The prime editing system based on CRISPR/Cas9 is a novel gene editing technology, which enables all 12 base-to-base conversions or transition, precise small insertion and deletion without requiring double-strand breaks or exogenous donor DNA templates. This review summarizes the development of gene editing approaches, the principle and characteristics of prime editing technique, as well as applications in animal and plant breeding and human gene therapy. It provides a reference for scientists to get insight into prime editing system and further use it in basic research and animal or plant breeding.

Copy number variation (CNV) is a type of structural variation with DNA fragment size ranging from 50 bp to 5 Mb in the genome. In recent years, with the development of detection technology, the application of detection methods of CNVs have expanded from widely used techniques such as CGH, SNP, and NGS to the emerging third-generation sequencing technology, leading to more CNVs that play important roles in areas such as the origin evolution and genetic breeding of livestock have been identified. However, comprehensive reviews on the research progress of CNV in livestock such as cattle, sheep, goat, pig and horse from the perspective of detection technology development are still lacking up to now. Therefore, this review introduces the main detection methods of CNN firstly. Thus, recent research progress in the detection of CNVs using CGH, SNP, and WGS technologies (including next-generation sequencing and third-generation sequencing) in the important livestock species-cattle, sheep, pig and horse-was comprehensively summarized; Finally, the problems of current research on CNV in domestic animals and its application prospects in animal genetic breeding in the future were proposed. This review is expected to lay the basis for future study on CNV in livestock.

Sex control is a technique in which a female animal produces offspring with a specific sex through human intervention. Since the separation of X sperm and Y sperm is the technical basis for the realization of sperm sexing in animals, this paper summarizes and compares the new techniques for the separation of sperm before fertilization, including flow cytometry, immunological approach of sperm sexing, pH separation, microfluidics and nanotechnology. The activation of TLR7/8 signaling pathway by R848 (Resiquimod) in immunological isolation is a potential method for sperm isolation, aiming to provide a theoretical basis for sperm sexing techniques.

Efficient estrus identification is an important way to improve the fertility of heifers and enhance the economic efficiency of cattle farms. With the increasing scale of dairy farming, the traditional manual observation of estrus identification method has been difficult to adapt to the needs of estrus identification of dairy cows in large-scale farms. Pedometer estrus identification is difficult to detect quiet estrus and does not improve estrus detection rates. Although the body temperature parameters of estrus identification potential, but the cow body hair, body surface temperature measurement is not ideal, and contact temperature measurement collection site is limited, wearable equipment research and development difficulties, different parts of the collection of cattle body temperature data vary greatly. For this reason, this paper summarizes the results of the previous use of different parts of the cow′s body temperature data estrus identification, comparative analysis of the relationship between the pattern of change of the body temperature of each part of the cow in estrus and estrus, for the research and development of efficient cow estrus identification technology to provide scientific reference.

Semen quality is an important index to measure the breeding value of male animals (poultry). Excessive reactive oxygen species (ROS) will be produced during the stress state of male animals (poultry) or semen freeze-thaw process, and when the antioxidant system of the animal or sperm itself cannot balance the excessive ROS, it is easily lead to the decline of semen quality. Vitamin E and selenium are two common antioxidants, and their moderate addition to animal diets or semen dilutions can alleviate the deterioration of semen quality caused by stress in males (poultry). In this paper, the application effect of vitamin E and selenium in regulating the semen quality of male animals (poultry) and the mechanism of their synergistic effect are reviewed, to guide production practice.

Artificial insemination (AI) is one of the core technologies accelerating the development of animal husbandry. Semen preservation is a key part of AI. Porcine semen is susceptible to bacterial contamination during preservation leading to a decline in sperm quality. With the advent of the era of total antibiotic ban, there is an urgent need to develop antibacterial alternatives with different activities to improve the effect of semen preservation. Antimicrobial peptides (AMPs) have become an important substitute for antibiotics due to their broad-spectrum antimicrobial activity and lower drug resistance. AMPs play an antibacterial role in the preservation of porcine semen by exerting antibacterial effects through cell membrane damage and non-membrane damage mechanisms. This article reviews the research progress of AMPs′ antibacterial mechanisms and their application in porcine semen preservation techniques. The aim is to provide a scientific basis for the development of antimicrobial peptide products preserved in porcine semen at room temperature.

Reducing the numbers of insemination, shortening pregnancy intervals, and increasing the pregnancy rate of cows are the important guarantee for improving the reproductive efficiency of dairy cows and reducing breeding costs. Estrous synchronization-fixed-time artificial insemination (ES-TAI) can not only meet the above requirements, but also skip the estrus identification process and directly carry out timed insemination, thus maximizing the reproduction rate of dairy cows. In the article, classic and new ES-TAI technologies of dairy cows were summarizes according to different processing methods, focusing on the principles, handling procedures and practical application effects of those technologies, which will provide some references for the further research and development of more efficient ES-TAI technologies in dairy cows and the reproduction management of dairy cows in large-scale farms.

Alterations of the gut microbiota may cause dysregulated mucosal immune responses, leading to the onset of inflammatory bowel diseases (IBD) in genetically susceptible hosts. Current effective treatments for IBD in animals are restoration of normal immune homeostasis of the gut microbiota by microbiota-targeted therapies including antibiotics, prebiotics, postbiotics, and fecal microbiota transplantation. In this review, the basis for promoting intestinal homeostatic immune responses through microbiota-targeted therapy, as well as recent advances in host-microbe interactions during the occurrence and development of IBD were discussed. Given that dysbacteriosis of the intestinal flora is a key feature in the establishment of chronic inflammatory events, it is hoped that in the near future the microbiota-targeted therapies will be appropriate to design new cost-effective, physiological, animal-oriented IBD treatment strategies to be applied in a personalized manner.

Staphylococcus aureus (S. aureus) is a common cause of infection in humans and animals. In recent years, S. aureus has become resistant to a variety of antimicrobial drugs, and studies on heteroresistance in S. aureus have been frequently reported. The broadest definition of heteroresistance is the presence of a heterogeneous population of bacteria with one subpopulation or several subpopulations that exhibit increased levels of antibiotic resistance compared with the main population. However, this definition includes phenomena with very different characteristics; for example, clonality of resistant subpopulations, the stability of the resistant subpopulations, the resistance level of the resistant subpopulations, and at a frequency greater than 1×10^-7. Heteroresistance is the intermediate stage of sensitive bacteria evolving into drug-resistant bacteria, which often leads to the failure of clinical therapy. This paper introduces the definition of heteroresistance, detection methods, etc., and reviews the prevalence of heteroresistance of S. aureus to glycopeptides, sulfonamides, β-lactams, and other antibacterial agents and the study of the mechanism of heteroresistance. It is aimed to strengthen the understanding on the heteroresistance of S. aureus, and to provide a reference for scholars to understand the prevalence and mechanism of heteroresistance of S. aureus.

Mitochondria will produce few amount of reactive oxygen species (ROS) in the process of oxidative phosphorylation. While mitochondria are stressed, mitochondrial depolarization and ROS production increase, causing either mitochondrial damage or an increase in the expression level of mitochondrial reactive oxygen species (mtROS). ROS, as an important medium for activating NLRP3, promotes the assembly and activation of NLRP3 inflammasomes, and triggers a variety of diseases in animal bodies, such as mastitis, endometritis, neurodegenerative diseases, etc. Mitochondrial autophagy, as a selective autophagy process, regulates the activity of NLRP3 inflammasomes by clearing damaged and excess mitochondria, and maintains normal physiological functions of cells. This article reviews the effects and mechanisms of mitochondrial autophagy regulating NLRP3 inflammasomes on animal health, in order to gain a deeper understanding of the impact of mitochondrial autophagy on NLRP3 inflammasome-related diseases and provide new prevention and treatment targets for animal health.

Cadmium is a toxic heavy metal that can accumulate in the body through the food chain, causing acute or chronic poisoning and posing a serious threat to the health of humans and livestock. Liver is the main target organ of cadmium toxicity injury. Therefore, it is of great significance to reveal the mechanism of cadmium hepatotoxicity and how to inhibit its toxic effect. Selenium is one of the essential trace elements in the human body and plays an important role in maintaining normal life activities. In recent years, many studies have shown that selenium has the effect of antagonizing cadmium hepatotoxicity. This article mainly summarizes the related studies on the mechanism of cadmium induced hepatotoxicity and the antagonism of selenium, hoping to provide reference for the prevention and treatment of cadmium pollution as well as the clinical application of selenium.

Intestinal tract is not only the largest and most complex organ in the animal body, but also the component of body′s defense forefront. The integrity of the intestinal barrier is extremely important for maintaining animal health, as it can effectively prevent the passage of antigens, bacterial toxins and harmful macromolecules. In recent years, it has been found that natural plant bioactive compounds(NPBCs)have many biological functions such as immune regulation, bacteriostasis, antioxidant, improving intestinal health, etc., and have wide application value in livestock production and disease prevention and control. In this paper, the effects of NPBCs on intestinal morphology, intestinal barrier function and intestinal flora structure of animals were reviewed, aiming to provide references for further understanding of the regulatory effects of NPBCs on intestinal health and their application in animal production.

Bovine respiratory syncytial virus (BRSV) is one of the viral causes of bovine respiratory diseases worldwide, mainly causing severe lower respiratory tract infections in calves under 1 year of age. BRSV encodes a total of 9 structural proteins, among which attachment protein (G protein) and fusion protein (F protein) are the main envelope glycoproteins on the surface of BRSV, which participate in the process of viral adsorption and fusion of host cells. G and F proteins contain a variety of identifying epitopes that stimulate the body′s production of neutralizing antibody responses and are often the focus of vaccine development for Bovine respiratory syncytial disease (BRSD). Exploring the mechanism of G and F proteins in BRSV infection is of great significance for the analysis of viral pathogenesis and vaccine development. This article aims to review the structural functions of BRSV G and F proteins and the development of related vaccines, in order to provide reference for the development of BRSV vaccine and the prevention and control of BRSD.

Infectious bursal disease (IBD) is an acute, highly contagious, and immunosuppressive avian disease caused by infectious bursal disease virus (IBDV). In recent years, with the emergence of IBDV novel variant strains, IBDV still threatens the poultry industry worldwide. It seems that the battle between host and IBDV will never end. Thus, it is urgent to develop a more comprehensive and effective strategy for the control of this disease. A better understanding of the mechanisms underlying virus-host interactions would be of help in the development of novel vaccines. Noncoding RNA (ncRNAs) are an abundant class of RNAs that do not encode proteins. NcRNAs include microRNA (miRNAs), long noncoding RNA (lncRNAs), circular RNA (circRNAs) and small nuclear RNA (snRNAs). Recently, many studies confirmed that noncoding RNA (ncRNAs) were widely involved in the interaction of IBDV and the host, and played a crucial regulatory role in IBDV infection. This review elaborated the role of miRNAs, lncRNAs, and circRNAs in IBDV infection, which will further provide theoretical guidance and new ideas for the development of novel IBDV vaccines or antiviral drugs.

Ovine haemonchosis is a highly pathogenic, blood-sucking parasitic disease caused by Haemonchus contortus, which mainly resides in the abomasum of small ruminants like sheep. The infected sheep can manifest as some chronic wasting symptoms, such as emaciation, anemia, weakness, and even death in an acute form. Haemonchosis is widespread in pastoral areas in China. Due to the absence of the safe and long-term vaccine and presence of serious anthelmintic resistance, high prevalence and incidence in grazing sheep flocks still persist, which led to significantly economic losses to the sheep husbandry. Accurate and reliable detection remains an essential component for diagnosing parasite infections, making treatment decisions, monitoring anthelmintic resistance and conducting epidemiological investigation. This paper reviews in detail the current research situations of existing detection methods for ovine haemonchosis, including pathogen detection, immunological detection and molecular detection. The advantages and limitations of the above detection methods were evaluated, and the developmental trend for future detection techniques was prospected as well. The purpose of this review is to provide some available information and guideline for the novel methods development and comprehensive prevention and control strategies against ovine haemonchosis.

As the most common gastrointestinal nematode disease in ruminants, Haemonchus contortus disease has brought significant economic losses to the farming industry, and the parasite has long developed serious resistance to the commonly used anthelmintic drug ivermectin, which has brought some obstacles to the prevention and treatment of the disease. In the research progress of resistance to ivermectin in Haemonchus contortus, three possible resistance mechanisms have been reported, which are targeting GABA-gated Cl^- channels, decreasing the frequency of glutamate-gated Cl^- opening, and binding to exocytosis transporter proteins. With the development of science and technology, regulatory non-coding RNAs (miRNAs and lncRNAs) have been reported in drug resistance studies in different organisms, and miRNAs and lncRNAs have been found to be associated with resistance mechanisms such as P-glycosylated transporter proteins, cytochrome P450 and GABA-gated Cl^- channels. However, this non-coding RNA is rare in the study of Haemonchus contortus resistance to ivermectin, so to break through the resistance problem, the non-coding RNA should be correlated with the reported possible resistance mechanisms, and in-depth study should be conducted to find out the key resistance targets. In this paper, we mainly summarize the research progress related to the mechanism of resistance to ivermectin in this disease, which will provide new ideas for the subsequent in-depth study of drug resistance.

The aim of this study was to screen out the most suitable hybrid combination of Houdan that exhibits good growth performance, excellent slaughtering performance and high meat quality, further to provide a scientific basis for the breed improvement of Houdan chicken. In this study, 20-week-old healthy Houdan chicken with similar body weights were selected as the female parents, while Yunong D line (D), Yunong L line (L), Yunong SHj line (SHj), Yunong C line (C), and Yunong H line (H) raised at poultry germplasm resource farm of henan agricultural university were used as the male parent for the crossbreeding test, and the hybrid offspring were named as D×HD, L×HD, SHj×HD, C×HD, and H×HD, respectively. The 50 healthy chickens were selected from each combination according to sex and reared until 12 weeks of age. Body weights were measured every two weeks. Three growth curve models, including Logistic, Gompertz and Von Bertallanffy, were employed to fit their body weights. A total of 6 males and 6 females at 12 weeks were randomly selected from each combination for slaughter. Their growth performance, slaughter performance, and meat quality were measured, and the data were then subjected to biostatistical analysis. The optimal model for growth traits is Gompertz with the fitness greater than 0.998. In terms of growth performance, the C×HD combination had significantly higher average body weight and body size than other combinations (P < 0.05), the highest feed consumption for hens being 2.59, and the SHj×HD combination of roosters had the highest feed consumption with 2.39. According to slaughtering performance, all combinations except for the hens in the L×HD and H×HD combinations, achieved more than 70% of semi-eviscerated rate and more than 60% of eviscerated rate. In terms of meat quality, the D×HD combination showed better meat quality than the other combinations, but there was no significant difference in drip loss among the combinations. Taken together, the C×HD combination exhibited excellent performance in growth, carcass, and meat quality traits, and it can meet consumers′ demand to some extent. Moreover, the research provided an important reference for the conservation, genetic improvement of Houdan chicken and strongly supported the efficient breeding and promotion of Houdan chicken.

The purpose of this study was to explore the biological characteristics of chicken fibroblast growth factor 6 (FGF6) gene, excavate the single nucleotide polymorphism (SNP) site located in FGF6 gene and analyze its correlation with chicken economic traits, and provide reference for the subsequent research on the effect of FGF6 gene on growth traits in chickens. Bioinformatics analysis was performed for FGF6 protein sequences. Based on the GBS data and phenotypic data of 768 individuals from Gushi-Anka chicken F₂ resource population, the SNP in the FGF6 gene including the promoter 2 kb region were screened, linkage disequilibrium and association analysis were conducted to explore the correlation of SNP and haplotype of FGF6 gene with economic traits of chickens. The results showed that the amino acid sequence of chicken FGF6 was 91.26% homologous to turkey; phylogenetic analysis showed that chicken was the most closely related to turkey, followed by green sea turtle, and had the greatest genetic distance from zebrafish. Conservative analysis showed that FGF6 was highly conservative among different species. Chicken FGF6 was a hydrophilic protein with one transmembrane region, and mainly localized in the cytoplasm. Its secondary and tertiary structures were consistent. Chicken FGF6 protein was modified by 22 phosphorylation sites and 1 N-glycosylation site, and potentially interacted with 5 proteins of its receptor family, FGFR1-4 and EGF. Six SNPs sites were screened in the promoter region of chicken FGF6, all of which were located in intron 1. Polymorphism analysis showed that the 6 SNPs sites had high polymorphic, and 5 of them were in Hardy-Weinberg equilibrium and strongly interlinked (D′=1, r²=1). The results of association analysis showed that rs73399071 was significantly correlated with 17 growth traits including body weight (BW), shank length (SL), shank girth (SG), and body slanting length (BSL), etc. at different stages of F₂ resource population (P < 0.05), and 10 carcass traits such as evisceration weight (EW), semi-evisceration weight (SEW), carcass weight (CW), and breast muscle weight (BMW), etc. (P < 0.05). The rs73399071 was not significantly correlated with meat quality traits (P>0.05), and significantly correlated with high-density lipoprotein (HDL) and choline esterase (ChE) (P < 0.05). The phenotype average values of most traits in CC genotype individuals were higher than those of GG genotype individuals. Haplotype combination analysis showed that body weights of individuals carrying with CCTTCCTTCC combinations at different stages were higher than those of other haplotype combinations. The results of this study provide a theoretical reference for further exploring the function of FGF6 gene in chickens. Five SNP sites in FGF6 gene were found to be significantly associated with body weight, body size, carcass traits and blood biochemical indexes of Gushi-Anka chicken F₂ resource population, providing candidate molecular markers for chicken breeding and a new idea for marker assisted selection in chicken.

The purpose of this study was to understand the molecular evolutionary relationships and expression level of the MYL gene family in sheep. In this study, the bioinformatics methods were utilized to identify and analyze the MYL gene family in sheep. The tissue samples were collected from 3 adult males of Junken white meat sheep, Xinjiang fine-wool sheep and Hu sheep, respectively. Quantitative real-time PCR (qRT-PCR) was used to detect the expression level of MYL1, MYL6B, and MYLPF genes in various tissue of Junken white meat sheep, Xinjiang fine-wool sheep and Hu sheep. The results showed that 12 MYL gene family members were located on 9 different chromosomes. They encoded hydrophilic and varying lengths proteins consisting of 147-211 amino acids. These proteins lacked signal peptides and transmembrane domains and predominantly exhibited α-helices as the secondary structure. Phylogenetic analysis revealed that MYLs were divided into 3 groups, and the members located in the same group had similar gene structures, conserved domains and motif distributions. Collinearity analysis indicated that MYL2 had segmental duplications with MYL10 and MYLPF, and experienced strong purifying selection. Protein-protein interaction network analysis revealed interactions between MYH10 and other members. Tissue expression profile analysis showed that the expression patterns of MYL genes had significant differences. The MYL1, MYL2, MYL6B, and MYLPF displayed high expression levels in skeletal muscle tissue. The results of gene cloning and sequencing showed that the full-length CDS of the target gene was amplified. The expression levels of MYL1, MYL6B and MYLPF in muscle tissues of Junken white meat sheep were significantly higher than those in other tissues (P < 0.000 1). And the 3 genes were dominant expressions in skeletal muscles of Junken white meat sheep than those in Hu sheep and Fine-wool sheep (P < 0.000 1). The results of the study provide valuable insights for further investigation into the functionality of the MYL gene family in sheep and the improvement of sheep meat performance traits.

The purpose of this study was to explore the key metabolites, metabolic pathways, and enzymes related to the flavor formation of Guyuan cattle beef, so as to lay a foundation for further analyzing the meat quality composition and improving the meat quality of Guyuan cattle beef. In this study, six 24-month-old healthy male Guyuan cattle were collected under the same growth conditions, and non-targeted metabonomics were used to analyze and identify the metabolites of eye meat, shoulder meat and hip meat. The differential metabolites were screened by principal component analysis, partial least squares discriminant analysis and cluster analysis, and the screening results were enriched and analyzed. Subsequently, the pathways and potential key metabolites regulating the synthesis and metabolism of unsaturated fatty acids were analyzed by FELLA, and the differential metabolites were analyzed by WGCNA. A total of 689 metabolites were identified in the eye meat, shoulder meat and hip meat, of which lipids and lipid-like molecules accounting for 32.08%, and 49 fatty acid metabolites. The results of cluster analysis showed that the samples of shoulder meat group and hip meat group were clustered together, while the samples of eye meat group formed a separate cluster. N6, N6, N6-trimethyl-L-lysine and maltotriose were identified as common differential metabolites among eye meat, shoulder meat and hip meat, with the highest content in eye meat. KEGG pathway analysis showed that differential metabolites were mainly concentrated in unsaturated fatty acid biosynthesis, purine metabolism, alanine, aspartic acid and glutamate metabolism. The modules related to three kinds of muscle tissue specificity were screened by WGCNA, and the key metabolites in each module were identified. In this study, through the metabonomic analysis of muscle tissues in different parts of Guyuan cattle, 10 key metabolites and 17 key enzymes related to meat quality differences and flavor formation were identified. It not only provides a theoretical basis for exploring the molecular regulation mechanism and molecular breeding of Guyuan beef flavor formation in the future, but also serves as reference for the quality breeding of other beef cattle.

The purpose of this study was to clone 6 pluripotency-related transcription factors Oct4, Sox2, Klf4, c-Myc, Nanog, Lin28 (OSKMNL) of yak and construct polycistronic lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry. In this study, the reproductive crest tissue of a 3-5-month-old healthy female yak fetus was used as the research material. The complete coding region sequences of 6 pluripotent related transcription factors OSKMNL were cloned by RT-PCR and analyzed by bioinformatics. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed by seamless cloning. The lentivirus was packaged with 293T cells, and the packaged lentivirus was infected with 293T cells and yak fibroblasts. The virus infection was detected by fluorescence expression and RT-PCR technique (the experiment was divided into virus infection group and blank control group, each group had 3 repeats). The results showed that the coding region of cloned yak OSKMNL gene was 1 083, 963, 1 434, 1 320, 903 and 618 bp, respectively. Sequence analysis showed that the amino acid sequence of OSKMNL in yak shared more than 99% homology with cattle, and the homology of Sox2, Klf4, c-Myc with cattle was 100%. The results of evolutionary tree showed that yak had the closest genetic relationship with cattle, zebu cattle and buffalo, but the farthest relationship with mice. Protein structure prediction showed that the 6 transcription factors OSKMNL of yak all had the corresponding protein functional structures of the gene family, such as POU domain, HOX domain, HMG domain, Znf-C2H2 domain, HLH domain, CSP domain and so on. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed. 293T cells infected by lentivirus were found to express red and green fluorescence. Yak fibroblasts infected with lentivirus showed red and green fluorescence in the experimental group, and RT-PCR results showed bands with expected size. In this study, 6 pluripotency related transcription factors OSKMNL of yak were successfully cloned. Lentiviral vectors FUW-teto-OSM-EGFP and FUW-teto-KNL-mCherry were constructed, which contain 3 transcription factors of yak, respectively. Lentivirus could infect yak fibroblasts. It is helpful to promote the application of pluripotent related transcription factor OSKMNL in yak stem cells and prepare for the follow-up study of yak iPSC.

The aim of this study was to analyze the molecular mechanism by which matrix metalloproteinase 14 (MMP14) regulated the differentiation of skeletal muscle satellite cells. Skeletal muscle satellite cells were isolated from 10 4-week-old C57/BL6 female mice by collagenase digestion. First, skeletal muscle satellite cells were induced to differentiate, and then qRT-PCR and Western blot were used to identify the expression changes of MMP14 during the proliferation and differentiation stages of satellite cells. siRNA was applied to inhibit the expression of MMP14 protein, which was divided into experimental group (si-MMP14) and control group (si-NC), with 3 replicates per group: firstly, the cells were induced to differentiate, and the level of cell differentiation between the experimental group and the control group was analyzed by immunofluorescence and qRT-PCR; furthermore, proteomic analysis was performed, combined with bioinformatics analysis to identify differentially expressed proteins and screen for key differentially expressed proteins and pathways regulated by MMP14. The results of this study show that: 1) The expression level of MMP14 was up-regulated in satellite cell proliferation stage and down-regulated in differentiation stage. Inhibition of MMP14 protein led to the obstruction of myogenesis, which was manifested by the decrease of myotube fusion index. 2) A total of 549 differentially expressed proteins were identified through proteomic analysis, including 66 up-regulated proteins and 483 down-regulated proteins. Differentially expressed proteins were mainly enriched in cell adhesion, fatty acid metabolism, and the AMPK pathway, and were involved in regulating biological processes such as cell fate determination, histone methylation, and chromatin structure. 3) MMP14 was found to directly interact with myogenic differentiation related proteins, lipogenesis related proteins, and heterochromatin structure regulation proteins, through protein interaction network analysis. Furthermore, the inhibition of MMP14 resulted in the down-regulation of myogenic differentiation transcription factors (PAX7 and MYOD) as well as H3-K9 methyltransferases (SETDB1 and SUV39H1), while leading to the up-regulation of lipogenic differentiation transcription factors (JUN and C/EBP-β). This study preliminarily analyzed the mechanism by which MMP14 regulates the differentiation of skeletal muscle satellite cells. MMP14 may be involved in the fate determination of satellite cells and the conversion of myogenic and lipogenic differentiation through H3-K9 histone methylation, which occurs before the initiation of satellite cell differentiation. The results of this study provide a theoretical basis for the study of skeletal muscle development.

Improving feed conversion efficiency in pigs is currently a key focus of pig breeding efforts. In this study, FCR values of 384 crossbred sows were ranked, and 20 pigs with extreme feed conversion ratio (10 with the highest FCR and 10 with the lowest FCR) were selected to form the HFCR group and LFCR group, respectively. Fecal samples were collected for 16S rRNA gene sequencing analysis. The results showed that the Richness index, Chao1 index, and ACE index of the LFCR group are significantly higher than those of the HFCR group (P < 0.01). The LEfSe analysis was used to identify microorganisms at the genus level with significant differences between the two groups. In the LFCR group, microorganisms with LDA>3 were mainly included Prevotella_1, Treponema_2, Prevotellaceae_NK3B31_group, Eubacterium_coprostanoligenes_group, p-1088-a5_gut_group, Prevotella_9, Akkermansia, Prevotellaceae_UCG_009, Ruminococcaceae_UCG-014, and Ruminococcaceae_NK4A214_group. In the HFCR group, microorganisms with LDA>3 were mainly included Megasphaera, Dialister, Catenibacterium, Pseudobutyrivibrio, Collinsella, Solobacterium, Subdoligranulum, and Streptococcus. The results of Spearman correlation analysis indicated that microorganisms significantly negatively correlated with FCR are mostly associated with short-chain fatty acid production, while microorganisms significantly positively correlated with FCR were mostly potential pathogenic bacteria. This study screened differential microorganisms in feces from pigs with high and low feed conversion rates, also conducted correlation analysis between the abundance of differential microorganisms and feed conversion rate traits, and ultimately identified fecal microorganisms significantly associated with feed conversion rate, thus provided a reference for the selection of intestinal microbial breeding markers for pig feed conversion rate traits.

The aim of this study was to reveal the effect and mechanism of epimedium on the estrus of gilts. A total of 32 gilts aged 210-220 days old, weighing (99.547±1.987)kg, mature and meeting breeding conditions were selected for the experiment. They were randomly divided into a control group and an experimental group, with 16 sows in each group and one sow in each replicate. Individuals in the control group was fed with a basic diet, while individuals in the experimental group was supplemented with 50 mg·d^-1 crude extract of epimedium in addition to the basic diet. Experimental feeding for 28 days. The results showed that gilts in the experimental group experienced early estrus, and serum FSH, LH, and E₂ significantly increased (P < 0.05). The ovarian transcriptome results showed that 477 upregulated differential mRNAs and 754 downregulated differential mRNAs were detected. GO enrichment analysis revealed that differential mRNAs was mainly enriched in processes such as transport vesicles, adipocyte differentiation, rhythmic behavior, and estrus cycle; KEGG enrichment analysis revealed that differential mRNAs was mainly enriched in tight junctions, carbohydrate metabolism, hedgehog signaling pathways, GnRH secretion, and PI3K-AKT signaling pathways. The results of ovarian metabolomics showed a total of 1 616 metabolites upregulated and 1 254 metabolites downregulated. KEGG enrichment analysis showed that differential metabolites were mainly enriched in α-linoleic acid metabolism, ATP transporters, linoleic acid metabolism β-pathways such as alanine metabolism, aminobenzoate degradation, and biotin metabolism. The results of network pharmacology analysis indicated that there were a total of 161 potential targets for the action of epimedium on the ovaries. The PPI interaction network obtained the top 10 protein grades as core targets, which were TP53, SRC, AKT1, CCND1, TNF, ESR1, EP300, ERBB2, JAK2, and PARP1 according to their scores. The GO analysis results indicated that the targets of epimedium on the ovaries were mainly involved in biological processes such as protein phosphorylation, positive regulation of MAPK cascade reaction, biological rhythm, positive regulation of gene expression, negative regulation of cell apoptosis process, positive regulation of intracellular calcium ion concentration, and positive regulation of RNA polymerase II promoter transcription. The KEGG analysis results showed that the target protein signaling pathway was enriched in PI3K-AKT signaling pathway, cell cycle, cell aging, HIF-1 signaling pathway, progesterone mediated oocyte maturation, and other pathways. The above results reveal the effects and pathways of epimedium on the estrus of gilts from the perspectives of in vivo, metabolism, transcription, and molecular level. This study found that feeding epimedium can change the transcription and metabolism patterns of sows′ ovaries, significantly increase FSH, LH, and E₂ levels, and promote estrus in gilts. The main components of epimedium can bind with TP53, SRC, AKT1, CCND1, TNF, ESR1, EP300, ERBB2, JAK2, and PARP1 to exert regulatory effects. This study provides a theoretical basis for the application of epimedium to improve the utilization rate of estrus in gilts.

The aim of this study was to investigate the effects of β-sitosterol (SITO), a natural plant-derived antioxidant molecule, on the in vitro maturation efficiency of porcine oocytes and the quality of early embryo development in vitro. In this study, SITO with different concentrations were added into porcine oocytes in vitro maturation medium, which was the experimental group, and the medium without SITO (containing 0.16% DMSO) was the control group, the concentrations in experimental groups were 10, 20 and 40 μmol·L^-1 respectively, and they were used to culture the oocytes in vitro collected from porcine ovaries in slaughterhouse. Different concentrations were repeated 3 times, and 150-200 oocytes were cultured in each group. After 46 h of culture, measured the maturation rate of oocytes, and mature oocytes were collected for reactive oxygen species, apoptosis, mitochondrial membrane potential staining and qRT-PCR of related genes; the adult oocytes were activated by parthenogenesis, the cleavage rate was calculated after 2 days, the blastocyst rate was calculated after 7 days, and the total number of blastocysts was calculated. The results showed that 20 μmol·L^-1 SITO treatment significantly improved the maturation rate of porcine oocytes in vitro, promoted the formation of blastocysts after parthenogenetic activation, decreased the ROS in oocytes, and increased the expression of anti-oxidative stress genes SOD and CAT. At the same time, SITO decreased the apoptosis level of oocytes, increased the expression of anti-apoptotic gene BCL-2, and decreased the expression of pro-apoptotic genes Caspase 3 and BAX. In addition, SITO could enhance mitochondrial membrane potential (ΔΨ m) and increase the expression of TFAM gene. In summary, the results of this study showed that adding 20 μmol·L^-1 SITO to porcine oocyte in vitro maturation medium can significantly promote the rate of porcine oocytes, and improve the in vitro development ability and embryo quality.

On the basis of feeding situation of dam and calf in yak industry, this study was conducted to investigate the effects of maternal nutritional supplementation in transition period and early weaning on the growth, serum biochemistry and immune function of yak calves, with the purpose of providing scientific basis for the efficient feeding of yaks. In this study, 18 healthy yaks in late gestation with similar body weight, day of pregnancy and 2-4 parities were randomly divided into 3 groups, including grazing feeding group (GF, n=6), nutritional supplementation group (SF, n=6) and nutritional supplementation and early weaning group (SW, n=6). Yaks in GF group were grazing feeding on the natural pasture throughout the transition period. The SF group were given a balanced nutritional supplementation with concentrate, hay and grazing feeding from 30 days before to 90 days after parturition according to the nutrition requirement of late pregnancy and early lactation, while yaks in the SW group were early weaned at 60 d after parturition besides balanced nutritional supplementation. The body weight and measurement of calves at birth and 90 d in GF, SF and SW groups were measured. Serum samples were collected at the age of 90 d to analyze the differences on serum biochemistry, growth related hormone secretion and immune function of yak calves with different maternal nutritional regulation. The results showed that the birth weights of calves in the SF and SW groups were significantly increased when compared to those in the GF group (P < 0.05). At 90 d of age, body weight, height and chest circumference of calves in the SF and SW groups were significantly higher than those in the GF group (P < 0.05), and there was no significant differences in calf weights and body measurements between SW and SF groups (P>0.05). Compared with the GF group, the serum glucose (GLU), globulin (GLB), and cholesterol (CHO) concentrations of calves in the SF and SW groups were significantly increased (P < 0.05), and calves in the SF group had significantly higher serum TP levels than those in the GF group (P < 0.05). For growth-related hormones, insulin-like growth factor I (IGF-Ⅰ) concentrations were significantly improved by the SF and SW treatment (P < 0.05). Serum growth hormone (GH) level in the SW group were significantly higher than that in the GF group (P < 0.05). Regarding to immune function, the serum secretory immunoglobulin A (sIgA) concentration of SF and GF groups was significantly higher than that in SW group (P < 0.05). In comparison with the GF group, the serum immunoglobulin A (IgA) level was significantly increased in the SF and SW groups (P < 0.05), and the serum immunoglobulin G (IgG) concentration was significantly higher in the SF group than in the GF group (P < 0.05). In conclusion, the nutritional supplementation in transition period provided sufficient nutrients to yak dam in late pregnancy and early-lactation, increased nutrient intake of yak calf thus enhanced the birth weight, serum glucose and nitrogen metabolite concentrations, and increased the secretion of growth-related hormone and immunoglobulin, as a consequence of that, the growth of yak calf was promoted. Meanwhile, early weaning with nutritional supplementation had no obvious negative effect on calf growth and immune function, and the secretion of growth hormone was further improved.

Since 2010, the appearance of the GII variant of porcine epidemic diarrhea virus (PEDV) has significantly increased the mortality of porcine epidemic diarrhea (PED) worldwide. Vaccine immunization has become an important strategy to prevent and control PED, and the neutralizing antibodies level induced by vaccines serves as a vital indicator to evaluate the immune effect and vaccine quality. However, the traditional PEDV neutralization assay on Vero-CCL81 cells depends on the addition of exogenous trypsin, which brings challenges such as unstable and unrepeatable results. This study aims to establish an accurate detection method to evaluate neutralizing antibodies in samples by constructing a recombinant viruses stably expressing a green fluorescent protein. In this study, part of ORF1a and ORF1b gene fragments and other structural genes of the GIIb subtype PEDV-GDU strain were cloned and inserted into the pUC57 vector. Furthermore, ORF3 gene was replaced by EGFP gene by using seamless cloning technology and an infectious cloning plasmid pUC57-PEDV-GDU-dORF3-EGFP was conducted. The recombinant virus with EGFP, which named rPEDV-GDU-dORF3-EGFP, was rescued. Furthermore, the biological characteristics of rPEDV-GDU-dORF3-EGFP and its parental virus were compared and evaluated by indirect immunofluorescence assay (IFA), Western blot and single-step growth curve assays. The rPEDV-GDU-dORF3-EGFP was further used to screen a cell line that supports the effective proliferation of PEDV without trypsin, and a neutralization assay was established with this cell line and rPEDV-GDU-dORF3-EGFP. The results demonstrated that the rPEDV-GDU-dORF3-EGFP was successfully rescued, and the rPEDV-GDU-dORF3-EGFP exhibited similar biological characteristics to parental virus in Vero-CCL81 cells. A subcloned Huh7 cells screened by the recombinant reporter virus can support infection and replication of PEDV without the addition of exogenous trypsin. Subsequently, a PEDV neutralization assay was successfully established using Huh7.10 cells and recombinant reporter virus. The analysis of correlation showed the level of correlation between the neutralization assay we established and ELISA was higher than that of traditional PEDV neutralization assay. In our study, the reporter virus rPEDV-GDU-dORF3-EGFP was successfully recued and the reverse genetic operation platform of PEDV-GDU strain has been successfully established, which provides a powerful tool for the rapid preparation of PED vaccine candidates. In addition, the neutralization assay based on the recombinant reporter virus on Huh7.10 cells can more accurately determine the level of neutralizing antibody in serum, which provides effective technical support for the evaluation of vaccine immune effect.

Porcine epidemic diarrhea (PED) is a porcine intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), characterized by watery diarrhea, vomiting and dehydration. It has caused huge economic losses to the global pig industry, but there are still no effective vaccines and treatments. Salinomycin (SLM) is a commonly used antibiotic with the advantages of low resistance, rapid excretion and very low residue. To explore the inhibitory effect of SLM on PEDV, firstly, the proliferation pattern of PEDV on Vero cells was detected by TCID₅₀, and then the CC₅₀ and IC₅₀ of SLM were measured by CCK-8 test and cytopathic effect (CPE). Finally, the co-culture models of SLM, PEDV and Vero cell were established. The effects of SLM on the replication cycle of PEDV were determined by RT-qPCR, Western blot and indirect immunofluorescent assay. The results showed that the highest virus titer (10^7.7·0.1 mL^-1) was observed in Vero cells at 24 hours after PEDV infection. The CC₅₀ of SLM on Vero cells was 7.698 μmol·L^-1, and the IC₅₀ for PEDV was 1.617 μmol·L^-1. The study showed that viral titer, N protein and N gene mRNA expression of PEDV gradually decreased with the increase of SLM concentration. SLM mainly inhibited the replication phase of PEDV, and had no significant effect on the adsorption, invasion and release phases of the virus. The research results could provide new ideas for further exploring PEDV inhibitory drugs.

Mycoplasma bovis (M. bovis) is an important pathogen that causes a variety of bovine diseases including bovine mastitis and calf pneumonia. Adhesion proteins are often thought to play key roles in the pathogenic mechanisms of M. bovis. Therefore, this study aimed to investigate four putative proteins of M. bovis Guizhou strain (GZ-2), namely M27, M32, M498, and M663, through prokaryotic expression in Escherichia coli BL21 (DE3) and purification, subcellular localization and adhesion studies. The results showed that recombinant M27, M32, M498 and M663 proteins were successfully expressed and purified; M27, M32 and M498 proteins all showed specific blotting bands in total proteins, cytosolic and cytoplasmic proteins of M. bovis, whereas M663 was undetectable; Immunostaining results showed that M27, M32, M498 and M663 proteins as well as M. bovis adhered to embryonic bovine lung (EBL) cells under confocal laser scanning microscopy, and the rabbit anti-M27, M32, M498 and M663 antibodies were able to inhibit the adherence of these proteins and M. bovis to the EBL cells. This was further confirmed by flow cytometry analysis with identical results. In conclusion, M27, M32 and M498 proteins are adhesion-associated proteins of M. bovis; of note, although M663 proteins were not detected in the Western blot analysis, it still is able to adhere to EBL cells and simultaneously inhibits the adhesion of M. bovis to EBL cells. This finding provides a basis of reference for the selection of target proteins for the diagnosis of M. bovis-associated diseases and the development of preventive vaccines.

Heat shock protein 70 (Hsp70) is an important membrane protein of Mycoplasma ovipneumoniae (Movi). It is highly conserved in the body but varies greatly in species and can be used as a candidate target gene for molecular biological detection. The purpose of this research is to establish a universal TaqMan real-time fluorescence quantitative PCR (qPCR) assay for the detection of Movi based on the Hsp70 gene and to further understand its genetic evolution. In this study, specific primers and probes were designed based on Hsp70 genes retrieved from GenBank to establish a qPCR method for the screening of Movi infection, after which eighty-eight goat nasal swabs and 43 lung samples of suspected Mycoplasmal pneumonia of sheep and goats (MPSG) were tested by the established qPCR method. Lung samples that tested positive for Movi were then used for pathogen isolation and identification, and the Hsp70 genes of the isolated strains were sequenced and analyzed. The results showed that the established qPCR method had a correlation coefficient of 1.00, and the amplification efficiency was 96%, slope was -3.411, Y-axis intercept was 37.29. The method was specific, and there was no cross reaction with other pathogens, such as Mycoplasma mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp.capripneumoniae(Mccp), Acholeplasmalaidlawii (AL), Mycoplasma agalactiae (Maga), Corynebacterium pseudotuberculosis (CP), Orf virus (ORFV), or Mycoplasma bovis (Mb). The method was sensitive, with a minimum detection limit of 5.72 copies.μL^-1. The method is reproducible, with intragroup and intergroup coefficients of variation both less than 1.00%. The length of the Hsp70 gene of the six Movi isolates were all 1 818 bp, and there were 96.0%~99.4% and 98.0%~100.0% homology of the nucleotide and amino acid, respectively, between the six Movi isolates with other Movi reference strains. Further analysis showed that goat-origin Movi had 1 more N-glycosylation site than sheep-origin Movi. The genetic analysis showed that they were both in the ClusterⅠA subgroup (goat-origin). In conclusion, this study established a specific, sensitive and reproducible qPCR method for the detection of Movi based on the Hsp70 gene. Sequence analysis showed that there was high nucleotide homology in the Hsp70 gene of Movi from different sources. Phylogenetic analysis confirmed that the Movi Fujian-origin strains had a close genetic relationship with the goat-origin strains. This study not only provides technical support for the clinical diagnosis of Movi but also provides reference for further understanding the genetic evolution of Movi.

Tuberculosis (TB) is a chronic respiratory disease caused by Mycobacterium tuberculosis (Mtb). There is mounting evidence that inflammatory response plays an important role in regulating the host immune responses. Aconitate decarboxylase 1 (ACOD1) is a stress-related inducible protein associated with inflammation and infection, many studies have identified ACOD1 as one of the most upregulated genes under various conditions associated with infection. However, the role of ACOD1 in the regulation of RAW264.7 macrophage inflammatory response induced by attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) infection remains unclear. In this study, we investigated the effect of ACOD1 on RAW264.7 cell inflammatory response during BCG infection. In addition, we explored the role of ACOD1 in regulating BCG-induced RAW264.7 cell inflammatory response using small interfering RNAs targeting ACOD1. The experiment used Western blot, Quantitative Real-time PCR (qRT-PCR) and immunofluorescence to detect the levels of ACOD1, cytokines and TLR4/MyD88/NF-κB signaling pathway proteins. The results demonstrated that BCG could induce macrophage inflammatory response and ACOD1 upregulation in a time and concentration-dependent manner; Knockdown of ACOD1 could attenuate BCG-induced inflammatory response in RAW264.7 cells. Moreover, we found that ACOD1 knockdown decreased the expression of IL-1β, TNF-α, IL-6, COX2, iNOS, and increased the levels of anti-inflammatory cytokines about IL-4 and IL-10; ACOD1 can also activate TLR4/MyD88/NF-κB signaling pathway, decreased the levels of TLR4, MyD88, TRAF6, p-NF-κB p65. These results indicated that ACOD1 could regulate inflammatory response caused by BCG through TLR4/MyD88/NF-κB signaling pathway.

In this study, two strains of Clostridium perfringens type A, named ZWCP209 and ZWCP210, were isolated from the tissues and organs of sudden death Hainan cattle. Genome assembly results showed that the genome size was between 3.4-3.5 Mb, and the number of tRNA and CDS was stable. Multilocus sequence analysis (MLST) showed that the sequenced strains belonged to the same new ST type. The genomes of 27 bovine C. perfringens strains were obtained from the NCBI database and analyzed by bioinformatics together with sequenced isolates: A total of 13 resistance genes were detected, among which the tetracycline resistance genes tetA(P) and tetB(P) had the highest carrying rate (79.4%). It is noteworthy that the two Hainan cattle isolates in this study carried more than 7 resistance genes, including optrA, which is resistant to oxazolidinone for non-veterinary use. Pan-genome analysis showed that the genomes of these strains contained a total of 7 345 genes, and the number of core genes accounted for 22.94%. In the phylogenetic analysis of the core genome and plc gene, the two Hainan cattle isolates were in the same branch and had the same virulence factors, with high homology. This study is the first complete genome sequencing and bioinformatics analysis of C. perfringens isolated from cattle in Hainan Province in China, which has a reference value for the prevention and treatment of C. perfringens disease in cattle and the study of genome.

The recombinant protein rEnApiAP2 of Eimeria necatrix alone and in combination with the recombinant proteins rEtGAM56 or rEtGAM59 of E. tenella were used to immunize chicks. The groups immunized with rEtGAM56 or rEtGAM59 alone, and the groups unimmunized and challenged (UC) or unimmunized and unchallenged were used as controls. The chicks were infected with E. necatrix or E. tenella, respectively. The survival rate, bloody stool, weight gain (WG), relative weight gain (RWG), lesion score (LC), oocyst reduction (OR), anticoccidial index (ACI) and serum antibody level (SAL) were used as indexes to evaluate the immune protective effect of rEnApiAP2 alone and in combination with rEtGAM56 or rEtGAM59, respectively. The results showed that the survival rate of all groups was 100%, the total number of bloody stools excreted by chicks in the immunized groups was less than that in the UC group, and the WG of the immunized groups were higher than that of UC group. Among the groups immunized with rEnApiAP2 alone, the RWG, LC and ACI in the group immunized with low dosage was the highest (82.36%, 73.88% and 155.26 respectively), and the LC was the lowest (1.71). Among the groups infected with E. necatrix, the ACI in the co-immunization groups was higher than that in the groups immunized with rEnApiAP2 or rEtGAM alone, of which the ACI in the group co-immunized with rEnApiAP2 and rEtGAM56 was the highest (169.83). Among the groups infected with E. tenella, the ACI in the group immunized with rEnApiAP2 (128.37) was lower than that of the group immunized with rEtGAM56 (130.12) and the group immunized with rEtGAM59 (151.88). The ACI in the group co-immunized with rEnApiAP2 and rEtGAM59 (141.62) was close to that in the group immunized with rEtGAM59 alone, and the ACI in the group co-immunized with rEnApiAP2 and rEtGAM56 (115.46) was lower than that in the group immunized with rEtGAM56 alone. The SAL of all immunized groups was significantly higher than that of the control group (P < 0.05). In conclusion, rEnApiAP2 protein had good immunogenicity and was able to induce effective immune protection against E. necatrix, and co-immunization with rEtGAM could enhance its immune protection against E. necatrix. These results laid a foundation to develop a recombinant coccidiosis vaccine.

The present study aimed to evaluate mice as a candidate of the experimental model of Mycoplasma ovipneumoniae (Mo) infection, and screen the optimal method of Mo infection mice model. Sixty healthy BALB/c mice were randomly divided into control group, Mo infection group 1, infection group 2, infection group 3 and infection group 4 (n=12). Mice in Mo infection group 1 were challenged once with 30 μL and 25 μL 10⁸ CCU ·mL^-1 NJ01 strain cells by nasal and intraperitoneal drip, respectively. Mice in Mo infection group 2 and 3 were intranasally inoculated twice and three times 30 μL 10⁸ CCU ·mL^-1, respectively. Mice in Mo infection group 4 were inoculated twice 50 μL 10⁸ CCU ·mL^-1 by throat spry. The control group were inoculated with the normal liquid culture. The body weight was respectively determined on the 0, 7th and 14th day post-infection (DPI). Mice in five groups were killed on the 14th DPI, blood and lung tissue samples were collected. To determine whether the infection model was established successfully and evaluate the optimal establishment method of M. ovipneumoniae infection model, the histopathological examination of lung tissue were observed by HE staining and histopathologic score, the payload of Mo in lung tissue was detected by quantitative PCR, and Mo IgG level in serum was determined by ELISA. On the 14th DPI, compared with control group, the body weight of mice in infection group 2 and group 4 significantly reduced 17.2% (P < 0.05) and 21.6% (P < 0.05). A mice died on the 13th DPI in infection group 2 and group, 4 respectively. On the 14th DPI, mice had inflammation in lung and lung histopathologic score was 3.5 and 3.3 in infection group 2 and group 4, respectively, which were significantly higher than both infection group 1 (P < 0.05) and 3 group (P < 0.05). No lung lesion was found in control mice. Histopathological examination showed that different degrees of interstitial pneumonia were observed in the lungs of the Mo infection groups. Inflammatory cell infiltration was seen in alveolar cavity. In the control group, the lung tissue structure was normal and intact, and there was no obvious inflammatory cell in alveolar cavity. The DNA copy numbers of Mo in infection group 1 and group 3 were 10^2.56 and 10^3.21 copies ·g^-1, respectively. The infection group 2 and group 4 were 10^3.84 and 10^3.77 copies ·g^-1 respectively. They were significantly higher than both group 1 (P < 0.05) and group 3 (P < 0.05). Control group was negative. The serum antibody OD_{450 nm} of Mo infection mice were 0.63, 1.05, 0.81 and 0.99 in infection group 1 group 2, group 3 and group 4, respectively, which were all significantly higher than control group (P < 0.05). Mo antibody level in infection group 2 and group 4 were significantly higher than infection group 1 (P < 0.05). Mice model infected M. ovipneumoniae were successfully developed in this experiment by nasal drip and throat spray 1×10⁸ CCU ·mL^-1 for continued two times Mo which can provide important basis for investigating the pathogenesis and therapy of mycoplasma pneumonia of sheep.

This experiment was conducted to study the antimicrobial resistance of Escherichia coli isolated from Mawang ducks to β-lactams, aminoglycosides, tetracyclines, amphenicols, fluoroquinolones and sulfonamides, and dissect the prevalence of β-lactamase genes and plasmid-mediated quinolone resistance (PMQR) genes in E. coli isolates. One hundred and forty six cloacal swab samples were collected from Mawang ducks at four farms in Youyang county, Chongqing municipality. E. coli was isolated and identified by selective enrichment and PCR amplification of the E. coli specific phoA gene. Antimicrobial resistance to 25 antimicrobials was determined using the disk diffusion method on Mueller-Hinton plates. The presence of β-lactamase genes and PMQR genes were detected by PCR. A total of 146 E. coli isolates were obtained from Mawang ducks. These isolates were severely resistant to ampicillin and cefazolin, with resistance rates of over 50%. However, they were completely susceptible to gentamicin, amikacin and ofloxacin. One hundred and eleven isolates (76.0%) and 83 isolates (56.8%) carried at least one type of β-lactamase genes and PMQR genes, respectively. The bla_TEM (74.0%, 108/146) and qnrS (54.8%, 80/146) genes were the most prevalent β-lactamase gene and PMQR gene, respectively. Sixty-three isolates carried β-lactamase gene(s) and PMQR gene(s) simultaneously. Mawang duck-derived E. coli was resistant to commonly used antimicrobials and carried β-lactamase genes and PMQR genes extensively.

In order to verify anticoccidial effect, optimal dosage and medication days of Changshan Oral Liquid (CSL), and lay the foundation for the application of new veterinary drug in future, the therapeutic effect analysis of experimental infected chicks and clinical cases of CSL on chicken coccidiosis were carried out. Healthy chicks were raised to 16 days under non-coccidiosis condition, and were randomly divided into six groups: healthy control group, infected control group, toltrazuril control group, and low dose group, medium dose group and high dose group of CSL (2.5, 5.0, and 10.0 mL·L^-1 drinking water respectively), 20 chicks were assigned in each group. Except for healthy control group, the rest groups were artificially infected with 6×10⁴ sporulated oocysts of Eimeria tenella (E. tenella) oocysts and the drugs were administered after 24 hours infection for 7 days. The clinical signs of chicks in each group were observed daily, such as spirit, feeding, drinking water and feces, etc. If there were dead chicks, they should be timely dissected to observe the anatomical changes, and anticoccidial index were calculated. Moreover, the chicks naturally infected with Eimeria oocysts were selected in the therapeutic effect analysis of clinical cases and divided into recommended dose group of CSL (5.0 mL·L^-1 drinking water) and toltrazuril control group, which were administered continuously for 4 days. The therapeutic effect analysis of experimental infected chicks showed that medium-dose and high-dose of CSL had good anticoccidial effect, anticoccidial index were 166.6 and 173.8 respectively. Besides, the doses above had good growth-promoting effect on chicks and relative weight gain were 119.8% and 101.6% respectively. The therapeutic effect analysis of clinical cases showed that CSL can effectively treat chicken coccidiosis. The cure rate and effective rate were 80.7% and 96.7% respectively. In the infected group, we found more Eimeria oocysts in cecal epithelial cells, lots of inflammatory cells and red blood cell in the tissues. In all drug groups, the chicken cecal structure were basically normal and intact. Our results suggest that CSL as a preparation of traditional Chinese medicine, has the characteristics of environmental protection and good anticoccidial effect, which has the potential to develop a new drug.

The purpose of this study was to investigate the role of renin-angiotensin system (RAS) in gut-vascular barrier injury in ulcerative colitis mice. Sixteen ICR mice were selected and randomly divided into control and DSS groups by equal volume saline gavage. Mice in the DSS group were supplemented with DSS (final concentration of 4%) in the drinking water starting on day 4 of the study to establish a murine ulcerative colitis model. During this period, body weight, fecal characteristics, and fecal occult blood of the mice were recorded daily for the calculation of DAI. Until day 8 of DSS drinking, all mice were necropsied after blood collection, then the colon length was measured, and colon tissue samples were collected. The following experiments were performed: 1) HE staining to observe the histopathological changes in the colon of each group of mice; 2) ELISA to detect the levels of VEGFA in blood and Ang1-7 and Ang Ⅱ in colon tissues; 3) Western blot to detect the expression of ACE2, ACE and PLVAP in colon tissues; 4) Spearman correlation analysis was used to analyze the correlation between ACE2, ACE expression changes and PLVAP expression changes, as well as the correlation between Ang1-7 and Ang Ⅱ content changes and VEGFA content changes. Results: 1) A DSS-induced ulcerative colitis model was successfully established in mice, which showed decreased body weight, highly significant increase in DAI (P < 0.01), highly significant shortening of colon (P < 0.01) and severe pathological changes in colon tissues; 2) Compared with the control group, mice in the DSS group showed colonic hemorrhage, highly significant increase in PLVAP and VEGFA protein expression in colon tissues (P < 0. 01); 3) Compared with the control group, ACE2 and ACE expression in colon tissues of mice in the DSS group were highly significantly upregulated (P < 0.01), and the contents of Ang1-7 and Ang Ⅱ were significantly and significantly increased (P < 0.05 and P < 0.01); 4) Correlation analysis showed that there were positive correlations between ACE2 and ACE expression changes and PLVAP expression changes, as well as Ang1-7 and Ang Ⅱ content changes and VEGFA content changes. The above results suggest that the colonic vascular barrier is damaged during DSS-induced colonic inflammation, the two pathways of RAS in the colon are activated or out of balance, and the activation of ACE/Ang Ⅱ pathways is dominant, suggesting that ACE/Ang Ⅱ is involved in the injury of intestinal-blood barrier in mice with colitis. Activating ACE2 or overexpressing ACE2 to enhance its degradation of Ang Ⅱ to alleviate the damage of intestinal blood barrier may be a new idea or way to treat or alleviate ulcerative colitis.

Metabolism is the cornerstone of all physiological activities, and amino acid metabolism, as one of the three major substance metabolisms, is involved in a variety of physiological activities of the host organism. It is closely related to the function of host immune cells and has an irreplaceable role in the development of diseases. To investigate the effect of Streptococcus uberis (S. uberis) infection on amino acid metabolism in mammary epithelial cells, S. uberis and mammary epithelial cell line (EpH4-Ev) were used for quantitative analysis of amino acids of cells by targeted amino acid metabolomics techniques in this study. The key enzymes expression of key amino acid (arginine) metabolic pathway was also validated by RT-qPCR and Western blot. The results showed that the levels of glycine, threonine, ornithine, asparagine and glutamine were significantly up-regulated (P < 0.05), and the levels of glutamate, proline, arginine, serine, histidine, valine, isoleucine, alanine, tyrosine, tryptophan, lysine and aspartate were significantly down-regulated (P < 0.05) in S. uberis-infected cells, with arginine content was most significantly changed (P < 0.000 1, VIP > 1.2). Two glycogenic amino acids, asparagine and glutamine, were significantly increased (P < 0.05), two ketogenic amino acids, lysine and leucine, were significantly decreased (P < 0.05). Further exploration of the arginine catabolic pathway revealed that S. uberis infection of mammary epithelial cells significantly upregulated the mRNA and protein expression levels of NOS2, a key enzyme of arginine metabolism (P < 0.05), and significantly downregulated the expression level of ARG1 (P < 0.05). Compared with the control group, the expression of pro-inflammatory factors IL-1β and TNF-α in the cells challenged with S. uberis were significantly up-regulated (P < 0.05). In summary, S.uberis infection caused disruption of amino acid metabolism in mammary epithelial cells, accelerated intracellular arginine depletion, and initiated inflammatory responses. The results suggest that arginine has an important role in host resistance to S. uberis infection, and can be used as a breakthrough for early diagnosis and future control of mastitis in dairy cows.

The aim of this study was to explore the mechanism of asiatic acid (AA) on LPS-induced pyroptosis in kidney cells of yellow-feather broilers through HMGB1/TLR4/NF-κB pathway. Forty one-day-old healthy broilers were reared to 7 days of age and randomly divided into four groups: control group (CON), LPS-induced model group (LPS), AA low-dose group (LPS+AA 15 mg·kg^-1) and AA high-dose group (LPS+AA 30 mg·kg^-1). Broilers in the AA treatment groups were given the corresponding dose of AA by intragastric administration for 14 days. Except the control group, all broilers were injected intraperitoneally 0.5 mg·kg^-1 LPS to establish the model of acute kidney injury at 16, 18 and 20 days of age. Broilers were killed on the 20th day after LPS was intraperitoneally injected for 12 h and renal samples were collected. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue. The mRNA expression levels of HMGB1, TLR4, NF-κB, IκB, NLRP3, Caspase-1, IL-18, IL-1β and TNF-α in renal tissue were detected by RT-qPCR. Protein expression levels of HMGB1, TLR4, P-NF-κB, NLRP3, GSDMD, IL-1β and Cleaved caspase-1/Caspase-1 were detected by Western blot. The expression level of P-IκB was detected by immunohistochemical method. The protein expression levels of HMGB1, NLRP3 and ASC were detected by immunofluorescence. The results showed that LPS induced vesicular degeneration of renal tubular epithelial cells and glomerular dysplasia, which could be ameliorated by treatment with AA. At the same time, the mRNA levels of HMGB1, TLR4, NF-κB, IκB, NLRP3, Caspase-1, TNF-α and IL-18 in renal tissue were significantly downregulated by AA(P < 0.05). Protein levels of HMGB1, TLR4, P-NF-κB, NLRP3, GSDMD, IL-1β, and Cleaved caspase-1/Caspase-1 decreased. The results of immunohistochemistry and immunofluorescence showed that AA could significantly reduce the expression and distribution of P-IκB, HMGB1, NLRP3 and ASC in renal tissue by LPS-induced. In conclusion, AA can alleviate LPS-induced pyroptosis in kidney cells of broilers by inhibiting the HMGB1/TLR4/NF-κB pathway.

This experiment was conducted to investigate the effects of different concentrations of asiatic acid (AA)on lipopolysaccharide (LPS)-induced acute myocardial injury (AMI) in broilers. All 50 broilers were selected to be reared to 7 days of age, and then divided into 5 groups, namely Control group, LPS group, LPS+AA 15 mg·kg^-1 group, LPS+AA 30 mg·kg^-1 group, LPS+AA 60 mg·kg^-1 group. At 7 days of age, all the AA groups were pretreated with corresponding doses of AA by gavage for 14 consecutive days. At 16, 18 and 20 days of age, all the LPS groups were intraperitoneally injected with 0.5 mg·kg^-1 LPS to induce AMI. Myocardial samples of broilers were collected at the age of 21 days. Histopathological changes of myocardium were observed by hematoxylin-eosin(HE)staining; Detection of markers of oxidative stress in myocardial tissue using glutathione peroxidase (GSH-Px)and malondialdehyde (MDA)kits; Real-time fluorescence PCR(qRT-PCR)and Western blot were used to detect the expression of genes and proteins related to oxidative stress and ferroptosis in myocardial tissues. The positive expression rate of Nrf2 and SLC7A11 in myocardial tissues was detected by immunohistochemistry. The positive expression rate of GPX4 in myocardial tissues was detected by immunofluorescence. The results showed that AA pretreatment alleviated the myocarial injury induced by LPS. Compared with LPS group, medium-dose AA pretreatment increased GSH-Px activity while all AA pretreatment decreased MDA content in myocardium significantly (P < 0.05 or P < 0.01). Importantly, AA pretreatment decreased the mRNA expression of Keap1, PTGS2 and HMGB1, and increased the expression of Nrf2, HO-1, NQO1, FTH1, SLC7A11, GPX4, GCLC and GCLM (P < 0.05 or P < 0.01). AA pretreatment also decreased the protein expression of Keap1 and HMGB1, promoted the protein expression of Nrf2, HO-1, NQO1, FTH1 and GPX significantly (P < 0.05). Immunohistochemistry and immunofluorescence results showed that AA pretreatment could significantly increased the expression of Nrf2 (P < 0.01), SLC7A11 (P < 0.01) and GPX (P < 0.01). The results of the current study showed for the first time that AA could alleviate LPS-induced AMI by regulating oxidative stress and ferroptosis. AA is expected to be a potential food additive for preventing LPS-induced AMI in broilers in the future.

Based on the genomic sequence of a prevalent Lumpy skin disease virus (LSDV) in China, we established an MGB probe fluorescence quantitative PCR method for detecting the ORF61 gene. In this study, a specific fluorescent quantitative PCR primer with an MGB probe was designed based on the LSDV gene sequence, and the specificity and sensitivity of this primer pair were screened and verified. Finally, we screened a pair of qPCR primers targeting the LSDV ORF61 gene. Using pCAGGS-ORF61, a eukaryotic expression plasmid, the standard curve y=-3.287 5x+47.87 was obtained, the linear correlation coefficient was R²=0.998 6, and the amplification efficiency reached 101%. The results of the specificity test, repeatability test, and sensitivity test showed that this fluorescent quantitative PCR primer pair had the advantages of high specificity, good repeatability, and high sensitivity. The minimum detection limit of this primer pair was 6.71 copies·μL^-1, and the coefficient of variation of intra-batch and inter-batch repeatability results was less than 2%. The above results indicate that we have established a fluorescence quantitative PCR method for the specific detection of LSDV, providing an effective detection method for preventing and controlling LSD.

In order to explore the effect of Toll-interacting protein (Tollip) on the proliferation of Foot-and-mouth disease virus (FMDV), CRISPR/Cas9 gene editing technology was used to construct a porcine kidney cell line (PK-15) with Tollip gene deletion, and the knockout effect was confirmed by PCR sequencing and Western blot, Then, CCK-8 kit was used to determine the change of cell viability after Tollip deletion, and there was no obvious difference between knockout cells and wild cells. Western blot, indirect immunofluorescence, real-time fluorescence and viral titer were used to determine the replication of viruses in knockout cell lines during FMDV infection. The results showed that knockout cells significantly promoted virus replication compared with wild cells during FMDV infection. In summary, Tollip deletion cell lines were successfully constructed on PK-15 cells, and the results showed that Tollip deletion contributed to FMDV replication. These results provide a theoretical basis for the subsequent study of Tollip function and antiviral mechanism.