Pluripotent stem cells (PSCs) have unlimited proliferation potential and the ability to differentiate into various types of tissue cells, making them the focus of current stem cell research. The research on PSCs in livestock not only holds fundamental theoretical significance, but also has important implications for application in animal breeding, biomedicine, food production and other fields. This review provides a comprehensive summary of the culture conditions, identification methods, and maintenance of pluripotency in pluripotent stem cells. Furthermore, it outlines the research progress of livestock pluripotent stem cells and provides a prospective outlook on its application prospects in order to offer references in further study of livestock pluripotent stem cells.

Spermatogenesis, oogenesis, and the development of a fertilized egg, are all crucial in mammalian reproduction. Currently, there is no shortage of studies on the maturation and development of mammalian oocytes, but the role of granulosa cells in the development of oocytes remains to be explored in depth. In female mammals, early ovarian granulosa cells are developed from the ovarian reticulum and ovarian epidermal cells; thereafter, the precursor granulosa cell stage surrounds the oocyte, providing nutrients and communicates information for the oocyte through gap junctions, and regulating oocyte maturation by relying on the regulation of reproduction hormones (such as AMH, FSH, and LH). In this paper, the origin of mammalian ovarian granulosa cells, the differentiation of granulosa cells at each stage of follicle development, and their functional mechanism during oocyte maturation were reviewed in order to provide help for the further study of the biological functions of mammalian ovarian granulosa cells, and provide optimization ideas for the in vitro oocyte maturation culture system.

Three-dimensional (3D) cell culture technology can accurately simulate the actual microenvironment of cells and reproduce the interactions between cells, so that the behavior characteristics of cells are more similar to the living state in vivo. Therefore, 3D cell culture technology has been widely used in the research of oocyte and embryo development in domestic animals, which has greatly improved the quality of oocyte and embryo development in vitro, and thus promoted the consistency and reliability of research results. This paper discusses the development of 3D in vitro culture from three aspects: materials, substrates and methods, and summarizes new classification methods based on the biological characteristics of cells, culture conditions, scaffold materials used, interactions between cells and the design of culture systems, namely: cell aggregation system, construction of structural scaffolds, construction of bionic scaffolds, and multidisciplinary intersections (extension). In the future, through the improvement and optimization of 3D model cell culture technology, it will provide important technical support and new possibilities for the research of in vitro culture of different domestic oocytes and embryonic development.

Ovarian granulosa cells (GCs) are the basic guarantee of follicle development in female animals. In the past, it was widely believed that apoptosis of GCs could regulate follicle development. However, recent studies have found that GCs autophagy can also regulate follicle development. In the animal husbandry industry, the quality of follicle development is closely related to the reproductive rate of female animals, which directly affects the economic benefits of livestock farms. At present, more and more studies have shown that miRNAs are one of the factors regulating autophagy in GCs. Therefore, this article will review the research progress on the origin, structure and function of GCs, the importance of GCs autophagy on the ovary, the miRNAs associated with GCs autophagy of the ovary and their regulatory effects on follicle development, in order to provide references for future related studies.

Although feed antibiotics can alleviate the decrease in feed intake and production performance of animals caused by inflammatory reactions, they can also lead to the occurrence of gut microbe drug-resistance and antibiotic residues in the animal body, posing safety risks. Therefore, the development of green and effective alternatives to feed antibiotics has become a top priority in the livestock industry. Glucosamine is a natural monosaccharide that can be obtained from chitin via enzymatic hydrolysis or strong acid hydrolysis. In recent years, research has demonstrated that glucosamine has anti-inflammatory, antioxidant, immune-enhancing, and reproductive performance enhancing functions, and is non-toxic and has no side effects. It is expected to become an ideal substitute for feed antibiotics. This article reviews the preparation methods, absorption and metabolism, function, and feeding effect of glucosamine on animals, aiming to provide a reference for its application in animal production.

Adverse early life experiences affect the developing nervous system, and shape the offspring's adult social behavior pattern. Maternal separation is an early life stress that lactating offspring often face, which has a lasting impact on their psychological and physiological development and can lead to neuroendocrine system disorders in offspring, and increases the risk of abnormal behavior and mental illness in the offspring's adult. Environmental enrichment is a therapeutic strategy used to improve the quality of life of captive animals, stimulate animal emotional expression, and reverse neurological damage or behavioral abnormalities in early stressed animals. The behavioral plasticity of offspring induced by early stress can be restored by environmental enrichment intervention in later life (adolescence or adulthood). Neurobiological changes and epigenetic modification are the main molecular mechanisms by which environmental enrichment reverses the abnormal behavior of early stressed offspring. Therefore, this paper reviews the abnormal behavior of offspring caused by maternal separation, the effects of environmental enrichment on alleviating the abnormal behavior of maternal separation, and the molecular mechanisms involved in regulating environmental enrichment effects. In order to provide effective intervention strategies for early life stress offspring abnormal behavior, and provide scientific basis for improving the welfare level of captive animals.

The protozoa of Apicomplexa include Cryptosporidium, Toxoplasma, Plasmodium, Eimeria and other parasites related to human and animal health. They can infect a wide range of hosts and are not easy to be found by the immune system, causing acute and chronic diseases. Due to the ethical and practical problems of its research in natural reservoir, as well as the complex life circle of apicomplexan protozoa and the difficulty of animal models, people's understanding of the biological characteristics of these pathogens is still limited. The recently used 3D system has promoted and expanded the research of apicomplexan protozoa, enabling us to simulate host pathogen interaction, providing a platform for vaccine development, drug-screening, parasitic biology research and other applications. This review summarizes the progress in the application of 3D cell culture system in the study of apicomplexan protozoa, with a view to providing reference for subsequent related researches.

Infection with Decapod iridescent virus 1 (DIV1) is a newly emerged crustacean viral disease caused by DIV1. The virus consists of two original isolates, namely shrimp hemocyte iridescent virus (SHIV 20141215) and Cherax quadricarinatus iridovirus (CQIV CN01). DIV1 is fast spreading, with a wide host range and high lethality, and it has become widely prevalent in shrimp and crab aquaculture in recent years, causing significant economic losses to the industry. Currently, various diagnostic techniques have been developed for infection with DIV1. This paper provides a summary of these techniques, with the aim of providing reference for the accurate diagnosis, prevention and control of this disease.

Brucellosis is a global zoonosis caused by Brucella infection. In recent years, the situation of brucellosis prevention and control is grim in China, posing a great threat to the cattle and sheep farming industry and public health security. Innate immunity serves as the first line of defense against pathogenic mocrobial infections and is crucial for maintaining homeostasis of the organism. Brucella can modulate innate immunity signaling pathways through a variety of strategies to establish persistent infections and also cause inflammatory diseases such as arthritis, hepatitis, encephalitis, and abortion. A comprehensive and in-depth understanding of the molecular mechanisms of Brucella interactions with innate immunity signaling pathways may provide new ideas for the development of safe and effective vaccines or drugs. Therefore, this paper reviews the roles and mechanisms of innate immunity signaling pathways such as Toll-like receptors(TLRs), cyclic guanosine adenylate synthetase(cGAS), stimulator of interferon genes(STING), NOD-like receptors(NLRs), AIM2-like receptors(ALRs), C-type lectin receptors(CLRs) and RIG-I-like receptors(RLRs) in the defense against Brucella infections, and summarizes the strategies of Brucella evade or hijacking these signaling pathways.

Brucellosis is an important zoonotic disease that is prevalent worldwide, while causing numerous economic losses to animal husbandry, it also poses a serious threat to human health and public health security. Brucella can use various strategies for immune system evasion, not only resisting the bactericidal effect of phagocytic cells, but also creating a favorable microenvironment in host cells for its survival, ultimately leading to persistent chronic infection. These characteristics pose significant challenges to the prevention and control of brucellosis. As a prerequisite for further understanding and preventing brucellosis, the virulence process, intracellular survival, and immune evasion of Brucella have always been the focus of attention. So, this work reviewed the Brucella pathogenesis research progress from perspectives of virulence factors, intracellular survival, and immune evasion mechanism, providing reference for future development of novel safe, efficient prevention and control products by elucidating Brucella pathogenesis.

Staphylococcus aureus (S. aureus) is an important zoonotic pathogen capable of causing various infectious diseases, posing a serious threat to human and animal health. It produces multiple virulence factors such as toxins, adhesion proteins, and immune evasion factors, and possesses a complex regulatory network for virulence. Due to issues like antibiotic resistance faced by traditional antibiotic treatments, the development of novel drugs aimed at reducing or eliminating the virulence of S. aureus has become a new direction in antimicrobial drug development. This article provides a comprehensive review of the latest advances in the regulation of S. aureus virulence and the development of antivirulence drugs, offering a reference and scientific basis for understanding the pathogenic mechanisms of S. aureus and further research on antivirulence drugs.

The CRISPR/Cas system is an adaptive immune system widely present in bacteria and archaea. It targets and cleaves specific nucleic acid sequences using RNA-guided nucleases to defend against foreign nucleic acid invasion. Based on the composition of effector protein complexes, the CRISPR/Cas system can be divided into two main classes: Class I systems utilize effector complexes composed of multiple Cas proteins, acting together with crRNA to exert target cleavage function, while Class II systems consist of single, multi-domain protein effector modules. Among them, the single-component effector protein-mediated Class II CRISPR gene editing technology is relatively straightforward and has been widely applied in synthetic biology fields such as bacterial gene expression regulation, genetic modification, metabolic pathway optimization, and pathogenic microorganism detection. Based on the differences in the Cas effector protein nuclease domains, Class II systems can further be categorized into subtypes II, V, and VI, each exhibiting differences in their applications in bacterial synthetic biology. This article reviews the immunological mechanisms, classification, and characteristics of Class II CRISPR-Cas systems, as well as their current applications and the latest developments in industrial bacteria, aiming to provide a reference for future research in bacterial synthetic biology to select, optimize, and explore more CRISPR/Cas systems.

This paper describes the principle, process and characteristics of loop-mediated isothermal amplification (LAMP) technology and its application in the detection of drug resistance genes.LAMP technology has been widely used in the field of molecular biology due to its high efficiency, specificity, sensitivity and simplicity. In addition to the basic LAMP detection methods, some novel LAMP technologies, such as IMS-LAMP, LAMP coupled with CRISPR/Cas system, LAMP coupled with Argonaute system, LAMP sensors, and LAMP coupled with microfluidic chip, are also introduced in this paper. In addition, for the detection of drug-resistant genes, LAMP technology shows a good application prospect in the detection of drug resistance genes, which provides an effective tool for rapid and accurate detection of drug resistance genes.

Antimicrobial resistance can increase difficult in treatment of infectious diseases, medical burden and mortality, posing a threat to global human and animal health. In the background of reducing and banning antimicrobials, it is urgent to develop new antimicrobials, alternatives, as well as antimicrobials adjuvants. It has been demonstrated that Traditional Chinese medicine and its active components have significant advantages in controlling of antimicrobial resistance in combination with antimicrobials and has broad prospects for development and application. In this study, we reviewed the latest research progress on traditional Chinese medicine and its active components for controlling antimicrobial-resistant bacteria through acting on drug-resistant plasmids, efflux pumps, cell membrane permeability, drug-resistant enzymes, drug-resistant genes and biofilms since 2017 when China banned the use of colistin as a growth promoter (Announcement No. 2428 of the Ministry of Agriculture of China), to provide reference for the development of effective new antimicrobials or their synergist against antimicrobial-resistant bacteria.

Optical coherence tomography (OCT) is a non-contact, non-invasive imaging technique widely used in human clinical ophthalmology and research. In recent years, with the rapid advancement of veterinary clinical medicine, OCT detection technology has been applied gradually in small animal medicine, comparative ophthalmology, and wildlife medicine. This article reviews the application and progress of OCT technology in the diagnosis and research of various ophthalmic diseases in different animals, aiming to provide meaningful guidance and reference for the clinical diagnosis of veterinary ophthalmic diseases and comparative medicine.

The study aimed to improve and optimize the annotation information of pig genome. In this study, the RNA-Seq technology was used, novel transcript was predicted, alternative splicing events, and SNP mutation sites in porcine myocardium were analyzed. Specifically, the myocardium tissue samples from six-month old healthy castrated Tibetan and Landrace boars (3 for each) were collected respectively, then novel transcripts, alternative splicing events and SNP variation sites were analyzed and their GO and KEGG enrichment analysis were performed based on RNA-Seq sequencing and bioinformatics. The results showed that a total of 930 new genes and 5 993 new transcripts were found. The enrichment analysis showed that the virion assembly and viral life cycle terms, NF-kappa B and Hedgehog signaling pathways were significantly enriched. Skipped exon (SE) accounted for the highest proportion of alternative splicing types, and 159 significant differential splicing genes were screened, mainly enriched in contractile fiber, AMPK signaling pathway and circadian rhythm. The variation loci of Tibetan pigs were greater than that of Landrace pigs, and the number of SILENT was 311 117 loci. There were 418 136 and 356 478 variation loci in EXON and UTR_3_PRIME, respectively. Under extreme environment, Tibetan pig myocardial tissue may regulate immune through NF-κ B and AMPK signaling pathways, participate in cell metabolism and apoptosis, improve myocardial injury, and inhibit hypoxia stress.

This study aimed to investigate the regulatory impact of TMEM182 on the proliferation and differentiation of porcine skeletal muscle satellite cells. This study utilized 3 each of healthy castrated boars, Mashen pigs and Large White pigs, housed under the same conditions for 180 days of age, were selected for collection of each tissue; one-week-old piglets were selected for isolation of porcine skeletal muscle satellite cells; qRT-PCR was used to investigate the expression profile of TMEM182 in different pig tissues, compare expression patterns in muscle tissues across various pig species, and analyze the temporal expression changes in skeletal muscle satellite cells during different stages of differentiation. By overexpressing TMEM182 (OE-TMEM182) and interfering with TMEM182 (si-TMEM182) in satellite cells, we utilized qRT-PCR to analyze the expression changes of proliferation marker genes and conducted EdU experiments to observe changes in the number of EdU-positive cells. Following the induction of myogenic differentiation in porcine skeletal muscle satellite cells, various techniques including qRT-PCR, Western blot, and immunofluorescence staining techniques were utilized to examine alterations in the mRNA and protein expression levels of genes associated with myogenic differentiation, as well as their impact on myotube fusion. The study results showed that TMEM182 had high expression levels in muscle tissues but was nearly absent in other tissues. In comparison to Mashen pigs, TMEM182 was notably more expressed in Large White pigs tissues (P < 0.05). As satellite cells differentiation progressed, at the 4th and 5th days of differentiation, there was an increase in TMEM182 expression. The expression level was the lowest on the 0th day of differentiation. However, at the 6th day of differentiation, there was a decrease in TMEM182 expression. Conversely, at the 7th day of differentiation, TMEM182 expression was at its peak(P < 0.05). The results of the intercalated proteins showed that the expression of CDH15, TMEM25, HS3ST1, and SEC11A was highly significantly reduced in the OE-TMEM182 group (P < 0.01), the expression of VWC2L was significantly reduced (P < 0.05).During the proliferation phase, overexpression of TMEM182 significantly down-regulated the expression of CDK1, PCNA, and Ki67 (P < 0.01), together with the number of EdU-positive cells was markedly decreased (P < 0.01). Conversely, interfering with TMEM182 resulted in an highly upregulation the expressions of CDK4, CDK1 (P < 0.01) and significantly upregulated the expression of Ki67 (P < 0.05), along with an increase in EdU-positive cells (P < 0.01). Furthermore, during myogenic differentiation, overexpression of TMEM182 led to a highly significant reduction in the expression levels of MyoG, MyHC (P < 0.01) and the expression level of MyoD was significantly decreased (P < 0.05). After interfering with TMEM182, the expression of MyoD, MyoG and MyHC were highly significantly increased (P < 0.01) and the expression of Myf5 was significantly increased (P < 0.05). At the same time, TMEM182 might regulate the proliferation and differentiation of porcine satellite cells by inhibiting PI3K-Akt signaling pathway. The study results indicate the crucial role of TMEM182 as a negative regulator in the proliferation and differentiation of porcine skeletal muscle satellite cells. This gene could be a key candidate gene influencing the growth and development of porcine skeletal muscle.

The aim of this study was to study the serum metabolomics of high and low resilience groups of porcine reprodective and respiratory syndrome (PRRS, blue ear disease) in Min pig, and to provide reference for the prevention and control of blue ear disease. In this study, from the perspective of metabolomics, 15 Min pigs aged 28 days with similar body weights were selected, the same feeding environment, and the same exposure to porcine reproductive and respiratory syndrome virus (PRRSV). Among them, 5 pigs were classified into the high-resilience group and 5 into the low-resilience group. Non-targeted metabolomic analysis of serum samples was performed using liquid chromatography-mass spectrometry (LC-MS). Differential metabolites in the serum of Min pigs with different resilience levels were screened through orthogonal partial least squares discriminant analysis (OPLS-DA) and cluster analysis, followed by pathway enrichment analysis. The results identified 39 differential metabolites between resilience groups (P < 0.05). Analysis revealed that sphingolipids, indigo carmine, arachidonic acid, and L-threonine were potential biomarkers. Further analysis indicated that pathways related to valine, leucine, isoleucine biosynthesis, and arachidonic acid metabolism might alleviate inflammatory responses by modulating cytokine levels and inflammatory mediators, thereby affecting resilience. In summary, this study identified sphingomyelin, indigo, arachidonic acid, and other metabolites associated with resilience to blue ear disease. Additionally, it provided a preliminary interpretation of the mechanisms underlying differences in resilience from a metabolic perspective. This research offers theoretical insights for both the prevention and control of blue ear disease and the breeding of Min pigs with high resilience.

The study aimed to generate gene edited embryos of single nucleotide polymorphisms (SNPs) of platelet-derived growth factor D (PDGFD) gene in Altay sheep using CRISPR/Cas9 technology. Five sgRNAs (PDGFD-314-sgRNA2, PDGFD-314-sgRNA5, PDGFD-410-sgRNA, PDGFD-397-sgRNA2, PDGFD-397-sgRNA7) were designed and synthesized based on 3 SNP loci related to lipid tail traits of PDGFD gene in Altay sheep, combined with Cas9 nuclease for in vitro activity analysis. Electroporation of Cas9-sgRNA ribonucleoprotein (RNP) in Altay sheep embryos 4-5 h after in vitro fertilization, 35-40 embryos per group, triple repetition. The blastocyst rate was counted on the 7th day of in vitro culture, and the genomic target gene loci were amplified and sequenced. PDGFD and MSTN genes (non-PDGFD gene) homologous recombination gene editing materials were electroporated into zygotes, and the blastocyst rate and PDGFD sgRNA off-target efficiency were analyzed on the 7th day of culture. All 5 sgRNAs had high cleavage activity in vitro. There was no significant difference in the survival rate and cleavage rate between the electroporated zygotes and control groups (P>0.05), but the blastocyst rate in the electroporation group was significantly lower than that in the control group (P < 0.05). The editing efficiency of PDGFD-397-sgRNA2 was higher than other sgRNAs in embryos. There were 19 off-target sites with one-base mismatch of PDGFD-397-sgRNA2 in the sheep genome, and the genes covering the exon region included CFDP1, APOO, PCDH11X, etc. There were 31 2-base mismatch sites, and TIMM17B, C1H1orf228 and TLL1 genes covered the exon region. PDGFD and MSTN genes homologous recombination editing materials were electroperforated respectively, there was no significant difference in the survival rate and cleavage rate between the two groups (P>0.05), but the blastocyst rate of the PDGFD group was significantly lower than that of the MSTN group (P < 0.05). The results of this study showed that CRISPR/Cas9 editing of SNP locus related to tail fat trait of PDGFD gene in Altay sheep significantly reduced the blastocyst rate of in vitro embryos, suggesting that PDGFD gene may affect the early embryo development of sheep. In conclusion, PDGFD gene may play an important role in the early development of ovine embryo.

The aim of this study was to investigate the effect of heat stress on m⁶A methylation modification in Hu sheep ovarian granulosa cells, so as to lay the foundation for revealing the regulatory mechanism of m⁶A methylation modification on the development and function of Hu sheep ovarian granulosa cells under heat stress. In this study, fresh ovaries of 2-year-old Hu ewes were collected, and granulosa cells in 3-5 mm follicles on the surface of ovaries were collected. They were randomly divided into two groups: control group (37 ℃) and heat stress group (42 ℃, 2 h per day for 3 consecutive days). The m⁶A peak was identified by methylated RNA immunoprecipitation sequencing (MeRIP-seq), and the gene expression data were obtained and analyzed. There were 135 significantly different m⁶A peaks in MeRIP-seq between the control group and the heat stress group, which were mapped to 130 differentially methylated genes, mainly enriched in the PI3K-Akt signaling pathway, glutathione metabolism and Wnt signaling pathway. RNA-seq identified 359 significantly differentially expressed genes, which were mainly enriched in steroid synthesis, cholesterol biosynthesis and other signaling pathways. The combined analysis of MeRIP-seq and RNA-seq data showed that there were significant differences in m⁶A modification and gene expression levels of five genes, DTX3L, MAGEF1, MAN1C1, CCDC77 and RAD51, which were mainly enriched in the Notch signaling pathway. This study results shows that heat stress can change the mRNA m⁶A modification level of Hu sheep ovarian granulosa cells, which may eventually affect cell proliferation, apoptosis, estrogen synthesis and other related pathways, providing a theoretical basis for further exploring the specific mechanism of m⁶A modification under heat stress.

The study aimed to investigate the expression pattern of miR-215-5p in tissues of Leizhou black ducks and verify the regulatory effect of miR-215-5p on the proliferation and apoptosis of follicular granulosa cells. Three 43-week-old egg-laying and three 51-week-old non-laying Leizhou black ducks with similar body weight and health conditions were selected. The expression levels of miR-215-5p in different tissues were detected using RT-qPCR. By constructing mimics and interference fragment vectors and transfecting them into follicular granulosa cells, cell proliferation and apoptosis were detected using CCK-8 and flow cytometry techniques, with each group containing 3 replicates. The results showed that the expression of miR-215-5p in egg-laying Leizhou black ducks was generally higher than that in non-laying ducks, with relatively high expression in the ovary, infundibulum, and breast leg muscles, and relatively low expression in the funnel part, uterus, and heart. CCK-8 and EdU assays showed that miR-215-5p inhibited the proliferation of follicular granulosa cells; flow cytometry detected that miR-215-5p promoted the apoptosis of follicular granulosa cells; RT-qPCR detection of proliferation and apoptosis marker genes expression showed that after transfection with miR-215-5p mimics, the expression levels of CCND1 and CDK2 were both significantly decreased (P < 0.01), and Caspase-3 was significantly increased (P < 0.01); while after transfection with miR-215-5p interference fragments, CCND1 was significantly increased (P < 0.05), CDK2 was significantly increased (P < 0.01), Caspase-3 was significantly decreased (P < 0.01), and TP63 was significantly decreased (P < 0.05). miR-215-5p in Leizhou black duck has the ability to restrain the proliferation of follicular granulosa cells and promote their apoptosis.

The purpose of this study was to establish Mengshan cattle induced pluripotent stem cells that can be stably passaged and meet the criteria for pluripotency. Initially, fibroblasts were isolated from the ear skin of adult Mengshan cattle bulls using enzyme digestion, and cell viability and growth curves were assessed. Meanwhile, a doxycycline-inducible, non-viral reprogramming system was used to induce the reprogramming of Mengshan cattle fibroblasts in 3i/LIF and NBFR culture systems. The pluripotency of the resulting induced pluripotent stem cells was subsequently evaluated. The results showed that the Mengshan cattle ear fibroblasts obtained in this study exhibited normal proliferation and good condition. The induced pluripotent stem cells from Mengshan cattle displayed a smooth-edged, dome-shaped colony morphology. Alkaline phosphatase staining was positive, and the expression of pluripotency marker genes was significantly higher than that of fibroblasts (P < 0.001). Immunofluorescence staining for pluripotency proteins OCT4, SOX2, and NANOG was positive, demonstrating a pluripotent state similar to that observed in bovine embryonic stem cells and induced pluripotent stem cells reported in previous studies. This achievement provides a new direction for the research and application of bovine induced pluripotent stem cells and helps to further explore their potential in the field of agricultural biotechnology. It lays a material foundation for in vitro gamete production and the restoration of endangered or extinct local cattle breeds.

To investigate the genetic basis of the body traits in Dezhou donkeys, this study collected blood samples from 139 healthy female Dezhou donkeys aged 2-3 years old as experimental materials. Body traits of the experimental population were measured, and descriptive statistical analyses were conducted. All individuals were genotyped using the 'Molbreeding Donkey No. 1' 40K liquid chip. The mixed linear model was used to perform a genome-wide association study (GWAS) on 11 body traits, and functional annotation and pathway enrichment analysis was performed on candidate genes. The fine mapping and linkage disequilibrium analyses was combined to detect the potential causal SNP loci associated with body traits. The GWAS results revealed 3 SNPs at the genome-wide significance level, 20 SNPs at the genome-wide suggestive level, and 41 candidate genes were identified as significantly associated with body traits. The GO and KEGG enrichment analysis indicated that candidate genes were significantly enriched in biological processes related to osteogenic differentiation, skeletal muscle development and lipid metabolism. Fine mapping and linkage disequilibrium analysis detected 4 significant SNPs with strong linkage disequilibrium within the candidate region on chromosome 16. The results of this study lay the foundation for the analysis of the genetic mechanism of body traits in Dezhou donkeys, and provide a reference for the application of molecular marker-assisted selection in donkey breeding.

In order to investigate the factors affecting the freezing tolerance of boar sperm, this study collected semen from 10 healthy Landrace boars aged 18-24 months old. The sperm motility, acrosome and membrane integrity, ATP levels, membrane fluidity, and fatty acid content of the semen in the good and poor freezing tolerance groups were detected after freezing and thawing. The results showed that the sperm total motility, progressive motility, curvilinear velocity, straight-line velocity, acrosome and membrane integrity, ATP level, and high membrane fluidity level of the thawed semen in the good freezing tolerance group were significantly higher than those in the poor freezing tolerance group (P < 0.05). The content of long-chain fatty acids such as oleic acid and linoleic acid in the sperm of the good freezing tolerance group was also significantly higher than that of the poor freezing tolerance group (P < 0.05). The sperm motility, progressive motility, linear velocity, frequency, acrosome integrity and membrane integrity, ATP level, and high membrane fluidity level in the treatment with 0.1% lipid mixture addition into semen of poor freezing tolerance group were significantly increased when compared to the those in control group (P < 0.05). In summary, this study detected boar sperm parameters before and after thawing, and found that the difference in freezing resistance of boar sperm may be due to the different content of long-chain fatty acids in sperm. Moreover, supplementing exogenous fatty acids can further improve the quality of boar post-thaw sperm, thus, it provided experimental basis for further improving the boar frozen semen production system.

This study aimed to investigate the effect of cyanidin-3-rutinoside added to the diluent on the cryopreservation of dairy sheep semen and to explore its mechanism of action. Fresh semen was collected from 6 East Friensian rams aged 2.5 to 3 years old and in good physical condition. After undergoing microscopic examination, the semen was mixed and divided into 6 groups. One group served as the pre-freezing group, which was diluted with a diluent without C3R. The remaining 5 groups were diluted and cryopreserved using diluents containing varying concentrations of C3R (0, 20, 40, 60, and 80 μg·mL^-1). Each group consisted of 3 replicates. The effect of C3R on the cryopreservation of dairy sheep semen was studied by evaluating related indicators of sperm quality, antioxidant capacity, and adenosine metabolism after thawing. The results indicated that the addition of C3R significantly improved sperm quality. The treatment group receiving 40 μg·mL^-1 of C3R exhibited the highest proportion of sperm forward movement and acrosome integrity, both of which were significantly higher than those in the control group (P < 0.05); The plasma membrane integrity rate in the 60 μg·mL^-1 C3R treatment group was the highest among all groups and was significantly higher than that of the control group (P < 0.05). The addition of C3R significantly enhanced the antioxidant capacity of sperm. The superoxide dismutase activity of sperm in the 40 μg·mL^-1 C3R treatment group was significantly higher than that in the other groups (P < 0.05), while the malondialdehyde content was significantly lower than that in the other groups. (P < 0.05); The total antioxidant capacity and catalase activity in the 60 μg·mL^-1 C3R treatment group were significantly higher than those in the other groups (P < 0.05); The active oxygen content in the 20 and 40 μg·mL^-1 treatment groups was significantly lower than that in the control group (P < 0.05). Therefore, under the conditions of this experiment, 40 μg·mL^-1 C3R demonstrated the best protective effect on dairy sheep sperm. Regarding adenosine metabolism, the activity of adenosine deaminase in the frozen group was significantly higher than that in the pre-frozen group (P < 0.05). Furthermore, the addition of 40 μg·mL^-1 C3R significantly inhibited the activity of adenosine deaminase and reduced the contents of adenosine and inosine (P < 0.05). Therefore, incorporating C3R into the freezing diluent can enhance the preservation efficacy of frozen semen from dairy sheep. When C3R is added at a concentration of 40 μg·mL^-1, it significantly boosts the antioxidant capacity of the sperm and decreases adenosine deaminase activity, thereby improving the quality of frozen semen from dairy sheep.

To investigate the metabolic response to acute heat stress of porcine sertoli cells, porcine sertoli cells were collected under 3 conditions: before acute heat stress (control group), immediately after acute heat stress (43℃, 0.5 h; HS0.5 group), and after a 36 hour recovery post-acute heat stress (HS0.5-R36 group), with 3 biological replicates per group. Metabolic profiling was detected using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Differential metabolites were validated via ELISA. The effects of key differential metabolite on cell viability, proliferation, and apoptosis were assessed using CCK-8, EdU, and Annexin V FITC/PI kits. Level of reactive oxygen species (ROS), mitochondrion distribution, and mitochondrion membrane potential were evaluated using ROS, RH123 and Mito Tracker staining. The results showed that 33, 57 and 115 significant differential secondary metabolites (P < 0.05; VIP>1.00) were identified respectively in HS0.5 vs. Control, HS0.5-R36 vs. HS0.5 and HS0.5-R36 vs. Control comparisons. The pathway analysis showed that significant differential metabolites were mainly enriched in taurine and hypotaurine metabolism and other pathways. ELISA verified that acute heat stress significantly decreased levels of taurine and gamma-aminobutyric acid, which were consistent to metabolomic results. Taurine level was significantly decreased around 5.7 μmol·L^-1 in HS0.5-R36 group as compared to control group. Treatment of porcine sertoli cells using taurine of 5.7 μmol·L^-1 for 48 h significantly increased cell viability, promoted proliferation, suppressed apoptosis and enhanced mitochondrial function (P < 0.05). These results suggest that acute heat stress could significantly alter metabolism, and the differential metabolite taurine partially mediate the damage exerted by acute heat stress on porcine sertoli cells. These results provide new insights for rescuing damage caused by acute heat stress on porcine sertoli cells.

The study aims to explore the change of rectum bacteria and virome before and after weaning and their interactions of Chalu black piglets through 16S rDNA and meta-virome sequencing. Feces were collected from the rectal of Chalu black pigs on day 7 before weaning and day 7 after weaning respectively. 16S rDNA and meta-virome sequencing were used to analyze the rectum microbes change of weaned piglets during the weaning transition. The results showed that, the α diversity of rectum microbiota was increased post-weaning and the β diversity analysis indicated a significant change in community structure (P < 0.05). What's more, the relative abundance of Firmicutes (P < 0.05), Lactobacillus (P < 0.01) and Limosilactobacillus (P < 0.01) were increased significantly, while the relative abundance of Bacteroidetes (P < 0.05) decreased significatly compared with that of pre-weaning; Meta-virome sequencing illustrated that there was no significant change in α diversity of the virus before and after weaning, but the virome structure changed significantly (P < 0.05). Especially, the relative abundance of Caudoviricetes and Microviridae are having a significant increase after weaning (P < 0.05); Moreover, the intestinal phages interacted with specific bacterial communities, and there was a significant positive correlation between the Akkermansiaceae and Caudovirales family phages (P < 0.05). In summary, the study demonstrated that the effects of weaning stress on the rectum microbiota and virome of piglets and provide new insights into the interactions between rectum microbiota and virome in local pig species, which is benificial in promoting the protection and utilization of local pig breeds in China.

This experiment was conducted to study the effect of low-protein diet structure on the growth performance and digestive charateristics in white-feathered broilers during the early growing period, and to provide references for the rational application of low-protein diversified diets in broiler production. Experiments were arranged as a 3×2 two-factor completely randomized design, in which the diet type factors included corn-soybean meal type (SBM), corn-soybean meal-cottonseed meal type (CSM) and corn-soybean meal-rapeseed meal type (RSM), and the corn gluten meal factor was divided into 2 types of no addition (0%) and addition (5%). A total of 390 male 1-day-old Arbor Acres (AA) broilers were randomly divided into 6 treatments, with 5 replicates per treatment and 13 broilers per replicate. The experiment included 2 phases, in the broiler brooding phase (1-14 days of age), each treatment was fed with the above 6 diets, respectively, resulted in 6 kinds of broilers (SBM0, CSM0, RSM0, SBM5, CSM5, RSM5), and in the growing phase (15-28 days of age), all the broilers were fed with the same diet. The growth performance of broilers in each phase was determined, as well as the nutrient digestibility, fecal scores, and in vitro nutrient digestibility of feces when broilers fed the same diet during the growing phase. The results showed as follows: 1) There was no significant interaction between diet type and corn gluten meal on the growth performance of broilers in each phase, the addition of corn gluten meal during the brooding phase significantly increased (P < 0.05) the F/G of broilers but did not significantly affect the other growth performance indicators. The final body weight and average daily gain of broilers fed SBM diet during the brooding and growing phase were greater than those fed the RSM diet (P < 0.05), but there was no significant difference on F/G among treatments. 2) A diet type×corn gluten meal interaction (P < 0.05) was observed for the digestibility of gross energy (GE), which was driven by broilers in the SBM0 group had higher (P < 0.05) digestibility of GE than those of RSMO、SBM5、CSM5 and RSM5 groups. Diet type had no significant effect on the digestibility of dry matter (DM) and crude protein (CP) of broilers, but the addition of corn gluten meal reduced (P < 0.05) the digestibility of DM and CP. 3) The addition of corn gluten meal increased (P < 0.05) fecal particle size, but had no effect on fecal scores. Broilers in CSM and RSM group had better (P < 0.05) fecal color, molding, and diarrhea scores compared with SBM broilers. 4) A diet type×corn gluten meal interaction (P < 0.05) effect was observed for the in vitro nutrient digestibility of broiler feces, the digestibility of DM and GE in feces in the SBM5 group was higher (P < 0.05) than that without the addition of corn gluten meal group. Additionally, effective energy value in broiler faeces was positively correlated (P < 0.05) with its ether extract content and negatively correlated (P < 0.05) with crude fibre content. In conclusion, the diet type and corn gluten meal had greater effect on the growth and digestive function development of broiler chickens. Especially in the early growing period, the addition of unconventional plant protein meal or corn gluten meal in corn-soybean meal diet will reduce the nutrient digestibility, thereby reducing the growth rate of boilers.

This study assessed the effects of lactulose (LAC) on the growth performance, apparent digestibility of nutrients, meat quality, serum biochemistry and antioxidant parameters in meat rabbit. A total of 192 35-day-old Ira male rabbits were randomly diveded into 4 groups which fed diets containing LAC 0%(control), 0.5%, 1% and 2% for 35 days, respectively, in which 7 d were prefeed period and 28 d were experimontal period. The results showed that 1) Compared to the control group, the average daily gain (ADG) of meat rabbits (P < 0.05) showed significant improvement in 2% LAC group from 1 to 28 d. Besides, the feed-to-gain ratio was decreased(P < 0.05) in 1% and 2% LAC treatments compared to control group; 2) The apparent digestibility of dry matter(DM), ether extract(EE), crude fiber (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF) and calcium(Ca) were increased (P < 0.05) in rabbits fed the 2% LAC diet compared with the control diet; 3) The serum glucose and malondialdehyde contents were remarkably increased(P < 0.01) and decreased in the 2% LAC group (P < 0.05), respectively. The level of serum total protein (TP) and globulin (GLB) were remarkably increased(P < 0.05) after feeding the 1% LAC diets compared to the 2%LAC group; 4) Compared to the control group, the pH of longissimus dorsi was improved (P < 0.05) in the 2% LAC treatment. The different addition of the LAC resulted in significant higher(P < 0.05) unsaturated fatty acids(UFA) and monounsaturated fatty acids (MUFA). 2% LAC treatment could significantly increase polyunsaturated fatty acid(PUFA) in longissimus dorsi (P < 0.05). The PUFA/SFA (P/S) ratio of longissimus dorsi was remarkablely increased (P < 0.05) in 1% and 2% LAC treatment. 2% LAC treatment could markedly increase the PUFA content and P/S of leg muscles in meat rabbits (P < 0.05). In conclusion, adding 1% and 2% LAC to the diet not only could improve the growth performance but also promote the antioxidant capacity and muscle quality of meat rabbits.

To establish a reverse genetic manipulation platform for the currently prevalent H9N2 subtype avian influenza virus (AIV), we isolated the A/Chicken/Guangdong/M/2021 (H9N2) strain (designated as M) from a diseased chicken farm in Guangdong. The full genome sequence was determined to identify the viral genotype and analyze differences from circulating strains. Plasmids for the eight genes were constructed using RT-PCR and seamless cloning, followed by co-transfection into 293T cells to rescue the virus, named rM. We assessed viral stability and growth characteristics, and conducted animal experiments to verify the consistency of rM with the original M strain in terms of transmission efficiency and pathogenicity. The M strain, classified as the G57 genotype, shares consistent biological properties with rM, including acid and heat stability, pathogenicity, and transmissibility. The reverse genetic system of AIV subtype H9N2 of the G57 genotype was successfully established, and the biological properties of M and rM were comprehensively verified to remain consistent from both in vivo and in vitro experiments.

H9 subtype of avian influenza (AI) poses a significant threat to the poultry industry, particular the broiler industry. As current inactivated vaccine cannot effectively prevent virus infection and replication in the upper respiratory tract of chickens, it is necessary to generate an mRNA vaccine targeting the hemagglutinin (HA) gene of H9 subtype avian influenza virus (AIV). Based on the HA gene sequences available on GenBank in 2022, full-length of the HA sequences (HA) and its extracellular domain (HAe) were optimized, and cloned into the pUC57 vector to construct transcription templates in vitro. After transcription in vitro, capping, and purification, the mRNAs were encapsulated in lipid nanoparticles (LNPs) and transfected into human embryonic kidney cells (293T), baby hamster kidney cells (BHK-21), and chicken embryo fibroblast cells (DF-1). HA protein expression was confirmed via Western blot. Twenty 3-week-old SPF chickens were randomly divided into four groups (n=5). Each chicken was immunized with 0.3 mL PBS, 25 μg HA mRNA LNP, 25 μg HAe mRNA LNP and 0.3 mL H9 subtype AI inactivated vaccine, respectively. Serum samples was collected 4 weeks post-immunization to assess hemagglutination inhibition (HI) antibody titers. All animals were intranasally and ocularly challenged with 0.2 mL of H9N2 subtype AIV (containing 10^6.0EID₅₀). Oral swabs were collected 5 days post-challenge to detect virus isolation, and peripheral blood mononuclear cells (PBMCs) were collected for the measurement of expression levels of IFN-γ and IL-4, alongside observing pathological changes of tracheal tissue. Western blot analysis showed that both 2 mRNA HA proteins were successfully expressed and could be effectively translated into cells following LNP encapsulation. The mean HI antibody titers (log2) post immunization with HA mRNA LNP and HAe mRNA LNP in SPF chickens were 5.6 and 3, respectively. The H9 subtype AI inactivated vaccine induced significantly higher antibody titer (9.8), compared to both two mRNA LNP groups (P < 0.01). Virus isolation rates of the challenge control, HA mRNA LNP, HAe mRNA LNP, and H9 subtype AI inactivated vaccine groups were 5/5, 1/5, 4/5, and 2/5, respectively. The HA mRNA LNP group exhibited significantly higher IFN-γ expression levels compared to other groups (P < 0.05), while the HAe mRNA LNP group showed significantly lower IL-4 expression levels (P < 0.05). The pathological changes of tracheal tissue indicated that immunization with HA mRNA LNP and H9 subtype AI inactivated vaccine effectively mitigated damage of H9 subtype AIV to tracheal epithelial cells in SPF chickens. The constructed mRNA vaccine with HA full-length of H9N2 subtype AIV could provide an effective protection to chickens on H9 subtype AIV attack, while mRNA vaccine with HA extracellular domain could not provide an effective immune protection.

Swine acute diarrhea syndrome coronavirus (SADS-CoV) was a new bat-origin swine enteric coronavirus that can cause diarrhea, vomiting, dehydration and death in newborn piglets. Nucleocapsid (N) protein is the most conserved structure protein, and has a good antigenicity. In the previous study, five monoclonal antibodies (mAbs, 1C10, 4B10, 6G1, 6F3 and 6E8, respectively) against the SADS-CoV N protein were obtained, among which 6E8 showed the strongest reactivity with SADS-CoV. In this study, truncated and deletion mutant plasmids of N protein were constructed using pET32a expression vector. The 6E8 epitope was located to amino acids 69-QKGQRK-74 by testing the 6E8 for reactivity with series of N truncation and deletion using Western blot. The 6E8 epitope was highly conserved among different SADS-CoV isolated strains and bat coronavirus HKU2. A similar epitope (VKGQRK) was found in porcine transmissible gastroenteritis virus (TGEV) and feline infectious peritonitis virus (FIPV), with one amino acid difference; however, it showed low homology with other coronaviruses. Specific tests showed that the 6E8 could not be reacted with porcine epidemic diarrhea virus (PEDV) and TGEV, despite only one amino acid different in the epitope recognized by 6E8 and TGEV, indicating that 69-Q was the key amino acid for 6E8 epitope. Further experiments showed that the 6E8 had a capacity to enhance the infection of SADS-CoV in Huh7 cells when the 6E8 incubated with SADS-CoV before infection, which was called antibody-dependent enhancement (ADE). In summary, the identification of the novel B-cell epitope in the SADS-CoV N protein provides new insights for designing novel SADS-CoV vaccines and developing epitope-related diagnostic reagents.

Salmonella Typhimurium is the most common serotype of Salmonella, which seriously endangers the development of animal husbandry and public health safety. Outer membrance protein OmpW, encoded by ompW, is a special nutrient transport channel that has iron ion efflux function and is closely related to drug resistance, pathogenicity and other factors. However, the function of OmpW in Salmonella has not been studied yet. To investigate the effect of OmpW in Salmonella Typhimurium, this experiment used Salmonella Typhimurium ATCC 14028 (WT14028) as a wild strain, constructed ompW gene deletion strain (14028ΔompW) using λ-Red homologous recombination technology and constructed complemented strain (14028ΔompW: : ompW). Growth characteristics, biofilm formation ability, environmental tolerance and antimicrobial resistance were detected to investigate the biological characteristics of ompW. And the impact on the pathogenicity was explored through mouse infection experiments. The results showed that there was no significant difference in colony morphology between the deletion strain and the wild strain. The deletion strain showed significantly slower in logarithmic growth period than the wild strain, but there is no significant difference in stable period. The biofilm formation ability of the deletion strain decreased by 31.73%±6.43%. In acidic (pH=3), alkaline (pH=10) and high salt (1 mol·L^-1 NaCl) environments, the survival rate of the deletion strain was significantly lower than that of the wild strain. But in high iron (>0.625 g·L^-1 FeCl₃) environment, the survival rate of the deletion strain was higher. The sensitivity of the deletion strain to aminoglycoside antibiotics such as gentamicin and kanamycin was significantly enhanced, but there was no significant change in sensitivity to other antibiotics such as ampicillin and tetracycline. The results of the mouse infection experiments showed that the deletion strain induced lower diarrhea rate, milder splenomegaly and intestinal damage compared with the wild strain. And the bacterial translocation ability of the deletion strain also significantly decreased. In summary, this study successfully constructed ompW gene deletion strain and complement strain of Salmonella typhimurium, and experiments showed that ompW is closely related to bacterial growth, environmental tolerance, drug resistance, pathogenicity, and other aspects. This study lays the foundation for further research on the pathogenic mechanism and drug resistance mechanism of Salmonella.

The aim of this study was to investigate the composition of the flora in the respiratory, intestinal and fecal tract of wild boars and the carriage situation of Clostridium perfringens (Cp). Respiratory, intestinal and fecal samples from twenty-seven wild boars were collected in Jiangxi region. Bacterial 16S rRNA gene sequencing technology was used to analyze the composition and differences of the flora in different physiological parts of wild boars. And the Cp genotypes of wild boar sources and the carriage of toxin genes cpa, cpb, cpb2, etx, itx, cpe and netb were also analyzed. The Clostridium perfringens in the intestinal and fecal samples were isolated and purified, in additon, the whole-genome sequencing were conducted on the isolates. The results of the flora analysis showed that the dominant phyla in all three groups were Firmicutes, Proteobacteria, Bacteroidetes and Actinobacteria, with the highest percentage of Firmicutes in the intestinal tract and feces and the highest percentage of Proteobacteria in the respiratory tract. Analysis of variance revealed highly significant differences in five genera in the three groups and significant differences in the remaining 10 genera; three Cp strains isolated from wild boar fecal samples were confirmed to be type A and were free of β2 toxin gene by PCR toxin genotyping and whole genome sequencing analysis; the carriage rate of Cp was 81.48% (22/27) in 27 samples, and the strains carried β2 toxin gene in these 22 positive samples were 9, representing 40.91% (9/22). No other toxin genes were detected. Combining the results of the toxin tests for each subtype, the Cp presented in the positive samples were all type A. In conclusion, based on the flora analysis, this study understood the microbial community composition of different physiological parts of wild boars and the carriage of Cp in wild boar, which provided theoretical clues for the prevention and detection of wild boar diseases.

The purpose of this experiment was to investigate the immunoprotection of a DNA vaccine targeting Eimeria necatrix gametocyte antigen gene against coccidiosis. In this study, a DNA vaccine of gametocyte antigen gene, pVAX-Engam59, was constructed and combined with CpG ODN2006 adjuvant to immunize the chicks. The level of specific antibody in each group was detected by indirect ELISA. The mRNA transcription of cytokine (IL-4, IFN-γ, and TGF-β4) was analyzed by qRT-PCR. Finally, each chick was infected with 2.5×10⁴ E. necatrix sporulated oocysts through the crop, and the clinical symptoms and bloody stools of each group were observed. The oocysts output, weight gains and lesion scores of each group were detected after 8 days post-infection. The results showed that the serum specific antibody and cytokine (IL-4, IFN-γ, and TGF-β4) levels of pVAX-Engam59 and pVAX-Engam59+CpG ODN2006 were significantly increased (P < 0.05), and the oocyst reduction ratios were 64.65% and 64.48%, respectively. Compared to challenged and unchallenged control group, the number of bloody stools and the lesion scores in the pVAX-Engam59 and pVAX-Engam59+CpG ODN2006 groups were significantly lower (P < 0.05), and the relative weight gain rates were significantly higher (P < 0.05). The ACI values were 156.02 and 159.72, respectively. In summary, we successfully constructed DNA vaccine targeting E. necatrix gametocyte antigenic gene, and both pVAX-Engam59+CpG ODN2006 and pVAX-Engam59 showed good immunoprotective effects, which were close to the anti-coccidial intermediate efficiency level, and could be candidates for a DNA vaccine against coccidiosis.

In order to understand the gastrointestinal nematode species and infections of three desert lizards in the Turpan-Hami Basin of Xinjiang, a total of 127 lizards of Laudakia stoliczkana, Eremias roborowskii and Phrynocephalus axillaris were examined for gastrointestinal nematodes using the dissection method to isolate gastrointestinal nematodes for morphological identification. Differential analyses of infection rates and intensity of infection among sexes, head and body lengths (Snout-vent length, SVL), and body weights of the three different desert lizards were performed using chi-square tests and one-way analysis of variance (ANOVA). The results showed that the total infection rate of gastrointestinal nematodes in the three desert lizards was 28.35%, with L. stoliczkana having an infection rate of 100%, E. roborowskii having an infection rate of 27.59%, and P. axillaris having an infection rate of 22.22%; the prevalence and intensity of gastrointestinal nematode infections in L. stoliczkana were significantly higher than those in E. roborowskii and P. axillaris, and the intensity of infection in males was significantly higher than that in females; the intensity of infection was higher in large-sized L. stoliczkana and P. axillaris. The three dominant species were identified as nematodes of the genus Abbreviata in the family Physalopteridae, nematodes of the genus Spauligodon in the family Pharyngodonidae, and nematodes of the genus Parapharyngodon in the family Pharyngodonidae, all of which are newly recorded genera in China. The present study investigated the infection status of the three species of gastrointestinal nematodes of desert lizards in the Turpan-Hami Basin of Xinjiang and analyzed their influencing factors. This study not only accumulates important data for the study of parasite diversity of desert lizards in Xinjiang but also provides important theoretical support for the conservation, development and utilization of desert lizards.

This study aimed to explore the mechanism of cannabidiol (CBD) in alleviating the apoptosis and autophagy of pig intestinal epithelial cells (IPEC-J2) induced by bisphenol A (BPA) through BRD4/AMPK/mTOR signaling pathway. In this study, the cell viability was measured by CCK-8 method, and the half inhibitory concentration of BPA on IPEC-J2 cells and the optimal dose of CBD antagonist for BPA were screened. Using the group comparison method, The experiment was divided into blank control group, BPA group (150 μmol·L^-1 BPA), BPA+CBD group (150 μmol·L^-1 BPA+10 μmol·L^-1 CBD), BPA+CBD+CQ group (150 μmol·L^-1 BPA+10 μmol·L^-1 CBD+20 μmol·L^-1 CQ) and CBD group (10 μmol·L^-1 CBD). The apoptosis rate of IPEC-J2 cells was detected by AO/EB staining and flow cytometry. The ROS levels in cells were detected by DCFH-DA method. Oxidative stress was detected by oxidative stress kit. The expression of LC3-Ⅱ protein was detected by immunofluorescence technique. Quantitative Real-time PCR (qRT-PCR) and Western blot were used to detect the expression of genes related to apoptosis, autophagy, intestinal tight-junction protein and BRD4/AMPK/mTOR signaling pathway. The results showed that apoptosis and autophagy levels of IPEC-J2 cells were increased after 150 μmol·L^-1 BPA treatment, and decreased after 10 μmol·L^-1 CBD treatment. CBD could significantly down-regulate the increase of ROS and MDA levels caused by BPA (P < 0.05), and up-regulate the decrease of GSH-Px activity (P < 0.05); CBD significantly down-regulated the increase of expression levels of apoptosis related genes (Bax and caspase-3), autophagy related genes (P62, LC3-Ⅱ/LC3-Ⅰ and ATG5) and BRD4/AMPK/mTOR path-related genes (AMPK) induced by BPA (P < 0.05). The expression levels of LAMP1, Bcl-2, BRD4, mTOR, ZO-1 and Claudin-1 were increased (P < 0.05); Immunofluorescence results showed that CBD could significantly reduce the expression and distribution of LC3-Ⅱ in IPEC-J2 cells induced by BPA (P < 0.05). In conclusion, CBD could antagonize BPA-induced apoptosis and autophagy of IPEC-J2 cells through BRD4/AMPK/mTOR signaling pathway.

This experiment was conducted to study the drug resistances of Staphylococcus aureus isolated from raw milk, the prevalence of heteroresistance of Staphylococcus aureus and explore the mechanism of heteroresistance. Thirty-six Staphylococcus aureus isolates were isolated from 179 batches of raw milk collected from Northeast, North China, East China, South China, Northwest and Southwest. The sensitivity and resistance of Staphylococcus aureus were identified using broth microdilution method. The combination of disk diffusion method and E-test method was used to screen suspected heteroresistant strains from strains with sensitive phenotypes, and then the suspected heteroresistant strains were confirmed by Population analysis profiling(PAP). The stability of the heteroresistant phenotype after serial passage was measured. Finally, whole-genome sequencing and whole genome re-sequencing analysis were used to determine the heteroresistant mechanisms. The results showed that 36 strains of Staphylococcus aureus were resistant to β-lactam antibiotics, the resistant rate to ranges from 11.11% to 61.11%, and had high sensitivity to tetracycline and aminoglycoside antibiotics. 19 combinations of Staphylococcus aureus and antibiotics with heteroresistant were identified through initial screening, and it was confirmed that the 3 combinations exhibited heteroresistant phenotypes, of which only amoxicillin-clavulanate-J23 heteroresistant stabled after serial passage. This study demonstrates that S. aureus of raw milk origin is high resistant to β-lactam and quinolone antimicrobials. Heteroresistant mechanisms to amoxicillin-clavulanic acid are associated with blaZ and metal β-lactamase expression. Heteroresistant mechanisms to tetracyclines is associated with tetracycline efflux pumps and resistance proteins.

Aflatoxin B₁ (AFB₁) contamination is wide spread worldwide and seriously endangers the health of livestock and poultry. At present, more than 34 countries and regions in the world have established limit standards for the content of AFB₁ in livestock and poultry feed. However, even exposure to AFB₁ below the limit standard may still have the risk to promote infections of certain pathogens. Therefore, in this study, 3D4/21 cells were used as a model. Firstly, the non-toxic concentrations of AFB₁ were screened, and then the relative NP protein expression and titers of swine influenza virus (SIV) were detected by Western blots and TCID₅₀, respectively, thereby determining the effect of non-toxic concentrations of AFB₁ exposures on SIV replication. Next, TMT technology under the proteomics platform was used to detect the differentially expressed proteins and enrichment pathways between SIV infection and SIV + AFB₁ groups, in order to explore the possible mechanism of AFB₁ promoting SIV replication. The results showed that 0.01 μg·mL^-1 AFB₁ had no cytotoxicities on 3D4/21 cells and significantly promoted the replication of SIV in 3D4/21 cells; TMT results showed that 242 proteins were differentially up-regulated while 327 proteins were differentially down-regulated in the SIV + AFB₁ group compared to SIV infection group. Further pathway enrichment analysis screened 137 pathways that affect the difference of AFB₁-induced SIV replication. The top five enrichment pathways were proteasome pathway, necroptosis pathway, multi-species apoptosis pathway, legionella disease pathway, and drug metabolism-other enzyme pathway, respectively. Among them, the apoptosis-related pathway may be the key pathway regulating AFB₁ promoting SIV replication, and the subsequent DAPI staining test confirmed that. The research results will lay a foundation for early prevention and control of SIV infection aggravated by AFB₁ pollution, and provide a reference for exploring the causes of the increase of other infectious diseases.

Mycoplasma hyopneumoniae (Mhp) colonizes to respiratory epithelial cells, causing the inflammatory injury of trachea, bronchus and lungs in pigs, which reduced the feed conversion rate and daily weight gain of pigs, resulting in serious economic losses to the swine industry. plantamajoside (PMS), as a commonly used anti-inflammatory traditional Chinese medicine, its role in inhibiting the inflammatory response caused by Mhp infection is unknown. The aim of this study was to investigate the effect and related mechanisms of PMS on inflammatory response induced by Mhp in vitro and in vivo. Mhp infected PK-15 cells and mice were used as infection models, respectively. First, PK-15 cells were treated with different concentrations of PMS to determine the maximum concentration of PMS that had no effect on cell morphology and viability. This highest concentration of PMS was then applied to PK-15 cells which infected with Mhp. The mRNA expression of tumor necrosisfactor-alpha (TNF-α), interleukin-6 (IL-6) and other inflammatory factors in PK-15 cells was detected by RT-qPCR. The results showed that the mRNA levels of IL-6, TNF-α, NF-κB and IκBα were significantly increased in PK-15 cells which infected with Mhp compared with the control group, while PMS could reduce the effect. Western blot and immunofluorescence were used to further analysis and the results showed that PMS could inhibited the phosphorylation of NF-κB P65 and IκBα, and inhibited the nuclear translocation of NF-κB P65, which induced by Mhp. Related studies on mouse models have found that PMS can improve the lung lesions, reduce the inflammatory cells of lung which induced by Mhp. At the same time, PMS can significantly reduce the content of TNF-α in serum. These results suggest that PMS may ameliorate Mhp-induced inflammatory response through NF-κB pathway, which provides important theoretical basis for novel prevention and treatment of Mhp infection.

This study aimed to investigate the bacteriostatic activity of Huning Powder against E. coli of chicken lung origin, as well as the anti-inflammatory and anti-oxidation effects on lipopolysaccharide (LPS)-induced damage model of chicken embryonic fibroblasts (DF-1). Huning Powder was prepared with 10 Chinese herbs including Morus alba L, Scutellaria baicalensis Georgi, and Taraxacum. The antibacterial activity of Huning Powder against chicken lung-derived E. coli was determined by Oxford Cup method, the minimal inhibitory concentration (MIC) and bactericidal concentration (MBC) of Huning Powder was determined by TTC method, and the bacterial growth curve was drawn. The contents of alkaline phosphatase (AKP), nucleic acid and soluble protein in bacterial culture medium were detected. The LPS-induced DF-1 cell damage model was established and drug was administered after the toxicity of LPS and Huning Powder to DF-1 cells was detected by CCK-8 method. The levels of the inflammatory factors IL-1β, IL-6 and IL-8 were determined by ELISA.The expression of key proteins of Toll-like receptor 4 (TLR4) and nuclear factor kappa B p65 (NF-κB p65) in the TLR4/NF-κB signaling pathway were determined by Western blot. The intracellular levels of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were determined by kits. The expression of key proteins of nuclear factor E2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein 1 (Keap1) in the Keap1/Nrf2 signaling pathway were determined by Western blot. Results were as follows: Chicken lung-derived E. coli was moderately susceptible to Huning Powder, and the MIC was 250 mg·mL^-1 and MBC was 500 mg·mL^-1. The growth of E. coli of chicken lung origin was inhibited and AKP, soluble protein and nucleic acid content in bacterial cultures were increased by Huning Powder. In cellular assays, the levels of inflammatory factors IL-1β, IL-6, and IL-8 in the supernatant of LPS-induced DF-1 cells were reduced by Huning Powder. The overexpressions of TLR4 and NF-κB p65 proteins were inhibited by Huning Powder. The levels of ROS and MDA were reduced, SOD activity and GSH content were increased by Huning Powder. The expression of Nrf2, NQO1 and HO-1 proteins were increased and the overexpression of Keap1 protein was inhibited by Huning Powder. The traditional Chinese medicine compound Huning Powder had good bacteriostatic activity against chicken lung-derived E. coli and could exert anti-inflammatory and antioxidant effects by regulating the expression of key proteins of the TLR4/NF-κB and Keap1/Nrf2 signaling pathways in LPS-induced DF-1 cells.

The study aimed to identify key genes and molecular markers that affect the freezability of bull sperm and apply them to molecular breeding of dairy cattle. In this study, 11 candidate genes related to bull sperm freezability were identified based on the previous omics study, such as ATP5F1A, DNASE2, and LPO. Primers were designed for the exons of these genes to detect genetic variation sites via PCR pooled sequencing. Subsequently, the Time-of-Flight mass spectrometry was used to genotype important loci in a resource population of 130 healthy bulls and association analysis was performed. Among them, there were 55 bulls in the high sperm freezability group, with an average fresh sperm motility of 0.68 and an average post-freeze motility of 0.42, and 75 bulls in the low sperm freezability group, with an average fresh sperm motility of 0.65 and an average post-freeze motility of 0.32. Finally, 14 significant SNPs were identified, among which the rs110909003 SNP (C/T) located in the exon of ATP5F1A was significantly associated with the bull sperm freezability (P=0.009). Western blot analysis was performed to detect the expression of ATP5F1A in sperm with different genotypes at the rs110909003 locus. The results showed a significant increase in ATP5F1A protein expression in sperm from bulls with the CC genotype compared to those with the TT genotype. This study identified ATP5F1A significantly associated with bull sperm freezability, as well as an important molecular marker, the rs110909003 SNP, located within this gene. This SNP may influence sperm freezability by affecting the mitochondrial oxidative phosphorylation process. These findings provide valuable information for the molecular breeding of frozen semen quality traits in Holstein bulls.

Canine circovirus (CanineCV) is a virus associated with canine gastrointestinal diseases. Since it was first identified from dogs in 2012 in the United States. Researchers have subsequently detected the presence of CanineCV in various wildlife species, including wolves (Canis lupus), badgers (Meles meles), red foxes (Vulpes vulpes), arctic foxes (Vulpes lagopus) and jackals(Lupulella mesomelas).In 2016, CanineCV was first reported in dogs in China. However, a systematic investigation of CanineCV infection in wildlife in China remains lacking. We acquired tissue specimens from an ill and deceased otter (Lutra lutra), and successfully identified the presence of CanineCV through the application of Polymerase Chain Reaction (PCR). We designed of CanineCV genome-wide primers for whole genome amplification, sequenced and analyzed the direction of genetic evolution. Result were as follows: Through PCR technology, we tested the otter tissue samples for CanineCV, and the results were positive. By whole-genome amplification, sequencing, and assembly, we successfully obtained a CanineCV whole-genome sequence with a length of 2 063 nt. Nucleotide homology analysis revealed that this strain had a gene sequence homology of 84.6%-98.2% with all other strains in GenBank. After constructing a genetic evolution tree, we found that this strain belonged to CanineCV-3. This is the first identification of CanineCV infection in an otter, and we have successfully obtained the first whole-genome sequence and genetic evolution information of CanineCV derived from wildlife in China. The above results illustrate the potential hazards of CanineCV infecting otters and their possibility of infecting more other animals, thus suggesting that we should strengthen the monitoring of CanineCV infection in different animals.