CRISPR gene editing technology can accurately and efficiently change the DNA source code and has been widely used in animal and plant breeding in recent years.In practice, the demand for multi-trait parallel improvement of agricultural organisms is increasing. The change of a single gene or locus alone cannot meet the above requirements.Therefore, it is desirable to establish a multi-gene simultaneous editing system to modify multiple genes. Simultaneous expression of multiple sgRNAs is critical to simultaneous editing of multiple genes. The commonly multi-sgRNA expression strategies include multiple single-cistron sgRNA parallel expression and multi-cistron sgRNA tandem expression. The frequently-used tandem expression tools include nuclease Csy4, tRNA system and self-cleaving ribozyme. This review analyzes and summarizes the advantages and disadvantages of the above multi-gene editing technology, discusses the future direction, and stresses its significance and application prospects.

Single-cell transcriptome sequencing technology(scRNA-seq) can identify individual cell types and cell states with high-precision resolution, breaking the dilemma that ordinary transcriptome sequencing unable to probe the specific expression characteristics of target cells, and playing an important role in the exploration of various fields, which is now widely used in many fields, such as human and mouse developmental biology, oncology, immunology, complex diseases, intestinal microbiome and clinical applications. In recent years, scRNA-seq has also carried out some pioneering research in the field of animal husbandry, mainly focusing on animal reproductive performance, embryonic development, analysis of key traits, etc. However, the application of scRNA-seq in other fields remains to be in-depth compared to its application in humans. In this paper, workflow of scRNA-seq and its application in domesticated animals is reviewed, intending to provide a reference for improving the efficiency of scRNA-seq analysis and its innovative application in domesticated animals.

Insulin is an essential hormone and plays crucial roles in a variety of biological processes including glucose and lipid metabolism, protein synthesis, growth, development, and trait formation. Chickens have natural insulin resistance, but the cause of chicken insulin resistance is still unknown. This review summarizes the research progress on the insulin signaling pathway in chicken liver, muscle and adipose tissue, and then proposes future research directions in chicken insulin signaling pathway and insulin resistance, with a view to providing new ideas for in-depth exploration of human insulin resistance and genetic improvement of chicken meat quality.

The coat color of the domestic horse is the main element and basis for identifying horses and registering breeds. Ancient DNA studies have shown that the first coat color appeared in the domestic horse was bay, followed by black and chestnut, which are collectively known as the three base coat colors. Like other mammals, pigment cells in the domestic horse produce different coat colors through different combinations of eumelanin and phaeomelanin. In this paper, we systematically summarize the 13 different coat color classifications, and review the progress of candidate genes related to the formation of different coat colors in the domestic horse over the past 30 years, so as to provide theoretical references and bases for the in-depth understanding of the genetic mechanism of the formation of different coat colors in the domestic horse, the reduction of the occurrence of the related genetic diseases, as well as the subsequent horse registration and breeding.

Bovine embryo in vitro production (IVP) is a modern reproductive biotechnology, which is widely used in livestock production and biological research and plays an essential role in promoting breed improvement and reproductive efficiency for both beef and dairy cattle. As development of molecular biology, biochemistry and cell biology, a great progress has been made in bovine IVP. However, the current bovine IVP is still facing bottleneck problems, such as low efficiency of in intro embryo production and poor embryonic development potential, seriously hindering the further large-scale promotion and application of this technology. In this paper, the research progress of classical and latest additives in bovine IVP was systematically reviewed, intending to provide references for optimizing and constructing the technical system of different additive combinations, and aiming to more efficiently obtain high-quality bovine in vitro embryos.

Endometritis in dairy cows is a reproductive disorder caused by pathogenic microorganisms infecting the uterus during or after delivery, which seriously affects the reproductive ability of dairy cows and restricts the development of dairy industry. Understanding the pathogenesis of endometritis in dairy cows is helpful for its efficient treatment. However, the causes of endometritis in dairy cows are more complex, such as bacteria, viruses, fungi, mycoplasma, etc. With the deepening of research, it has been proposed that the disorder of intestinal flora may also affect the occurrence and development of endometritis in dairy cows. Some studies showed that probiotics can improve endometritis in dairy cows by regulating the host gut microbiota. This article reviews the relationship between gastrointestinal flora and endometritis in dairy cows and the application of probiotics in endometritis in dairy cows, in order to provide new ideas for the management of endometritis in large-scale dairy farms.

Dairy products, as one of the essential human foods, have attracted widespread attention due to the presence of active proteins and peptides. This review summarizes the bioactive functions of active proteins and peptides in five types of specialized milk (goat, sheep, donkey, camel and yak), including their roles in regulating immune function, antioxidation, antimicrobial activity, and anti-tumor effects. Furthermore, based on relevant studies, the current review provides a prospective outlook on their potential applications in disease prevention and health promotion. This review contributes to a more comprehensive understanding of the functional mechanisms of active proteins and peptides in specialized milk, offering valuable references for future research.

Avian metapneumovirus disease is an emerging viral infection caused by avian metapneumovirus (aMPV), an infectious disease characterized by acute upper respiratory tract infection and a severe decrease in egg production and eggshell quality of laying chicken. aMPV causes septicemic and exudative diseases of domestic ducks, geese and turkeys, leading to economically devastating to poultry industries. aMPV can infect chickens, ducks, pigeons and other birds, and is widely prevalent around the world, causing huge economic losses to the poultry industry. In this paper, we review aMPV in terms of genomic structure and its encoded proteins, epidemiological status, pathogenicity, infection-induced host immune response, and its diagnostic techniques and vaccines to provide a scientific basis for the diagnosis and prevention of the disease in the future.

Calicivirus is a single-stranded positive-sense RNA and non-enveloped viruse, which has a wide host range and thus seriously threaten the health of humans and many other vertebrates. Due to the variety of virus species and epidemic strains, easy transmission and regional infection, public health diseases caused by calicivirus frequently broke out worldwide, and its incidence of infection has increased significantly in recent years. Although different genera infect different types of hosts, their main capsid proteins have similar structures. The major capsid protein of calicivirus contains critical regions of immunogenicity, which can bind to host receptors and may mediate the infection process. Moreover, amino acid-related immunogenicity and variation of receptor binding sites are recognized as driving forces of viral evolution. In this paper, we reviewed the progress of research on the genomic structure, immunogenicity, receptor binding and genetic evolution of the major capsid proteins of calicivirus, with a view to providing references for subsequent scientific research and effective prevention and control of related diseases.

The genus of Eimeria parasite is the most common parasite in poultry and livestock, causing severe digestive system diseases in animals such as chickens, rabbits, sheep, and cattle, which affect animal health and the economic benefits of animal farming. With the rapid development of molecular biology technologies, researchers have established genetic manipulation platform for Eimeria, including transient transfection, stable selection, and gene editing. These techniques have realized gene overexpression, gene knockout, and gene editing in Eimeria, greatly advancing the understanding of Eimeria's basic biology and its potential as a novel eukaryotic vector vaccine. However, challenges still remain in this field. This article systematically reviews and summarizes the key technologies, innovative designs, technical bottlenecks, and application prospects of Eimeria's genetic manipulation platform, providing references for the establishment of genetic manipulation platform for other livestock parasites and offering new insights for the development of new control measures against coccidiosis.

Manganese is an important trace element that participates in various physiological processes, including skeletal, reproductive, energy metabolism, and normal functioning of the nervous and immune systems. Compared with soluble manganese ions, nano-manganese can maintain the continuous release of manganese ions at the injection site, induce local immune responses, and enhance the cellular uptake of antigens by dendritic cells (DCs), promoting the activation and maturation of DCs in the body. This article reviews the relevant issues of nano-manganese from the perspectives of their mechanism of action, preparation methods, functional characterization, problems with nano adjuvants, and solutions. The application of nano-manganese as vaccine adjuvants was emphasized and the problems and solutions of nano-manganese were proposed.

Pathogen inactivation is the most critical and basic technology for producing inactivated vaccines. The ideal inactivation method is to completely inactivate the pathogen and maintain the immunogenicity of the antigen without chemical residues. Compared with traditional inactivation methods, the main advantages of irradiation technology in vaccine development are that it can penetrate the pathogen but causes less damage to the pathogen surface antigens, and does not require the removal of any chemical residues after inactivation. Therefore, irradiation technology is more suitable for developing safe and effective vaccines. This article briefly reviews and summarizes the history and progress of using irradiation technology in vaccine development, and discusses potential strategies for developing vaccines using radiation technology.

The aim of this study was to explore the characteristics of porcine transmissible gastroenteritis virus (TGEV) infection mediated by porcine aminopeptidase N (pAPN) gene knockout intestinal porcine epithelial cell line J2 (IPEC-J2) and to provide theoretical basis for further understanding the mechanism of pAPN gene in the process of TGEV infection. The study was divided into 3 groups, pAPN gene knockout IPEC-J2 group (IPEC-J2-KO group), wild-type IPEC-J2 group (IPEC-J2-WT group), and wild-type IPEC-J2 group not inoculated with TGEV (Mock group), with 3 replicates set up in each group. Firstly, quantitative real-time PCR (qPCR) was used to determine the best time point for collecting cell samples following inoculation with TGEV strain. Secondly, the off-target effect of IPEC-J2-KO was detected. Then, the infection characteristics of IPEC-J2-KO, IPEC-J2-WT inoculated with TGEV and Mock were analyzed by qPCR, western blot (WB), indirect immunofluorescence assay (IFA), and 50% tissue culture infective dose (TCID₅₀). Finally, the expression of NF-κB p65 and its phosphorylated protein pp65 in Mock, IPEC-J2-WT and IPEC-J2-KO were detected by WB. The results of qPCR showed that 24 hours after exposure was the best time point to collect cell samples to evaluate the impact of TGEV on IPEC-J2; The off-target analysis results showed that no off-target effects were detected in IPEC-J2-KO; The results of virus infection characteristic analysis showed that the virus copy number and virus titer in IPEC-J2-KO were significantly lower than those in IPEC-J2-WT (P < 0.001). Compared with the Mock, there was no significant difference in virus copy number and virus titer in IPEC-J2-KO (P>0.05), and no expression of TGEV-N protein was detected in IPEC-J2-KO. In addition, compared with the Mock, after inoculation with TGEV, the phosphorylation level of NF-κB p65 was significantly increased in the IPEC-J2-WT group (P < 0.001), while there was no significant difference in IPEC-J2-KO group (P>0.05). This study results showed that IPEC-J2-KO can effectively resist TGEV infection, and TGEV did not affect the activity of the transcription factor NF-κB in the innate immunity-related signaling pathway in IPEC-J2-KO. This study provided the evidence that IPEC-J2 could serve as a cell model for the study of TGEV infection characteristics, and laid the foundation for elucidating the mechanism of pAPN gene in TGEV invading host cells and researching new disease-resistant pig varieties.

This study aimed to investigate the effects and mechanisms of transient starvation on the metabolism and autophagy of porcine skeletal muscle satellite cells (SMSCs), to analyze the role of autophagy in skeletal muscle development, and to help design strategies for improving meat production traits in domestic pigs. Resuscitate the SMSCs cell lines isolated and preserved in the laboratory, and divide the cells into 5 groups based on the serum concentration: 20% serum group (control group), 15% serum group, 10% serum group, 5% serum group, and 0% serum group, to form varying degrees of hunger stress. When the cells fuse to 70%-80%, culture for 24 hours for detection, with 4 replicates in each group. Cell apoptosis, membrane potential, reactive oxygen species(ROS) levels were detected by flow cytometry; ATP levels were measured using a reagent kit, and the expression of autophagy marker proteins(LC3B-Ⅱ, p62) and pathway-related proteins(AMPK, mTOR) was detected by WB; transmission electron microscopy was used to detect changes in mitochondrial morphology and organelles in cells. The cell viability, level of p62 protein, and the p-mTOR/mTOR ratio decreased with the decrease of serum concentration; apoptosis rate, level of ROS, level of ATP, the level of LC3B-Ⅱ protein, the p-AMPK/AMPK ratio, the number of autophagy lysosomes and the abnormal rate of nucleus and mitochondria increased; Compared with the 20% serum concentration group, the 15% group showed a significant increase in membrane potential, while the 5% and 0% groups showed a highly significant decrease. This indicates that short-term serum starvation can induce autophagy induced by the AMPK/mTOR signaling pathway and accelerate cell metabolism, but at the cost of some toxic effects such as accelerating cell apoptosis and inhibiting cell proliferation.

This study aimed to investigate the effects of low temperature on adipose tissue morphology, lipid metabolism-related gene expression and enzyme activities, and AMPK/PGC-1α pathway in Hezuo pigs. Ten Hezuo pigs with similar body weight and good health condition at 75 days of age were selected and randomly divided into a control group ((23±2)℃) and a low-temperature group ((-15±2)℃), each with 5 pigs, after being reared for 7 d at room temperature, for 15 d of the experimental period. At the end of the experiment, the adipose tissue was collected from the axilla and groin of the Hezuo pigs, and the morphology of the adipose tissue was observed by HE staining; the activity of lipid metabolism-related enzymes was detected by ELISA; the expression of adipose browning heat-producing genes and lipid metabolism-related genes was detected by fluorescence quantitative PCR; and the expression of the AMPK/PGC-1α pathway proteins was detected by Western blot. The results showed that, compared with the control group, the axillary and inguinal fat of the Hezuo pigs showed browning changes, the number of adipocytes became larger, and the cross-sectional area was reduced after the low-temperature treatment; LPL activity, IL-6 mRNA expression and AMPK, P-AMPK and PGC-1α protein expression were highly significantly increased in axillary and inguinal fat (P < 0.01), and LEP mRNA expression and FAS activity were highly significantly decreased (P < 0.01); UCP3 and HSP70 mRNA expression were significantly increased in axillary fat (P < 0.05), and PPARγ mRNA expression was significantly decreased (P < 0.05), AMPK and TNF-α mRNA expression was highly significantly increased (P < 0.01); AMPK and TNF-α mRNA expression was significantly increased (P < 0.05) in inguinal fat, UCP3 and HSP70 mRNA expression was highly significantly increased (P < 0.01), PPARγ mRNA expression was highly significantly decreased (P < 0.01); PGC-1α, ADPN, FAT mRNA expression and HSL activity were significantly increased (P < 0.05) in inguinal fat but not in axillary fat (P>0.05) in Hezuo pigs after low-temperature treatment. In summary, the axillary fat and inguinal fat of Hezuo pigs under low temperatures underwent browning, and the area of adipocytes decreased, the number increased, the body heat production increased, and the key genes of lipid metabolism LEP, ADPN, PPARγ, FAT and the AMPK/PGC-1α pathway might be involved in the lipid metabolism of the organism.

The aim of this study was to characterize the structural variations (SVs) in the genome of Xinjiang brown cattle, and on this basis, to explore the structural variations affected during the breeding process of Xinjiang brown cattle. Based on the whole genome resequencing data, the structural variation in the genomes of Xinjiang Brown cattle, their parents (Swiss brown cattle and Kazakh cattle) and Chinese Simmental cattle was examined using 3 software programs, Manta, Delly and Lumpy, and then analyzed the genomes of Xinjiang Brown cattle in comparison with the remaining 3 breeds using principal component analysis, construction of phylogenetic tree and fixation index (F_ST). The results showed that a total of 54 969 SVs were detected in the 4 cattle breeds, with the highest percentage of deletion-type variants (56.59%) and the lowest percentage of insertion-type variants (0.03%). Quantitatively, the distribution of these SVs on chromosomes showed a tendency of decreasing with larger chromosome numbers. Shared and breed-specific variants existed among different breeds, with the local breed Kazakh cattle possessing the highest number of specific SVs. The results of principal component analysis and phylogenetic tree revealed that Xinjiang brown cattle were closely related to Kazakh and Swiss brown cattle. A number of key genes related to milk production traits (PI4K2A, ELOVL3, ECHS1, SCD, TCF7L2, PNLIPRP2, BTRC, PLCE1), growth and development (BMP6, TLL2, MAPK9, ROR2), adaptability (PRKCB) and immunity (GSTO2, GSTO1) were mined out by selective signaling analysis, gene annotation and enrichment analysis. This study analyzed the characteristics of Xinjiang brown cattle in terms of structural variation and revealed some highly differentiated genes in the breeding process of Xinjiang brown cattle, which provided basic information for promoting the genetic improvement of Xinjiang brown cattle.

The purpose of this paper was to evaluate the amplification effect of different single cell whole genome amplification (scWGA) systems on bovine trace blood genomic DNA by next-generation sequencing, and to establish a trace DNA whole genome amplification system. In this study, the whole genome of 1 ng blood genomic DNA of Huaxi cattle was amplified by MDA (multiple displacement amplification) and MALBAC (multiple annealing and looping-based amplification cycles) scWGA systems, and then the sequencing library was constructed based on the amplification products of the two systems and the original undiluted blood DNA. The whole genome sequencing (WGS) was performed using the DNBSEQ-T7RS sequencing platform. The amplification efficiency was evaluated by comparing the fragment's size, concentration and total mass of the amplified products. The amplification effects of the two systems were evaluated by analyzing GC content, average depth, consistent rate of typing and call rate. The results showed that the fragment of the amplified product in MDA system was larger than that in MALBAC system (8 kb vs.0.2-2 kb), the concentration and total mass of the product were significantly higher than those in MALBAC system (P < 0.05). Based on the sequencing data, the genome coverage of MDA system was significantly higher than that of MALBAC system at 1×and 5×sequencing depth(P < 0.05). In addition, the consistency of typing rate and call rate of MDA system were significantly higher than MALBAC system, while the allele drop rate and false positive rate of MALBAC system were significantly lower than MALBAC system(P < 0.05). In summary, this study revealed that the characteristics of two amplification systems of MDA and MALBAC were based on the DNA amplification of Huaxi cattle 1 ng blood genome, which provided a theoretical basis for improving the key amplification techniques in Huaxi cattle embryo genome selection and promoting the progress of Huaxi cattle genetics and breeding.

This research aimed to explore how connective tissue growth factor influences the growth and lactation differentiation of mammary epithelial cells in dairy cows within an in vitro culture setting. In this study, the effects of CTGF recombinant protein and overexpression of CTGF gene on cell proliferation, apoptosis, milk fat and milk protein synthesis and secretion and signaling pathways were analyzed. The EdU kit and flow cytometry were used to identify cell proliferation and apoptosis; the Nile red dye and triglyceride kit were used to count intracellular lipid droplets and milk fat levels in the culture solution; qRT-PCR and Western blot were used to detect the expression of β-casein, crucial signaling molecules in milk fat and protein synthesis, and the extracellular matrix cell surface receptor β1 integrin. Furthermore, the protein interaction between CTGF and β1 integrin was examined using co-immunoprecipitation. In vitro culture, both CTGF recombinant protein and overexpression of CTGF were found to enhance the growth of adherent cells by promoting proliferation and inhibiting apoptosis. Real-time quantitative PCR and Western blot results demonstrated that CTGF upregulated the expression of pro-proliferative CyclinD1, MAPK, Bcl2, while downregulating the expression of pro-apoptotic Caspase3 and Bax at both transcriptional and translational levels. Furthermore, CTGF upregulated the expression levels of FASN, SREBP1, PPARγ key genes involved in milk fat synthesis-leading to increased intracellular lipid droplets synthesis and extracellular triglyceride secretion. Additionally, it also upregulated the expression levels of PRLR, STAT5, p-STAT5, the key genes involved in milk protein synthesis-significantly promoting β-casein synthesis. Moreover, CTGF was found to upregulate the expression level of β1 integrin with Co-IP confirming a protein interaction between CTGF and β1 integrin. Ultimately, CTGF promoted the growth and lactating ability of cultured dairy cow mammary epithelial cells; CTGF coexists with β1 integrin in the adhesion complex and can achieve some of its biological effects by influencing the β1 integrin signaling pathway.

The purpose of this study was to investigate the effect of erythropoietin (EPO) on apoptosis factors in yak renal interstitial fibroblasts (RIFs). Firstly, the kidney tissues of 3 healthy yaks and cattle aged 4 years were collected respectively, immunohistochemistry and Western blot were used to detect the exact distribution of EPO and its expression in the kidneys of yaks and calves. Meanwhile, the yak RIFs were identified by primary culture, and different drug treatments were applied to regulate the EPO expression level in the RIFs, and the effects of EPO on the apoptosis-related genes Bax, Bcl-2, and PCNA at mRNA and protein expression levels were detected. Immunohistochemical results showed that EPO was positively expressed in collecting duct epithelial cells, renal tubular epithelial cells, and peritubular mesenchymal fibroblast-like cells in the kidneys of both yaks and calves. qRT-PCR and Western blot results showed that the expression of EPO in the kidneys of yaks was significantly higher than that in the calves (P < 0.001). Subsequently, the EPO-generating RIFs were cultured in primary culture and the expression of EPO was regulated, and the results showed that the activation group significantly elevated the expression of Bcl-2 and PCNA and decreased the expression of Bax in the RIFs (P < 0.001).The EPO-inhibition group significantly decreased the expression of Bcl-2, PCNA and up-regulated the expression of Bax in the RIFs (P < 0.001). In conclusion, it was showed that EPO was positively expressed in collecting duct epithelial cells, renal tubular epithelial cells and peritubular mesenchymal fibroblast-like cells in the kidney of yak and calf, and the high expression of EPO in the kidney of yak was able to promote the expression of Bcl-2 and PCNA and decrease the expression of Bax in the RIFs, which indicated that EPO could play an anti-apoptotic role in the kidney of yak.

The study aimed to investigate the effect of autophagy-related protein 14 (ATG14) regulate hair follicles growth and development by autophagy progress in rabbit dermal papilla cells (DPCs). In this study, healthy 6-month-old Angora rabbits were selected to collect dorsal skin to isolate and culture DPCs. The coding sequence (CDS) of ATG14 was cloned, and biological characteristics of ATG14 was analyzed by bioinformatics. After the overexpressed and knockdown of ATG14 in DPCs, and the expression of autophagy related protein and hair follicle growth and development related genes was detected, and the effect of ATG14 on the cell proliferation was investigated. The results showed that the length of CDS was 1 479 bp for the ATG14 gene, which could code 492 amino acids. Bioinformatics analyses indicated that the ATG14 protein didn't have potential signal peptides and transmembrane regions, which belong to the unstable proteins localized in the nucleus, and exhibited high homology in different mammals. After the overexpression and knockdown of ATG14 in DPCs, the WB results showed that ATG14 could upregulate the protein expression of autophagy related protein LC3 and Beclin1, but downregulate the autophagy inhibitor P62 protein expression. ATG14 could increase the fluorescent expression of pEGFP-LC3B in DPCs, indicating that ATG14 could activate the expression of LC3B. What's more, the overexpression of ATG14 could upregulate the mRNA expression of BCL2, CCND1, FGF2, WNT2 and LEF1, downregulate the gene expression of SFRP2 and TGFβ-1 (P < 0.05), the knockdown of ATG14 could downregulate the gene expression of BCL2, CCND1, FGF2, WNT2 and LEF1, upregulate the mRNA expression of SFRP2 and TGFβ-1 (P < 0.05). ATG14 could upregulate the expression of LEF1 and CCND1. The overexpression of ATG14 could promote the cell proliferation in DPCs (P < 0.01). In this study, the rabbit ATG14 gene regulating the autophagy process in DPCs was analyzed, which provided the theoretical basis for elucidating the regulatory mechanism of hair follicle growth and development in rabbit.

The aim of this study was to establish methods for the isolation, culture, identification and adipogenic differentiation of pigeon preadipocytes in vitro. Subcutaneous adipose tissue was collected from 3 healthy 1-day-old silver king pigeons. Pigeon preadipocytes were isolated by type I collagenase digestion. The isolation method was improved based on the conventional isolation method of preadipocytes. Primary and passage cultures were performed, and cell morphology was observed. The preadipocytes were identified by immunofluorescence staining using specific marker DLK1. Adipogenic differentiation was induced by adding insulin and sodium oleate to the culture medium. The distribution of lipid droplets in the cells was indicated by staining with BODIPY493/503. The triglyceride content in the cells were measured by the triglyceride assay kit. Quantitative real-time PCR (qPCR) and Western blot were used to detect the expression of adipogenic-related genes during preadipocytes differentiation. The results showed that the pigeon preadipocytes displayed spindle-shape. The modified isolation method yielded more preadipocytes compared to the conventional isolation method. Compared with 37 ℃, the number of preadipocytes was significantly increased at 41 ℃ (P < 0.001). Immunofluorescence staining confirmed positive DLK1 expression, indicating the obtained cells were indeed preadipocytes. The results of BODIPY493/503 staining revealed abundant lipid droplets in cells after 6 days of differentiation. The relative triglyceride content in the cells was significantly increased with differentiation time (P < 0.01). The qPCR data indicated that the expression of PPARγ, SCD, DGAT2, PLIN2, FASN, AFABP, and LPL genes was significantly upregulated after 2 days of adipogenic induction (P < 0.05), and continued to increase with longer differentiation time. The expression of SREBF1 gene was significantly up-regulated after 2 days of differentiation (P < 0.05), and remained unchanged thereafter. The expression of ACACA gene was significantly increased after 2 days of differentiation (P < 0.05), and reached its peak after 4 days of differentiation. The Western blot results showed that the relative expression of PPARγ, LPL and PLIN2 were significantly up-regulated after 2 days of differentiation (P < 0.05), and then further increased with the prolongation of differentiation time. In conclusion, this study successfully modified the conventional isolation method to isolate pigeon preadipocytes and screened the optimal culture temperature. The obtained pigeon preadipocytes were efficiently differentiated into mature adipocytes after induction with insulin and sodium oleate. This study provides a good cell model for investigating the molecular regulation mechanism of fat metabolism of pigeon, and also provided seed cells and technical guidance for the preparation of pigeon cell cultured meat.

The purpose of this article was to explore the serum proteomic characteristics of early pregnancy in Yili horses using proteomic techniques, screen for differential proteins, and analyze their roles in pregnancy. In this study, 20 healthy Yili mares with the same age and similar weight were selected for artificial insemination after natural estrus. Pregnancy diagnosis was performed on the 13th day after ovulation in the mare. Three pregnant mares and three non pregnant mares were selected based on ultrasound results, and their serum samples were collected. Using 4D-DIA technology for protein quantitative and qualitative analysis of serum samples from Yili mares, screening differential proteins, and conducting bioinformatics analysis such as GO and KEGG. The results showed that a total of 823 proteins were identified, with a molecular weight range of 8-1 010 ku; Based on the threshold of absolute multiple of differences greater than 1.5 times and P < 0.05, 53 proteins with significant differences were screened, of which 26 proteins were upregulated and 27 proteins were downregulated. Differential proteins mainly participated in biological processes such as cellular metabolism, biological regulation, metabolism, response to stimuli, cellular component organization, or biogenesis; The KEGG enrichment pathways included proteasomes, arginine biosynthesis, PI3K Akt signaling, ECM receptor interactions, and other pregnancy related pathways. Based on biological analysis such as GO and KEGG, six significantly different proteins were preliminarily screened for possible relationship with early pregnancy, and protein interaction network diagrams were drawn using them. The results showed that CTSS, MMP1, SERPINA5, and others were at key nodes. Six pregnancy specific proteins have been preliminarily screened in this study, providing basic data for screening pregnancy specific proteins and molecular regulatory mechanisms in equine animals in the future.

High throughput sequencing was performed on granulosa cell(GC)layer at early stage of laying interval(LI)to screen key genes involved in pigeon follicle development and selection. Eighty pairs of 12-month-old White King pigeons, with similar laying patterns and body weights were selected in this study. Three female pigeons were selected at first day (LI1) and third day (LI3) of LI, respectively, the GC layer of F1 and SF1 follicles were collected, followed by transcriptome analysis using RNA-seq. Differentially expressed genes (DEGs) among 4 groups: L1F1/L1SF1 group, L3F1/L3SF1 group, L1F1/L3F1 group, and L1SF1/L3SF1 group were analyzed. Gene Ontology (GO) functional annotation and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs were performed, protein-protein interaction (PPI) network analysis using the STRING database and visualization using Cytoscape software were conducted to selected key genes. Five DEGs from each group were randomly selected for RT-qPCR to verify the reliability of transcriptome. The 77, 2 736, 5 698 and 3 864 DEGs were obtained in the 4 groups, respectively. GO analysis results showed that DEGs in all groups were mainly enriched in cellular processes, cellular anatomical entity and binding. KEGG analysis result showed that DEGs in L1F1/L1SF1 and L3F1/L3SF1 groups were enriched in the steroid hormone biosynthesis and ovarian steroidogenesis signaling pathways. DEGs in L1F1/L3F1 and L1SF1/L3SF1 groups were enriched in the glycosaminoglycan degradation, synaptic vesicle cycle and oxidative phosphorylation signaling pathways. In addition, DEGs closely associated with follicle development and selection were screened by PPI.Five DEGs were randomly selected from each group for RT-qPCR verification, and the expression trends of these DEGs were consistent with the sequencing results. RNA-seq technology was applied to identify key genes associated with pigeon follicle development and selection, which including EEF2, DDX5, PLK2, IGF2R, LHCGR, HSD3B1, CYP19A1 and StAR. These results will provide the theoretical basis for further exploring the molecular mechanisms of follicle development and selection in pigeon.

The aim of the study was to investigate whether apolipoprotein A1 (apolipoprotein A1, Apoa1) mediates the reduction of testosterone synthesis in mouse Leydig cells line (TM3) induced by bisphenol A (BPA) exposure from the perspective of lipid metabolism. TM3 cells were randomly divided into 7 groups with different concentrations (0, 5, 10, 20, 40, 60, and 80 μmol·L^-1), and 0 μmol·L^-1 BPA was the control group (CON); After 24 h of treatment with the different concentrations, cell viability was detected using theby CCK-8 method to determine the optimal dose of BPA. Testosterone (testosterone, T) synthesiscontent in TM3 cell supernatant was detected by ELISA; mRNA expression levels of lipid metabolism-related genes Apoa1, Apoa2 (apolipoprotein A2) and Apoc3 (apolipoprotein C3) genes were measured in TM3 cells usingby RT-qPCR. APOA1 protein expression level was detected usingby Western blot and immunofluorescence method. Intracellular lipid droplet accumulation was observed usingby oil red O staining. The results showed that 20 μmol·L^-1 BPA treatment for 24 h had no significant effect on the viability of TM3 cells; However, an extremely significant inhibition of TM3 cell viability was observed followingin treatment with 40 μmol·L^-1 BPA for 24 h (P < 0.01); In addition, after 20 μmol·L^-1 BPA treatment on TM3 cells for 24 h, the testosterone content in the culture supernatant was extremely significantly lower than that in the CON group (P < 0.01), the mRNA expression level and protein expression of Apoa1 gene were extremely significantly elevated (P < 0.001), but there was no significant change in the mRNA expression level of Apoa2 andor Apoc3 genes; The accumulation of lipid droplets in TM3 cells was extremely significantly reduced by 20 μmol·L^-1 BPA treatment for 24 h compared with the CON group (P < 0.000 1). In conclusion, BPA can reduce the lipid droplets accumulation in TM3 cells by up-regulating Apoa1 expression levels, enhancing reverse cholesterol transport (Reverse cholesterol transport, RCT), leading to a decrease in testosterone synthesis in TM3 cells.

The purpose of this experiment was to determine the colonization of fungal diversity in rumen and feces of Inner Mongolia white cashmere goats at different ages by high-throughput sequencing technology, and to provide data basis for subsequent research on early nutritional regulation of cashmere goats. Three female lambs of Inner Mongolia white cashmere goats of each days of age (10, 30, 50 and 150) with body weight of (4.99±0.13), (7.71±0.52), (13.83±0.18) and (20.60±0.81) kg at 10, 30, 50 and 150 days of agerespectively were selected according to the normal production management and feeding in the farm area, each lamb was a replicate, all together 12 lambs, a total of 12). They were slaughtered and rumen fluid (group R) and feces (group F) were taken. The ITS region of rumen and fecal fungi was amplified by specific primers ITS1F/ITS2R, and sequenced by Illumina Miseq PE300 platform. The results showed that: 1) A total of 1 636 055 clean tags were obtained from 24 samples in this experiment, with an average of (68 168±24 317) clean tags per sample and an average length of (244±28) bp per sequence. 2) With the increase of age, the total OTU of rumen and fecal fungi and the OTU shared with the next day-of-age decreased slightly at first, but then increased gradually. The fungal OTUs shared by rumen and feces at 10, 30, 50 and 150 days of age accounted for 34.22%, 20.87%, 27.63% and 20.53% of the total number of OTUs detected in rumen and feces at the same age, respectively. 3) There was no significant difference in Shannon, Simpson, Ace and Chao indexes of rumen fungal community with the increase of age (P>0.05), but the Shannon and Simpson indexes of fecal fungi at 50 and 150 days of age were significantly higher and lower than those at 10 days of age, respectively (P < 0.05). At 50 days of age, the Shannon and Simpson index of fungi in rumen were significantly higher and lower than those in fecal group (P < 0.05). 4) The β diversity of rumen fungi at 50 days of age and 150 days of age was significantly clustered into one group (P < 0.01), and was significantly separated from the β diversity of rumen fungi at 10 days of age and 30 days of age (P < 0.01). The β diversity of fecal fungi of 50-day-old and 150-day-old were significantly independent distribution and were seperated from 10-day-old and 30-day-old were significantly independent distribution (P < 0.01). In addition, the similarity of fungi β diversity in rumen and feces groups at 150 days of age was higher than that in other age groups. 5) At 10, 30, 50 and 150 days of age, the composition of rumen and fecal fungi was similar at the phylum level, which were Ascomycota, Unclassified_k_fungi, Neocallimastigomycota, Basidiomycota and Mortierellomycota, among which Ascomycota was dominant. At the genus level, the composition of rumen and fecal fungi was slightly different. The species with the highest abundance in rumen were Unclassified_k_fungi at 10 and 50 days of age, Sporormiella and Saccharomyces at 30 and 150 days of age, respectively. The species with the highest abundance in feces were Unclassified_k_fungi and Neocallimastix at 10 and 50 days of age, respectively, and Cleistothelebolus and Cladosporium at 50 and 150 days of age, respectively. 6) Further LefSe analysis at the phylum level showed that Neocallimastigomycota was significantly enriched in the rumen aged 150 days and in feces of Inner Mongolia Cashmere Goat aged 50 days(P < 0.05). When comparing the rumen and fecal fungal communities at the same age, Unclassified_k_fungi and Ascomycota were significantly enriched only in the rumen and feces at 150 days of age. 7) Based on FUNGuild function prediction, rumen fungi were dominated by Unclassified and Pathotroph at 10 and 50 days of age, respectively, and Saprotroph was dominant at 30 and 150 days of age. Fecal fungi were mainly Pathotroph-Saprotroph-Symbiotroph at 10 and 150 days of age, and Saprotroph at 30 and 50 days of age. It can be seenconcluded that with the development of Inner Mongolia Cashmere Goat lambs, the rumen fungal community is gradually conducive to the degradation of fiber in the feed, and the digestion ability is significantly enhanced. There are still many high-abundance and unclassified species in the rumen and feces of young age, and their functions are not yet clear, so further research is needed.

The aim of this experiment was to reveal the intramuscular fat deposition pattern of sika deer at different ages, and to study the effect of intramuscular fat content on flavor quality. The main tests wereDeer at 2, 3, 4 years old were selected to detect the muscle tissue characteristics, then according to the content of intramuscular fat (IMF) the samples were divided into high IMF group (IMFH group) and low IMF group (IMFL group), to determine its effect on inosinic acid and fatty acid content, and to analyze the expression of four genes, namely, fatty acid binding protein 4 (FABP4), fatty acid synthetase (FASN), sterol regulatory element-binding transcription factor 1 (SREBF1), and acetyl-coenzyme A carboxylase (ACACA) expression of four genes. The results showed that the intramuscular fat content of sika deer increased gradually with age (P < 0.001), with the highest intramuscular fat content in the ligamentum teres (P < 0.001) and the lowest content in the anterior tendon (P < 0.001); As the age of the sika deer grows, the diameter and number of roots of the muscle fibers increased significantly (P < 0.05), the density of the muscle fibers decreased significantly (P < 0.05), and the contents of myosinic acid, palmitoleic acid (C16∶1), C18 fatty acids, saturated fatty acids and monounsaturated fatty acids content increased significantly (P < 0.05); Myofiber diameter area and stearic acid (C18∶0) were slightly lowerwas significantly higher in the high intramuscular fat samples than in the low intramuscular fat samples (P < 0.05). The number of myofiber roots and the content of myosinic acid at 4-year-old, myofiber density, myosinic acid, C16∶1, oleic acid (C18∶1), and linolenic acid (C18∶3) content at 3-year-old in IMFH group were higher than that in the low intramuscular fat samples (P < 0.05), while thy myofiber density was nighen; FASN and ACACA gene expression was slightly higher in high intramuscular fat samples than in low intramuscular fat samples (P>0.05), FABP4 and SREBF1 gene expression was significantly higher than in low intramuscular fat samples (P < 0.05), and intramuscular fat was negatively correlated with polyunsaturated fatty acids (P < 0.05) and positively correlated with monounsaturated fatty acids (P < 0.05).In this experiment, it was concluded that the meat tenderness of sika deer decreased with age and the content of flavor substances increased, the higher the intramuscular fat content, the more tender the meat, and the efficient expression of four genes, FABP4, FASN, SREBF1 and ACACA, and the content of monounsaturated fatty acids had a promotional effect on the deposition of intramuscular fat.

The aim of the experiment was to investigate the mitigatingregulating effect of Lactobacillus plantarum on diarrhea lambs in indexes including the damage of anti-oxidative capacity in serum, jejunal microbiota, and jejunal mucosal barrier damagein diarrhea lambs. Twenty-four newly weaned lambs were selected and distributed into four groups using a completely randomized experimental design: the control group (C), the diarrhea group (D), the group of antibiotic-treated under diarrhea (DA), and the group of Lactobacillus plantarum-treated under diarrhea (DL), with six replicates in each group. Lambs in groups D, DA and DL were dosed with 20 mL of ETEC K99 starting on the first day of the formal trial for 8 d consecutively. On the morning of the ninth day, lambs in group DA were injected regulated with florfenicol, and lambs in group DL were regulated by dosinggavaged with Lactobacillus plantarum, and the regulationabove treatment lasted for 4 consecutive days. The blood was collected fasting on days 1, 9 and 13 of the trial for the detection of serum antioxidant indexes as well as diamine oxidase (DAO) and D-lactic acid (D-LA) concentration in lambs. After slaughter the jejunal content was collected to determine the composition of bacterial community. The jejunal mucosal were collected to explore the expression of intestinal mucosal barrier-associated proteins and mucus proteins. Alisin Blue staining was uesd to study the changes of acidic mucus. The results showed that: 1) After 8 d of ETEC K99 treatment, the feces of lambs in D, DA and DL were all shapeless, and the concentrations of DAO and D-LA in the serum of lambs in the three groups were significantly higher than those in group C (P < 0.05), which indicated that the model of diarrhea had been successfully constructed; 2) On the 9^th day, compared with that of group C, the T-AOC and SOD in serum of lambs in D, DA and DL groups decreased significantly (P < 0.05), and the MDA content increased significantly (P < 0.05). Compared to on the 9^th day, the antioxidant capacity of lambs in DA and DL groups increased significantly on the 13^th day (P < 0.05), but there were significant decrease on the concentration of DAO, D-LA and MDA in serum of lambs in these two groups (P < 0.05);3) Compared to group C, the expression of Occludin, Claudin1 and MUC2 protein in group D decreased significantly (P < 0.05). Compared with group D, the expression of Occludin protein in jejunal tissues of DA lambs was significantly higher (P < 0.05), but the expression of Claudin1 and MUC2 proteins reduced slightly (P>0.05). The expression of Occludin, Claudin1 and MUC2 protein in DL group were significantly higher than that of DA group (P < 0.05). 4) Compared with group C, the Chao and Shannon values of the bacterial community in the jejunal content of lambs in D and DA decreased significantly (P < 0.05), and while increased in DL group (P>0.05).The abundances of Firmicutes in the jejunum of lambs from C, D and DL groups were significantly higher than in DA group (P>0.05). The abundances of Actinobacteriota in the jejunum of lambs was significantly higher in C, D and DA than in DL group (P < 0.05). The abundances of Proteobacteria in the jejunum of lambs was a little higher in D group than in C and DL groups (P>0.05), while the abundances of Proteobacteria in the jejunum of lambs in group DA was significantly higher than in C, D and DL groups(P < 0.05). Correlation analysis revealed that more microorganisms associated with MUC2 expression and the phylum Proteobacteria abundance was significantly negatively correlated with the expression of MUC2 protein (P < 0.05), while the phylaabundance of Firmicutes and Verrucomicrobiota were significantly positively correlated with the expression of MUC2 protein (P < 0.05). NK4A214_group had a significant positive correlation with MUC2 expression (P < 0.05) and Escherichia-Shigella was significantly negatively correlated with MUC2 expression (P < 0.05). In conclusion, ETEC K99 induced diarrhea in lambs, decreased serum antioxidant capacity and microbial diversity of jejunal content, and inhibited the expression of jejunal mucus barrier protein. Compared to antibiotic therapy, gavaging Lactobacillus plantarum increases the diversity of jejunal contents flora and promotes the expression of jejunal acidic mucus and mucus tissue barrier proteins. Above all, Lactobacillus plantarum had a better effect in improving lamb diarrhea.

In order to investigate the epidemic variation of porcine reproductive and respiratory syndrome virus (PRRSV) strain in Tianjin area, a new epidemic strain of PRRSV (named TJ-C6) was isolated and identified from weaned piglets with severe respiratory symptoms by means of isolation and culture of porcine alveolar macrophages (PAMs), purification by limited dilution method and identification by IFA. The whole genome amplification and its molecular characteristics were analyzed. The results showed that the whole genome length of this strain was 15011bp, and the nucleotide homology with NADC30 strain was the highest (91.7%), which was NADC30-like PRRSV (lineage 1.8). Nsp2 had 131 discontinuous deletion features ("111+1+19aa"). GP5 N-glycosylation site analysis showed that there were three glycosylation sites (N32, N43, N50). Recombination analysis showed that this strain was a recombinant PRRSV with NADC30-like (lineage 1.8) as the skeleton and NADC34-like (lineage 1.5), and its recombination region was located in 12213-14628nt (ORF2a-ORF6). In this study, a recombinant PRRSV-2 strain of lineage 1.8 and lineage 1.5 was isolated and identified, which can provide reference for the prevention and control of porcine reproductive and respiratory syndrome in Tianjin.

The purpose of this research is to determine the pathogen of piglet diarrhea in pig farms in Hezhou area of Guangxi in the spring of 2023 by using the Meta-transcriptomics sequencing technology to screen the incidence and infection of pigs in this area, so as to provide theoretical basis for the prevention and control of piglet diarrhea diseases in local pig farms. In March 2023, swabs and feces of six adjacent pig farms with simultaneous outbreak of diarrhea within a range of 10 kilometers were collected in Hezhou, Guangxi, and Meta-transcriptomics sequencing was performed by MGISEQ 200 sequencing platform for porcine pathogenic infectomes research. The pathogenic spectrum of piglet diarrhea in this area was systematically screened and the abundance of each pathogen was quantitatively analyzed. The most important viruses were confirmed by Real-time RT-PCR, after which the identified viral gene sequences were selected for phylogenetic analysis.Pearson analysis was used to analyze the correlation of pathogenic co-infection. The results showed that a total of 17 pathogenic bacteria, 12 viruses, and 3 parasites were detected in these samples by Meta-transcriptomics sequencing. The predominant viruses were diarrhea-associated viruses represented by the porcine epidemic diarrhea virus (PEDV), including porcine sapovirus (SaV), porcine kobuvirus (PKV), porcine astrovirus (PAstV), porcine sapelovirus (PSV), and rotavirus A (RVA). The bacteria mainly consisted of three kinds of Enterobacteriaceae, three kinds of Lactobacillaceae, and two kinds of Bacillaceae. The parasites included two kinds of Trichomonads and Iodamoeba. The phylogenetic analysis of spike gene (S gene) of PEDV from five farms showed that the strains all belonged to the G2c cluster group, that the nucleotide similarity between the strains was 99.99%, and that S gene was recombined. Additionally, partial VP4 gene of rotavirus A, of the P[6] genotype, was detected in one of the farms, which had the highest nucleotide similarity with human rotavirus. In addition, more than 2 piglet diarrhea-related viral infections were detected in 4 pig farms. Phylogenetic analysis showed that there was a multi-lineage co-epidemic of diarrheal pathogens in this region, and even different lineages of the same virus were circulating in the same farm. Pathogen correlation analysis showed that there was a significant negative correlation between PEDV/PSV (P < 0.001), PEDV/porcine torovirus (PToV) (P < 0.001) and PEDV/PAstV (P < 0.05), while PEDV/PKV had a significant positive correlation (P < 0.05). The above findings indicate a diverse pathogenic spectrum of piglet diarrhea in adjacent breeding regions of this area, with PEDV identified as the primary pathogen. Furthermore, a correlation between PEDV and other diarrhea-related viruses was observed in the samples. This study offers a comprehensive overview of the infection spectrum of diarrhea pathogens in piglets in this region, pinpointing the most direct causes of diarrhea. Additionally, it enables the analysis of the relationship between diarrhea-associated pathogens in small-scale breeding areas, providing precise guidance and reference for the prevention and control of piglet diarrhea in this region.

The aim of this study was to establish a convenient, efficient and specific recombinase-aid amplification (RAA) assay based on the porcine TGEV S gene. Comparing the genome sequence, selecting specific segment of TGEV S gene to construct the recombinant plasmid pUC57-S as templete. RAA primers and probes were designed and screened. RAA assays were established. The sensitivity, specificity and repeatability of the method were evaluated. Verifying the established test method by clinical samples of diarrhea, and compare the consistency with the testing results of the fluorescence quantitative PCR assay. Our results showed that the best primer pair F2/R1 and probe were screened out. The assay can be completed in 30 min at 40 ℃. The lowest detection limit of fluorescent RAA assay was 1.34×10¹ copies·μL^-1. There was no cross reaction with other virus nucleic acid, as porcine epidemic diarrhea virus, porcine delta coronavirus, porcine respiratory coronavirus and other common swine viruses. A total of 107 clinical samples were tested using this method, and positive rate was 5.61% (6/107), which was consistent with the test results of fluorescence quantitative PCR. This study successfully established a sensitive, specific and visual RAA test method, which can get rid of the dependence of expensive equipment, the professional and technical detection technology, and provided a reliable technical basis for the clinical detect of TGEV.

In order to screen effective antiviral drugs against pseudorabies virus (PRV), we here constructed the recombinant strains PRV-mCherry expressing red fluorescent protein mCherry based on the human-originated isolate hSD-1/2019 and classical strain Ea. Specifically, mCherry reporter gene was inserted into the site before the terminator of PRV UL35 through homologous recombination technique. Based on the constructed recombinant fluorescent viruses, a high-throughput drug screening platform indicated by fluorescence intensity was established and applied for testing 1 621 drugs in the natural product library for screening effective antiviral drugs. Properties of the drugs were preliminarily explored. The results showed that mCherry was inserted in the correct position and was stably inherited. The fluorescence intensity of the recombinant strain PRV-mCherry was representative of the viral proliferation. The strains PRV-mCherry showed similar proliferation curves as the parental strains, supporting the usability of the PRV-mCherry strains in drug screening. Three drugs that could effectively inhibit PRV proliferation in vitro were screened out from the natural product library by using PRV-mCherry. Based on the determined 50% cytotoxic concentration and 50% inhibitory concentration, the selection indices of the three drugs were calculated to be 203.63-564.58 μmol·L^-1, indicating that they had good drug-forming properties. The research provided new ideas in antiviral drugs for the treatment of PRV infection.

Aichivirus C is a newly confirmed virus associated with caprine diarrhoea. The purpose of this study was to isolate goat Aichivirus C and analyze the genomic characteristics. Nine fecal samples and 13 tissue samples from a dead kid were collected from a goat farm with an outbreak of diarrhoea in Sichuan province, and used for testing the common pathogens associated with diarrhoea. Virus was isolated from Aichivirus C positive samples using Vero cells and identified by PCR, indirect immunofluorescence and electron microscopy. The complete genomes of the isolates were amplified and analyzed. Results were as follows: 8 fecal samples with diarrhoea and 8 tissue samples from a lamb were detected as positive for Aichivirus C by RT-PCR, and no other common diarrhoea-causing pathogens were detected in the above samples. Two Aichivirus C strains were successfully isolated from positive samples using Vero cells, and the virus titers were 10^6.5TCID₅₀·mL^-1 and 10^5.9TCID₅₀·mL^-1, respectively. The nearly full-length genomic sequences of both strains were obtained, which shared 78.8%-97.4% nucleotide identity with the known goat Aichivirus C genomes from GenBank and 97.0%-97.4% nucleotide identity with the SWUN/F11/2019 isolate from China. Furthermore, the VP0, VP3 and VP1 genes were successfully amplified from 5 clinical positive samples. Phylogenetic analysis based on the genome revealed that the two isolates were clustered with the SWUN/F11/2019 strain. Unique amino acid mutations were also revealed in the VP0 and VP1 genes. In summary, two strains of goat Aichivirus C were successfully isolated and the nearly full-length genomic sequences were obtained. Phylogentic analysis demonstrated that these two strains are closely related to the SWUN/F11/2019 strain from China, with a unique molecular characteristics in the VP0 and VP1 genes. The results enrich the molecular genetic information of Aichivirus C from goats in China and laid the foundation for further studies on the pathogenicity and molecular biological characteristics of goat Aichivirus C.

A disease characterized by short beak, long tongue, and poor growth was observed in a cherry valley duck farm in Shandong, suspected to be infected with short beak and dwarfism syndrome virus (SBDSV). This study aimed to isolate and identify the virus, and to test its pathogenicity to cherry valley ducks. First, the infection of short beak and dwarfism syndrome virus was confirmed by clinical symptoms, pathological changes, and PCR detection of the diseased materials. Second, after isolating and culturing the virus in SPF duck embryos, it was observed by electron microscopy and analyzed by homology and phylogenetic tree, and then the EID₅₀ of the virus allantoic fluid was tested. Finally, in the animal reversion test, 2-day-old cherry valley ducks were orally administered with allantoic fluid, and the pathogenic characteristics were observed, as well as the virus distribution was detected by PCR and indirect immunofluorescence assay. The results showed that SPF duck embryos died at 96-120 hours post-inoculation, and virus particles of 20-50 nm in size were observed by electron microscopy. The allantoic fluid had an EID₅₀ of 10^-4.85·0.2 mL^-1. The allantoic fluid was positive for SBDSV by PCR, confirming the successful isolation and cultivation of SBDSV from duck embryos. The main clinical manifestations of this case were short beak and weight loss. The virus was shed continuously for two weeks after infection. The VP2 fragment had a 99.8% similarity with the sequence of duck short beak and dwarfism syndrome virus, indicating that it was caused by the infection of this virus, which was named SBDS-SD strain. In the animal challenge experiment, clinical symptoms similar to those of the natural case, such as short beak, diarrhea and significant weight loss, were observed. This experiment successfully isolated a strain of short beak and dwarfism syndrome virus and verified its pathogenicity to cherry valley ducks, providing a basis for further research on this disease.

This study aimed to investigate the expression of chicken sodium hydrogen exchanger isoform 1 (chNHE1) in different types of chicken tissues, the tissue tropism of subgroup J avian leukosis virus (ALV-J), and the changes in mRNA of chNHE1 after ALV-J infection. We used qPCR to detect the transcription of chNHE1 in the organs of commercial broilers, commercial layers, and SPF chickens, as well as the viral load in various tissues and the changes in chNHE1 transcription in tissues after ALV-J infection. The results showed that chNHE1 was transcripted in the heart, liver, spleen, lung, kidney, cecal tonsil, thymus, and bursa in all three types of chickens, but the transcription levels varied greatly, with relatively higher transcription levels in the immune organs. After SPF chickens were infected with ALV-J, the viral load was relatively higher in the heart, spleen, kidney, and cecal tonsils, while it was relatively lower in the liver, lung, thymus, and bursa. The transcription of chNHE1 in the spleen, kidney, and cecal tonsils was significantly upregulated after ALV-J infection, but there was no significant change in the transcription in the heart compared to the control group. This study analyzed the tissue transcription characteristics of chNHE1 in three types of chicken and the effect of ALV-J infection on the transcription chNHE1 expression in tissues, providing an important theoretical basis for exploring the relationship between the expressiontranscription of the receptor chNHE1 and the tissue tropism of ALV-J.

In order to understand the etiology of abortion in foxes, the aborted fetuses in a fox farm in Hebei Province was analyzed by dissection, pathogen isolation and cultivation, 16S rRNA sequencing, virulence gene detection, serotype detection, drug susceptibility analysisassay and pathogenicity analysis assay in this study. As a result, 6 strains of Klebsiella pneumoniae were isolated. Twelve virulence genes (wabG, fimH, ent, icuA, icuB, mrkA, ycf, ecp, uge, ureA, iroB and ybtA) and three serotypes (K20, K54 and K57) were determined, and the K57 was the dominant serotype. Through K-B drug susceptibility testing, it showed that the isolated K. pneumoniae was moderately or highly sensitive to aminoglycosides, cephalosporins, quinolones, polypeptides and other antibacterial drugs, and multiple drug resistance to macrolides, tetracycline, penicillin and other antibacterial drugs was found. According to the pathogenicity analysis, the K. pneumoniae isolate had certain pathogenicity to mice, and the median lethal dose (LD₅₀) was 1.2×10⁷ CFU·mL^-1. In general, K. pneumoniae isolate showed certain pathogenicity, drug resistance, and carrying a variety of virulence genes in this study, and it might be one of the factors causing abortion of fox fetuses. The findings might provide hints for prevention and control of fox fetal abortion.

Vaccination is an essential measure in preventing lumpy skin disease (LSD). However, the induction of humoral immune responses post-vaccination varies, and there is currently a lack of effective methods to evaluate vaccine immunogenicity. This study employed Meta-analysis using R software to statistically analyze published data on immune antibody monitoring for LSD, including subgroup analyses, to comprehensively assess the significance of immune antibody monitoring. A computer search was conducted in both Chinese and English databases, including CNKI, Wanfang Data, VIP, PubMed, and Web of Science. The search was up to July 2023, collecting relevant studies on immune antibody monitoring for LSD. Based on predetermined inclusion and exclusion criteria, a total of 24 articles were included. Among these, 13 articles were related to sheep/goat pox vaccine, and 13 articles were related to LSD vaccine. Two articles covered both sheep/goat pox and lumpy skin disease vaccines, with a total of 8, 638 samples, including 2, 782 positive samples. The outcome measure was the positive rate of immune antibodies. Meta-analysis results indicated an immune antibody level of 41% (95%CI: 27%~55%) for sheep/goat pox vaccine and 73% (95%CI: 64%~86%) for LSD vaccine. The homogeneity among studies was low, and there was no significant difference between the two vaccines (P=0.59). The literature related to sheep/goat pox vaccination(P=0.12) and LSD vaccination(P=0.077) both suggested no apparent publication bias. Subgroup analysis was based on immunization sites and detection methods, the mesults showed that there were no significant differences between different immunization sites(P=0.84) or detection methods(P=0.40), indicated non-heterogeneity in both subgroups. These findings suggest that there is significant uncertainty in evaluating immune protection effectiveness using immune antibody monitoring methods, comprehensive analysis and judgment will be necessitating in practical applications.

The aim of this study was to investigate the community structure diversity of psychrophilic bacteria in raw milk in northern China by metagenomic sequencing. A total of 14 raw milk samples were collected from 14 pastures in Inner Mongolia and Beijing, China in winter. The culturable psychrophilic bacteria were isolated and purified, total microbial DNA in raw milk was extracted as well. Moreover, the psychrophilic bacteria in raw milk were analyzed by 16S rDNA sequencing and metagenomic sequencing, respectively. The results showed that no significant difference was found in community structure of psychrophilic bacteria in raw milk between Beijing and Inner Mongolia. Pseudomonas, Stenotrophomonas and Brevundimonas were the dominant psychrophiles bacteria in raw milk from the two regions. The global and overview profiles, amino acid metabolism and carbohydrate metabolism were the most important functional metabolic pathways, and psychrophilic bacteria contained a variety of antibiotic resistance genes. In conclusion, this study used traditional culture and metagenomic sequencing to accurately determine the species and functional information of the psychrophilic community in raw milk, which is helpful to understand the life activities of psychrophilic bacteria in raw milk and provide basic information for risk assessment of psychrophilic bacteria. Furthermore, it lays down foundational knowledge pivotal for the effective prevention and management of pasture-related issues in Beijing and Inner Mongolia, China.

Overall 35 strains of Staphylococcus aureus (S. aureus) isolated from one pig slaughterhouse and different pig farms were used as experimental materials to analyze the correlation between the susceptibility of benzalkonium bromide and the biofilm formation ability. Sensitivity to benzalkonium bromide and ability to form biofilms of S. aureus strains were determined using the micro-dilution broth method and the crystal violet staining test, respectively. The multi-locus sequence typing (MLST) and the carrying of genes related to disinfectant resistance and biofilm formation of 26 strains of S. aureus from the slaughterhouse were analyzed based on whole-genome sequences. The results showed that among the 35 strains, the minimum bactericidal concentration (MBC) of benzalkonium bromide ranged from 1 to 16 mg·L^-1, and there were 16 strains with strong biofilm-forming ability, accounting for 45.71% (16/35) of the total, of which 93.75% (15/16) were samples collected from downstream in the slaughter chain. ST7 was the dominant type of S. aureus with strong biofilm-forming ability (93.75%, 15/16). The benzalkonium bromide MBC value of S. aureus was positively correlated with biofilm formation ability (Correlation coefficient=0.690), the disinfectance has nothing to do with the gene. In conclusion, biofilm formation may increase S. aureus resistance to disinfectants, weaken the disinfection effect of benzalkonium bromide, thus enhance the risk of bacterial contamination and transmission in breeding and slaughtering.

The purpose of this experiment was to study the effects of mdv1-miR-M4-5p analog encoded by Marek's disease virus (MDV) on proliferation and apoptosis of MDCC-MSB1 cells. In this study, mdv1-miR-M4-5p mimics, inhibitors and their negative controls were transfected into MSB1 cells for 48 h. Real-time quantitative fluorescent PCR (qRT-PCR) was used to detect the transcription of mdv1-miR-M4-5p, TGF-β1, Smad2, caspase-9, caspase-3, cyt-c, cyclinD1, Bcl-2 et al other molecules related to proliferation and apoptosis in MDCC-MSB1 cells. Cell proliferation was detected by CCK-8 and cell cycle and apoptosis were detected by flow cytometry. The results showed that compared with the control group, the transcription levels of mdv1-miR-M4-5p, cyclinD1 and Bcl-2 in MDCC-MSB1 cells transfected with mdv1-miR-M4-5p mimics was significantly up-regulated. The transcription of TGF-β1, Smad2, cyt-c, caspase-9 and caspase-3 was down-regulated, the proliferation of MDCC-MSB1 cells was promoted, the the number of G1 phase cells were decreased, the S and G2 cells were increased, and the apoptosis rate was decreased. Compared with the control group, the results of the proliferation and apoptosis of MDCC-MSB1 cells and the expression of related molecules transfected with mdv1-miR-M4-5p inhibitor were opposite to those transfected with mdv1-miR-M4-5p mimics. The results showed that Mdv1-miR-M4-5p could promote the proliferation and inhibit the apoptosis of MDCC-MSB1 cells. This may be conducted by inhibiting the TGF-β1/Smad2 signaling pathway.

Endometritis is a prevalent disease in large-scale pig farms, but its pathogenic mechanism has not been fully clarified. This study aimed to analyze the diversity of vaginal microflora and its correlation with serum pro-inflammatory cytokines in sows with endometritis, with the ultimate goal of identifying the principal pathogenic bacteria responsible for endometritis in sows. Seven healthy (group C) and seven endometritis (group E) postpartum sows from a pig farm in the suburb of Beijing city were selected, respectively. Serum pro-inflammatory cytokines interleukin (IL)-1α, IL-1β, IL-6, IL-8, and tumor necrosis factor-α (TNF-α) levels were measured using enzyme-linked immunosorbent assay (ELISA). Genes in vaginal microflora of sows were sequenced through Illumina NovaSeq 6000 sequencing and microbial diversity were analyzed using the OmicStudio platform to evaluate the correlation between microbiota and pro-inflammatory factorscytokines. The results showed that concentrationsthe levels of IL-1α and IL-6 in group E were significantly higher than those in group C (P < 0.01). Vaginal microbial diversity was significantly lower in group E compared with group C (P < 0.05). At the phylum level, the relative abundance of Firmicutes in group E was drastically reduced compared with group C, while that of Proteobacteria was significantly increased in group E compared with group C (P < 0.01). At the genus level, the most dominant genera in group C and group E were Fusobacterium and Escherichia-Shigella, respectively. In group E, IL-8 was positively correlated with Ruminococcus and Aerococcus (P < 0.05), there was a significant positive correlation between IL-6 and Escherichia-Shigella (P < 0.01). In sows with endometritis, beneficial bacteria in the vaginal microflora were decreased, pathogenic bacteria were increased, serum pro-inflammatory cytokine levels were elevated compared with those in healthy sows. Serum pro-inflammatory cytokine levels were closely correlated with the number of certain vaginal microflora. This study can provide new insights for the prevention and therapy of endometritis in sows.

This study aimed to investigate the transfer of antibiotic resistance genes (ARGs) carried by ICE_Prophage in Streptococcus suis. The whole genome sequencing of S. suis SC124 was performed using the Illumina MiSeq and Pacbio RSII platforms. The whole genome sequence of SC124 isolates was analyzed by online websites RAST, ICEberg 3.0, PHASTER and NCBI database. MICs were determined by broth microdilution. The transferability of ICE_Prophage carrying ARGs in S. suis was investigated by conjugation. The present of circular intermediates was examined by inverse PCR. Competition was used to evaluate whether the transconjugant had fitness cost. The results showed that there was an ICE_Prophage (ICESsuSC124_ΦSsuSC124) carrying tetracycline, macrolide and aminoglycoside resistance genes in S. suis SC124. ICESsuSC124 carrying tetracycline resistance genes was transferred at a frequency of 5.52×10^-9, and no prophage transfer was found. Inverse PCR revealed that ICESsuSC124 formed a circular intermediate with a size of about 3.8 kb. Compared with S. suis BAA, the transconjugant containing ICESsuSC124 did not have a competitive advantage under the action of drug-free pressure selection, and the transconjugant can produce a certain degree of fitness cost. In conclusion, the ICE_Prophage carrying tetracycline, macrolide and aminoglycoside resistance genes in S. suis SC124, in which ICESsuSC124 could be horizontally transferred, and the transconjugant could produce a certain degree of fitness cost.

Canine mammary gland tumors are a prevalent neoplasm in dogs, with around 50% of cases exhibiting malignancy. They have a conspicuous inclination to spread to other parts of the body and have the potential to readily result in the demise of animals. When the PREX1 protein in the Rho-GTPase family is overstimulated, it can make Rac-GEF less effective, which can mess up the cell cycle and change how cells work. Alterations in PREX1 can potentially facilitate the development of cancerous cells. This study aimed to investigate the influence of PREX1 on the physiological functions of canine mammary tumor cells. In this study, shRNA was used to silence the expression of PREX1. A Western blot test was then used to see how the expression of PREX1 protein changed in the cells after the suppression. The CCK-8 test and colony formation test were used to see how the substance affected the ability of the CHMp canine mammary carcinoma cell line to divide. Additionally, the scratch test and Transwell test were utilized to examine the influence of silencing the substance on the invasiveness of the cell line. Research indicates that reduced expression of PREX1 can impede the proliferation and invasion capacity of CHMp cell lines.

This study takes the AMPK-SIRT3 positive feedback loop as the starting point. The purpose is to explore the mechanism of improvement of bilobalide on inflammatory damage induced interleukin-1β (IL-1β) in ATDC5 chondrocytes. Using IL-1β to induce inflammatory damage of ATDC5 chondrocytes to construct an osteoarthritis model in vitro. Randomly divided into 5 groups (a control group, IL-1β group, IL-1β group cotreated with bilobalide) which cotreatment group is further divided into low, medium, and high dose groups based on the application concentration of bilobalide (15, 30 and 60 μmol·L^-1). Western blot and real-time fluorescence quantitative PCR (qRT-PCR) methods were used to detect the protein and mRNA expression of ADAMTS4, PGC-1a, Collagen Type Ⅱ, MMP-3, NRF-1, and Fis1 in each group. A reagent kit was used to detect the ATP content in each group. The expression level of SIRT3 was detected by immunofluorescence. Western blot was used to detect the levels of AMPK-SIRT3 positive feedback loop related proteins (p-AMPK, AMPK, and SIRT3). Construct an AMPK-SIRT3 signaling pathway blockade model by treating ATDC5 chondrocytes with Compound C and 3-TYP. Mainly detecting downstream related protein changes (PGC-1a, NRF-1, and Fis1). The results showed that bilobalide down-regulated the expression of ADAMTS4 and MMP-3 (P < 0.05), promoted the expression of Type Ⅱ collagen (P < 0.05), regulated ECM metabolic balance, and promoted ATP synthesis. Mechanistically, after intervention with bilobalide, p-AMPK, SITR3, PGC-1a and NRF-1 levels (P < 0.05) significantly increased while Fis1 levels (P < 0.05) significantly decreased. After pre-treatment with Compound C and 3-TYP in ATDC5 chondrocytes, PGC-1a, NRF-1 and Fis1 protein levels were inhibited to varying degrees. In summary, bilobalide activates the expression of PGC-1a through the AMPK-SIRT3 positive feedback loop and regulates mitochondrial biogenesis to improve inflammatory damage in ATDC5 chondrocytes.

The objective of this experiment was to investigate the effects of dietary supplemented with dandelion and akebia extract on growth performance, diarrhea, intestinal morphology and structure, intestinal mucosa tight junction protein gene expression, drug transporter gene expression in liver and intestine of weaned rabbits, and to explore whether the combination of dandelion and akebia extract can improve the bioavailability of Chinese medicine extract. A hundred and twenty 35-day-old weaned rabbits were randomly divided into 4 groups with 10 replicates per group and 3 rabbits per replicate. Four groups including control group (basic diet), dandelion group (basic diet +0.5% dandelion extract), akebia group (basic diet +0.5% akebia extract), and dandelion+akebia group (basic diet+0.5% dandelion extract+0.5% akebia extract).The trial lasted for 28 days. The results were showed as follows: 1)At the first week of the experiment, compared with control group, the ADG in dandelion group and akebia group was significantly increased (P < 0.05), the value of F/G was significantly decreased (P < 0.05). 2) The villus height of jejunum in dandelion group was significantly higher than that in akebia group (P < 0.05). 3) The content of total flavonoids in jejunal mucosa of dandelion+akebia group was significantly higher than other groups (P < 0.05). 4) The relative expression level of zonula occluden 1(ZO-1) gene in ileum of group dandelion+akebia group was significantly higher than that of dandelion group and akebia group (P < 0.05). 5) The relative expression levels of multidrug resistance-associated protein 2(MRP2) and organic anion transporting polypeptide 2B1(OATP2B1) genes in jejunum of akebia group was significantly lower than those of control group (P < 0.05), and the relative expression level of OATP2B1 in jejunum of dandelion+akebia group was significantly lower than control group (P < 0.05). The relative expression of multidrug resistance-associated protein 3(MRP3) gene in ileum of dandelion group and akebia group was significantly lower than control group (P < 0.05). The relative expression levels of breast cancer resistance protein(BCRP) and MRP3 genes in liver of dandelion+akebia group were significantly lower than those of the control group (P < 0.05), and the relative expression levels of OATP2B1 gene in liver of akebia group and dandelion+akebia group were significantly lower than control group (P < 0.05). In conclusion, dietary supplementation of dandelion or akebia extract can improve the growth performance of weaned rabbits, combined supplementation of dandelion and akebia extract can increase the total flavonoid content in jejunum mucosa, and dietary supplementation of dandelion and akebia extract can regulate the expression levels of drug transporter genes in liver and intestinal mucosa of weaned rabbits.

The aim of this study was to establish a rapid detection method of Schmallenberg virus (SBV). A real-time reverse transcription recombinase acid amplification (RT-RAA) was developed with specific primers and probes based on of SBV S gene. The results showed that, through optimizing experimental conditions, the assay could detect SBV specifically within 8 minutes at 38 ℃. There was no cross-react with foot-and-mouth disease virus(FMDV), pestedes petites ruminants virus(PPRV), African horse distemper virus(AHSV), Seneca valley virus(SVV), or vesicular stomatitis virus(VSV). The detection of limit(LOD)was 10³ copies/reaction. At the same time, the co-efficient of variations in intra- and inter-assay were both less than 5%. The detection rate for the spinked positive samples and negative sheep samples was 100%, which was consistent with SBV RT-qPCR results. This study successfully established a fast, sensitive, and specific real-time RT-RAA assay for detection of SBV, which provided a new technical method for prevention and control of SBV infection.