MDA) and ROS were significantly increased (P < 0.05), and total antioxidant capacity (T-AOC) tended to decrease in the OA group (P=0.083). After adding 4 mmol·L^-1 betaine, the T-AOC level was higher than that in OA group, and the MDA content was significantly lower than that of OA group (P < 0.05). RT-PCR results showed that the expression levels of antioxidant related genes including Nrf2, HO-1 and NQO1 in the OA group were significantly lower than those in the Con group (P < 0.05). Compared with the OA group, the addition of 4 mmol·L^-1 betaine significantly increased the mRNA abundance of Nrf2, HO-1, NQO1, CAT, SOD-1 and GPX-1 (P < 0.05). The present study showed that 4 mmol·L^-1 betaine alleviated oleic acid-induced lipid deposition and oxidative stress in CEL cells, which will provide a theoretical basis for the application of betaine in the livestock and poultry industry.

Mycobacterium avium subsp. paratuberculosis (MAP) is the pathogen causing Johne's disease in ruminants and brings inestimable economic losses to the global livestock industry every year. Due to the difficulty in treatment, timely diagnosis and early culling of diseased animals are currently the main means of MAP prevention and control. The gold-standard culture detection cycle for MAP is long, while the qPCR technology, which directly detects nucleic acids in samples, can quickly identify the pathogen and is currently widely used. This study aims to establish a triplex TaqMan qPCR method for MAP detection targeting non-insertion sequence (IS) of MAP, which can improve the sensitivity and specificity of detection. Design primer probes for the MAP2765c, F57 and hspX genes in the MAP genome. Taking the MAP strains isolated in this laboratory as a control, a triple qPCR detection system was established. A total of 181 samples of dairy and beef cattle were clinically collected, and a compliance test was carried out using the triple qPCR method and the national standard method (GB/T 27637—2011). The results showed that the sensitivity of the triple qPCR method established in this study could reach 10 copies·μL^-1; the coefficient of variation of the repeated results was all less than 5%; in the specificity experiment, only MAP nucleic acid showed an amplification curve; the R² of the standard curves were all greater than 0.999 and the amplification efficiency was between 97% and 100%; the positive rate of clinical samples detected by the triple qPCR method was 22.65% (41/181), which was higher than that of the national standard method (15.47%), and the detection coincidence rate of the two methods was 76.28% (138/181); according to the detection requirements of the ranch, the samples were classified and analyzed: the detection rates of the national standard method and the triple qPCR method in "high-MAP-risk samples" were 25% (17/68) and 44.12% (30/68) respectively, while the positive rates of the two methods for detecting samples with lower MAP risk were both 9.73% (11/113). The triple qPCR method designed in this study has good sensitivity, specificity and repeatability, high linearity and quantitative accuracy, and can detect MAP more sensitively in high-MAP-risk samples than the national standard method. The research results provide an optimal solution for dairy and beef cattle farms to carry out monitoring, early warning, control and purification of paratuberculosis.