Muscle tissue is divided into 3 types:skeletal muscle, cardiac muscle and smooth muscle. Among them, skeletal muscle is the largest organ of livestock and poultry, accounting for about 40% of the body mass, and it consists of muscle fibers with different sizes, shapes and contents of muscle contractile proteins, which are essential for maintaining body posture, respiration and thermoregulation. A complex combination of extrinsic and intrinsic mechanisms regulates myogenesis at all stages of muscle fiber development, and relevant signaling mechanisms play a decisive role in this process. Skeletal muscle of livestock and poultry is the main source of meat products and the improvement of meat quality traits is an important goal of every livestock farm, a comprehensive understanding of myogenesis and related mechanisms is necessary. In this paper, the occurrence, types and regulatory mechanisms of muscle fibers were reviewed by integrating the literature related to muscle fiber research at domestic and international from 1987 to 2022, highlighting the genes and signaling pathways related to the developmental patterns of muscle fibers in livestock and poultry, and presenting the problems in current research on the regulatory mechanisms of muscle fibers in livestock and poultry to provide an outlook on the future research directions.

Embryonic development in pigs requires a series of crucial physiological stages such as fertilization, cleavage, hatching, morphological transformation, implantation, and cellular differentiation. While a tightly regulated and accurately directed genome is a determining condition for normal embryonic development, recent studies have revealed that appropriate DNA methylation modification also plays an essential role in this process. DNA methylation is a wide and crucial epigenetic modification that does not alter the primary sequence of DNA. However, it contains heritable information and plays an indispensable role in the transcriptional regulation of genes. DNA methylation appears to be a highly dynamic process, influenced by maternal nutrition and developmental conditions in porcine embryonic development. This review describes the effects of DNA methylation on embryonic development from three aspects:early embryonic development, somatic cell nuclear transfer and maternal nutrition during pregnancy, in order to provide references for further research on the mechanism of DNA methylation in porcine embryos during development and to improve the viability of cloned embryos.

Microbial colonization in the digestive tract in early life can affect the animal body and have long-term health effects. A detailed understanding of the colonization of early rumen microorganisms is of great significance to animal health, growth and development. After birth, ruminants begin to contact with external microorganisms, and their rumen microbial flora structure changes dramatically, which is easily affected by animal age, species and dietary structure. Rumen contains complex microbial flora, which is mainly composed of anaerobic bacteria, archaea, fungi and protozoa. The present article reviews the colonization composition and changes of rumen bacteria, archaea, fungi and protozoa in young ruminants, and expounds the effects of feed composition and feed additives on the rumen microbiota of young ruminants, so as to provide a theoretical basis for the phased nutritional regulation of young ruminants.

Scarcity of feed protein source and nitrogen emission pollution are important problems in poultry industry. For this reason, low-protein diets for laying hens came into being in recent years and was gradually widely used. Low-protein diets of laying hens in recent studies were mainly formulated according to the standard ileal digestible amino acid content of feedstuffs, in order to reduce protein resources consumption, feeding cost, and nitrogen emission. Here, the theoretical basis of low-protein diet and its research progress in laying hens were briefly reviewed, aiming to improve the application of low-protein diet in laying hens.

“Cross resistance” (CR) is a phenomenon in which the bacteria have evolved to resist different types of antimicrobial molecules with similar structure or mechanisms of action, while "collateral sensitivity" (CS) can be defined as a situation where the bacteria have developed resistance to one antibiotic showed increased susceptibility to another antibiotic. The evolution of bacterial resistance is a complicated process that can either lead to cross resistance or collateral sensitivity under the selection pressure from antibiotics. Recently, CS has been proposed as a promising alternative approach to counteract the rising problem of antibiotic resistance. To better understand the mechanisms by which bacterial collateral sensitivity has been evolved and the application prospect of CS, in this review, we summarized our current knowledges on key factors influencing the bacterial collateral sensitivity and the regularities of CS. We also discuss directions and challenges for developing CS-informed treatment strategies.

Toxin-antitoxin (T-A) system exists widely in bacterial genomes and plasmids, and regulates various physiological activities in bacteria. Bacterial biofilm formation is a survival strategy adopted by bacteria to adapt to stressful environments (adverse environments), which has strong antibiotic resistance and immune escape, and widely exists in nature. Biofilms have extensive harm and seriously threatens to the health of livestock, poultry and human. In this review, the roles and regulatory mechanisms of different types of T-A system in biofilm formation were summarized, aiming to better understand and master the roles and regulatory mechanisms of T-A system in biofilm formation, and lay a foundation for the removal and control of biofilm formation.

CRISPR/Cas9-based gene editing technology, the newest generation of gene-editing technique, can target and edit nearly any kind of genes mediated by a single guide RNA (gRNA) to realize the direct genomic deletions, mutations, and/or insertions. In recent years, the application of CRISPR/Cas9 system in the large genomes of DNA viruses, especially in those of herpesviruses, has become the latest international research hotspot. Since the first report that using CRISPR/Cas9 system to edit avian herpesvirus genome such as Marek's disease virus (MDV) in 2016, this technology has been widely applied in editing the viral protein-coding genes and non-coding RNA genes, constructing gene knocked-out or recombinant vaccines, antiviral therapy as well as disease resistant breeding in recent five years. Herein, this paper reviewed the latest progresses on CRISPR/Cas9 gene editing-mediated avian herpesvirus gene function studies and vaccine developments. The prospect of CRISPR/Cas9-based gene editing is also discussed for providing important reference for future virology research.

Toxoplasma gondii is an important zoonotic parasite with a wide range of intermediate hosts, complex life history, diverse modes of transmission and global distribution, which can infect almost all warm-blood animals, including humans, causing zoonotic toxoplasmosis. About one third of the world human population are chronically infected with T. gondii, which poses a serious health threat to immune deficiency patients, pregnant women and pregnant animals. Therefore, T. gondii is an important zoonotic parasite. Dense granule protein family secreted by the dense granules, one of the important subcellular secretory organelles of T. gondii, plays important roles in many biological processes, such as regulating the host cell cycle and immune response by modulating the signal pathways, gene expression, participating in the protein transport and localization, nutrient uptake, the formation and stability maintenance of intravacuolar network (IVN), as well as egress and chronic infection of T. gondii. In this review, the basic biological functions of the dense granule proteins are summarized in order to provide new research ideas for the pathogenesis of T. gondii, the discovery of potential drug targets against T. gondii and the development of anti-T. gondii vaccines.

This experiment was conducted to study genotype distribution of SNPs associated with important economic traits, and inter-population genetic structure in 7 local pig breeds in Sichuan province and Tuhe black pig in Shandong province. The single nucleotide polymorphism (SNP) of 8 pig breeds was measured using the porcine 50K SNP bead chip, which include 23 Chenghua pigs, 26 Ya'nan pigs, 60 Qingyu pigs, 57 Neijiang black pigs, 151 Yacha pigs, 57 Wujin pigs, 51 plain Tibetan pigs, 109 plateau Tibetan pigs and 28 Tuhe black pigs (562 healthy pigs in all). Next, data quality controls and genotypes related to 7 economic traits were performed by employing Plink and R software. Cluster analysis and principal component analysis (PCA) were conducted by Mega X and Plink softwares, respectively. The population genetic differentiation index (Fst) was calculated by VCFtools software and to analyze the genetic relationship between populations. The results showed that the proportion of non-advantage genotypes of feed intake, sour meat and stress traits were all lower than 2.20% in local pig breeds in Sichuan province, while the proportion of non-advantage genotypes of resistance to piglet diarrhea, FCR, boar semen quality and multi-rib were 8.99%, 11.80%, 73.97% and 95.32%, respectively. In Tuhe black pigs, q allele of stress trait wasn't existed, the proportion of non-advantage genotypes of feed intake and sour meat traits both were 3.57%, while the proportion of non-advantage genotypes of boar semen quality, resistance to piglet diarrhea, FCR and multi-rib were 39.29%, 50.00%, 82.14% and 92.86%, respectively. Clustering and PCA showed that a relatively distant genetic distance appeared between Tuhe black pigs and Sichuan local pig breeds, and Yacha, Wujin, Neijiang and Tibetan pigs (plain and plateau) were clustered into one group respectively, indicating that the population hierarchy among breeds was obvious. In addition, Ya'nan, Chenghua and Qingyu pigs were clustered together. The Fst index showed that there was a high genetic differentiation between Yacha and Qingyu pigs, both of which belong to Huchuan mountain pigs. The proportion of non-advantage genotypes of feed intake, sour meat, stress, resistance to piglet diarrhea and FCR traits were low, while that of boar semen quality and multi-rib traits were high. In Tuhe black pigs, the q allele of stress trait wasn't existed, and the proportion of non-advantage genotypes of feed intake and sour meat traits were low, while that of boar semen quality, resistance to piglet diarrhea, FCR and multi-rib traits were high. Population genetic structure results showed that the Yacha pigs and Qingyu pigs were classified as Huchuan mountain pigs is still debatable.

The study aimed to explore the efficiency of low-density liquid-based chips in practical animal breeding and reduce breeding costs. A total of 3 761 healthy Large White pigs with about 160 days of age and 110 kg of body weight were used in this study, 100 out of these Large White pigs were randomly selected to serve as a validation population for imputation, in which a 10K SNP panel was generated by sampling markers from the 50 K panel, according to the marker information in 10K SNP panel. Meanwhile, 800, 2 000, 3 600 individuals were randomly selected from the remaining pigs as the reference populations, respectively. Beagle 4.1 was used to impute the 100 validation pigs to 50K SNP panel, and 10 replications were carried out. Genotype consistency and genotype correlation coefficients were used to evaluate the accuracy of genotype imputation. The results showed that the average linkage disequilibrium (r²) of the 10K and 50K SNP panel were 0.227 and 0.258, respectively, with little difference. Minor allele frequency (MAF) of 0.05 was the inflection point for the accuracy of genotype imputation, the accuracy significantly increased after removing markers with MAF less than 0.05. With the reference population size increased from 800 to 3 600, the imputation accuracy was improved from 0.90 to 0.95, and the standard deviation of imputation accuracy among 10 replicates was also decreased from 0.006 to 0.002. For small reference population with 800 individuals, the genotype imputation accuracy of each chromosome fluctuated obviously, while with enlarging the reference population, the imputation accuracy of each chromosome became very close. The study result demonstrates that the imputing from 10K to 50K liquid-based SNP panel is feasible and could be helpful for the application in genomic selection on a large scale, dramatically reducing the breeding cost of genomic selection.

The objective of this study was to clone different alternative splices of pig CPB2 gene, so as to predict the functional properties of the corresponding proteins and study the expression characteristics of different transcripts. Three 12-month-old Bama miniature pigs with the same health status and body weight were used as experimental animal, the CDS region and part of non-coding region of CPB2 gene were amplified by RT-PCR and gene flat terminal cloning technology, while the basic biological characteristics of CPB2 coding protein were predicted by bioinformatics tools. The expression characteristics of CPB2 gene in liver, heart, kidney, subcutaneous fat, mesenteric fat, biceps femoris muscle and longissimus dorsi muscle were detected by RT-PCR. qRT-PCR was used to detect the expression characteristics of CPB2 gene in liver of metabolic disease susceptible pigs prepared in the laboratory under the effect of nutrient-rich diet.Three kinds of alternative splicing isoforms (CPB2-1, CPB2-2 and CPB2-3) of pig CPB2 gene were cloned successfully. CPB2-1 was identical with the NCBI sequence XM_001929144.6. CPB2-2 and CPB2-3 were newly discovered transcripts. Sequence alignment showed that alternative 5' splice site (A5SS) events occurred in CPB2-2 and CPB2-3 compared with CPB2-1. Bioinformatics showed that CPB2-1 encoded an acid unstable protein with 423 amino acids, and CPB2-2 and CPB2-3 encoded an alkaline unstable protein with 281 amino acids; Compared with CPB2-1, CPB2-2 and CPB2-3 lacked signal and activating peptides, but had the same carboxypeptidase activity domain. The three CPB2 transcripts were only expressed in liver and kidney, no expression was found in other tissues. In liver of metabolic disease susceptible pigs induced by nutrient-rich diet, CPB2-1 (P<0.01) and CPB2-2 (P<0.01) significantly reduced, CPB2-3 was not significantly reduced (P=0.14). In conclusion, three kinds of alternative splicing isoforms of pig CPB2 were successfully cloned in liver in this study, suggesting that CPB2-1 is a normal transcript of fibrinolysis and coagulation. Newly discovered transcripts of CPB2-2 and CPB2-3 may have carboxypeptidase activity and important physiological functions when retained in liver and kidney. CPB2 gene may be associated with metabolic related chronic liver disease.

The aim of this study was to investigate the effect of holocarboxylase synthetase (Hlcs) on glycolytic gene expression and to lay the foundation for further studies on cellular glycolysis. In this experiment, C2C12 cells were used as the experimental material, and siRNA technology was used to interfere with the Hlcs gene, and the effect of Hlcs gene interference on the glycolysis gene expression was detected by fluorescence quantitative PCR and Western blotting. The results showed that, during the differentiation of C2C12 cells, the expression trends of Hlcs genes was consistent with Pfkm, Pkm, Hk-2 genes, suggesting that Hlcs genes were correlated with glycolysis. After interfering with Hlcs, the number of myotubes formed during cell differentiation was significantly less than that in the control group, the mRNA level of the glycolytic rate-limiting enzyme Pfkm was significantly decreased (P<0.05), and the mRNA and protein levels of the Pkm were significantly increased (P<0.05). As a coenzyme of carboxylase, biotin could significantly up-regulate the mRNA levels of key enzymes Pkm and Hk-2 (P<0.05). After interfering with Hlcs, the number of lipid droplets formed by cell differentiation was obviously less than that in the control group, the mRNA level of Pfkm was significantly increased (P<0.05), and the mRNA level and protein expression level of the Pkm were significantly increased (P<0.05); The addition of biotin could significantly up-regulate the mRNA level of Pfkm and Pkm (P<0.05). In summary, during the differentiation of C2C12 cells, Hlcs genes were expressed in the same trend as glycolytic genes, and interfering with Hlcs genes could inhibit the expression of Pfkm and Hk-2 genes and promote the expression of Pkm in myogenic differentiated C2C12 cells; while promoting the expression of Pfkm and Pkm and inhibiting the expression of Hk-2 genes in lipogenic differentiated C2C12 cells.

This study aimed to compare the prediction accuracy for chicken residual feed intake (RFI) trait estimated by genomic estimated breeding value (GEBV) estimates combined with prior marker information from genome-wide association study (GWAS) and genomic best linear unbiased prediction (GBLUP) method, and provide theoretical and technical support for improving the accuracy of genomic selection. In this study, 2 510 individuals from 3 generations of Guangxi Jinling flower chicken were selected, including 1 648 roosters and 862 hens, using residual feed intake from 42 to 56 days of age as target trait. The experimental population was randomly divided into two groups, one of which was used as a priori marker information discovery group for GWAS analysis, and the most significant top 5%, top 10%, top 15% and top 20% SNPs were selected as prior marker; in another group, the genetic parameters and prediction accuracy were estimated using different prior marker information. The accuracy was obtained by 10 repetitions of the five-fold cross-validation method, and then the two groups were cross-validated. The results showed that the heritability of RFI calculated by GBLUP was 0.153, and the prediction accuracy was 0.387-0.429. The heritability of RFI calculated by the genomic selection method combined with GWAS prior marker information was 0.139-0.157, and the prediction accuracy was 0.401-0.448. Incorporating the top 10%-top 15% SNPs with the most significant P values in the GWAS results as priors into the genomic selection model can improve the prediction accuracy of RFI by 2.10%-5.17%.

The study aimed to explore the effect of HNRNPAB (heterogeneous nuclear ribonucleoprotein AB) on the proliferation and differentiation of bovine skeletal muscle satellite cells. In this study, primary skeletal muscle satellite cells of Luxi cattle fetus isolated and cultured in vitro were used as experimental materials. The myogenic differentiation in vitro was induced, and the cells before differentiation and on day 1, 2, and 3 after differentiation were obtained. RNA (4 replicates per group) or protein (3 replicates per group) were extracted, qRT-PCR and Western blot were used to detect the expression of HNRNPAB before and after myogenic differentiation. Then the interfering RNA of HNRNPAB was synthesized and transfected into bovine skeletal muscle satellite cells to interfere with the expression of HNRNPAB, and a negative control group was set up; In the experimental group and the control group, EdU was used to detect cell proliferation, qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of HNRNPAB, proliferation marker factor Pax7, Cyclin D1 and differentiation marker factor MyoG and MyHC. The mRNA expression of HNRNPAB in the process of myogenic differentiation of bovine skeletal muscle satellite cells showed a trend of first increase and then decrease, the highest expression was on the first day of differentiation, and the protein expression levels before and after differentiation were also significantly different. After interfering with HNRNPAB, the positive rate of EdU cells increased significantly (P<0.01). The mRNA levels of proliferation marker factor Pax7 and Cyclin D1 significantly decreased compared with the control group (P<0.01). The protein expression level of Pax7 increased significantly (P<0.01). Compared with the control group, the mRNA expression levels of differentiation marker factor MyoG and MyHC were significantly increased (P<0.01), and the protein expression levels of them were also significantly increased (P<0.05). The results of this study showed that interfering with HNRNPAB decreased the transcription level of the proliferation marker factors of bovine skeletal muscle satellite cells, increased the protein expression level of Pax7, and promoted the myogenic differentiation process of bovine skeletal muscle satellite cells.

The aim of this study was to conduct in-depth analysis of mRNA expression profile data from transcriptome sequencing in bovine mammary epithelial cells (BMECs) of dairy cows with high and low milk fat percentage, and to identify key candidate genes affecting milk fat metabolism in dairy cows. Transcriptome sequencing of BMECs from Holstein dairy cows (4 cows in high and low milk fat percentage groups,respectively) was performed by Illumina PE150 method, and the differentially expressed genes were screened with P<0.05 and|log₂FoldChange| ≥ 1.5. KOBAS website was used for functional enrichment analysis. Finally, the accuracy of the sequencing results and the tissue expression profiles of differentially expressed genes related to milk fat metabolism were analyzed by real-time quantitative PCR (qRT-PCR) technology. The results show that there were 578 differentially expressed genes between the high and low milk fat groups, including 332 differentially expressed up-regulated genes, 246 differentially expressed down-regulated genes. Functional enrichment analysis identified 366 significantly enriched GO items including biological process (BP), cellular component (CC) and molecular function (MF) (P<0.05). Among them, GO items closely related to lipid metabolism included long-chain fatty acid transport, positive regulation of adipocyte differentiation, mammary alveolar development, arachidonic acid binding, etc. The differential genes were significantly enriched in 47 KEGG pathways (P<0.05), and there were 15 pathways involved in lipid metabolism, including regulation of lipolysis in adipocytes, phospholipase D signaling pathway, Hippo signaling pathway, etc. Among them, ID2, PRKAA2, FABP4 and ADCY5 were the key candidate genes regulating milk lipid metabolism. Tissue expression profile analysis showed that the expression level of FABP4 was the highest in mammary gland tissue, and the expression levels of ID2, PRKAA2 and ADCY5 were also at a high level compared with other tissues. In this study, 4 important candidate genes affecting milk fat metabolism in dairy cows were screened, which provided an important theoretical basis for studying on the molecular regulation mechanism of milk fat metabolism in dairy cows in the future.

The study aimed to analyze the regulatory action of miR-138 during the yak preadipocytes differentiation. In this study, miR-138 mimics and inhibitor were designed and synthesized to overexpress or suppress miR-138 in preadipocytes. The oil red O staining assay were performed to analyze the effects of miR-138 on lipid deposition, while CCK-8, scratch test assays and flow cytometry were performed to analyze the effects of miR-138 on proliferation. Furthermore, the potential target genes of bta-miR-138 in the process of lipid deposition were screened by bioinformatics analysis; And the mRNA expression levels of miR-138 target genes were detected by RT-qPCR technology, in addition, dual-luciferase report assay were used to verify the interaction between miR-138 and its target. The results showed that overexpression of miR-138 significantly reduced lipid deposition level compared with the control (NC) group (P<0.01), and significantly increased in lipid deposition when miR-138 suppression (P<0.01). RT-qPCR results showed that, overexpression of miR-138 also significantly inhibited the expression of adipose differentiation markers (PPARγ and c/EBPα, P<0.01), whereas the expression of PPARγ and c/EBPα were enhanced when miR-138 suppression (P<0.05). In addition, overexpression of miR-138 significantly down-regulated the mRNA expression of potential targets PTPN11, CREB1, ADCYAP1R1, PGC-1α and SNAP25 (P<0.05 or P<0.01), while the mRNA expression of MXLIP, SNAP25, PGC-1α and PTPN11 significantly in creased (P<0.05 or P<0.01) when miR-138 suppression. The results of CCK-8 and scratch test showed that the cell proliferation activity decreased significantly (P<0.05) when miR-138 overexpression 24 to 36 h, whereas the proliferation activity was increased after miR-138 inhibition. The flow cytometry analysis results indicated that cell cycle were arrested at G1 to S phase, and mRNA level of cell cycle-related genes CCND1, CCNB1 and the proliferation marker gene Ki67 were significantly decreased after miR-138 overexpression. A total of 263 common targets of miR-138 were predicted by bioinformatics analysis. GO annotation results showed that those target genes significantly enriched in "Positive regulation of transcription from RNA polymerase II promoter", "Nuclear chromatin", "Chromatin DNA binding", and "Type I pneumocyte differentiation", KEGG enrichment results showed that these target genes mainly participated in "Axon guidance", "Insulin resistance", "Insulin secretion" and "RNA degradation" pathways. The dual-luciferase report assay showed that co-transfection of miR-138 mimics and PGC-1α-Wt-PGL3-basic significantly reduced the cell fluorescence activity (P<0.01). The present results suggest that miR-138 can decrease the mRNA expression level of PGC-1α by binding its 3' UTR sequence, and inhibit the differentiation and lipid deposition of intramuscular preadipocyte in yak, thus decrease the proliferation of cell. The current study may help clarify the mechanism underlying yak meat characters formation at molecular level.

Genomic mating (GM) is the optimized selection and matching using genomic information, which can effectively control the level of population inbreeding and maximize genetic gain. Genomic mating is based on all individuals in the population, which is a little contrary to the actual breeding work. This study simulated the basic group data of 9 000 individuals with a heritability of 0.5. According to GEBV, 30 sire and 900 dam were selected for each generation as breeding individuals, and then 4 different mating schemes were used:genomic mating, positive assortative mating, negative assortative mating and random mating. Among them, 3 different schemes were given in genomic mating, namely, the maximum genetic gain, the minimum inbreeding, and the maximum variance between families. Five generations were breeding for each scheme, and the average GEBV, genetic gain, inbreeding coefficient, and genetic variance of the offspring population were compared, and the average was taken for 5 times. The ΔG of the 3 genomic mating schemes were significantly higher than those of random mating and positive assortative mating (P<0.01). The ΔG of the genomic mating scheme with the maximum genetic gain was 4.3% higher than that of positive assortative mating. The ΔF of the 3 genomic mating schemes of were 22.2%-94.1% lower than those of positive assortative mating. Among them, the ΔF of the minimum inbreeding scheme selected in genomic mating was 11.8% lower than that of negative assortative mating. The genetic variance of positive assortative mating decreased rapidly and was significantly lower in the 5th generation than all schemes except the scheme that selected the maximum genetic gain in genomic mating (P<0.05). The genetic variance of the 3 schemes of genomic mating was 10.8%-32.2% higher than that of positive assortative mating. The result indicate that genomic mating can not only obtain higher genetic gain than positive assortative mating, but also effectively reduce the rate of inbreeding, and slow down the rate of genetic variance reduction to ensure a certain genetic variation. As an effective sustainable breeding method, genomic mating is very necessary in livestock and poultry breeding.

The maturation rate of oocyte can be used as the index to measure the quality and development ability of oocytes cultured in vitro. The purpose of this study was to investigate whether PLC-γ1 was involved in in vitro maturation of sheep oocytes and its effects. Sheep oocytes were cultured in mature medium containing different concentrations of U73122 (PLC inhibitor) and m-3M3FBS (PLC activator), with 150 cells per concentration. The experiment was repeated 3 times. The cell maturation rate, cleavage rate and morula rate were calculated to screen out the optimal inhibitory and promoting concentration of PLC-γ1. The mRNA levels of PLC-γ1, BAK, BAX, CASP3, CASP8, P53 and BCL6 in oocytes in treatment of 0.5 μmol·L^-1 U73122 and 0.5 μmol·L^-1 m-3M3FBS were detected by qPCR for 48 h. Western blot was used to detect the expression of PLC-γ1, BAK, BAX, CASP3, CASP8, P53 and BCL6 proteins in oocytes in treatment of 0.5 μmol·L^-1 U73122 and 0.5 μmol·L^-1 m-3M3FBS for 48 h. The experiment was repeated 3 times with 150 cells per concentration. In sheep oocytes, 0.5 μmol·L^-1 U73122 was the optimal concentration to promote maturation, and 0.5 μmol·L^-1 m-3M3FBS was the optimal concentration to inhibit maturation. The cleavage rate and blastocyst rate in 0.5 μmol·L^-1 U73122 treatment group were lower than those in control group. Cleavage rate and blastocyst rate in 0.5 μmol·L^-1 m-3M3FBS treatment group were higher than those in control group. The qPCR results showed that, compared with the control group, the mRNA expression levels of BAK, BAX, CASP3, CASP8 and P53 genes in U73122 group significantly increased, while the mRNA expression levels of PLC-γ1 and BCL6 genes significantly decreased, these were contrast in m-3M3FBS group. Western blot results showed that, compared with the control group, the protein expression levels of BAK, BAX and CASP8 significantly increased in U73122 group, while the protein expression levels of PLC-γ1 and BCL6 significantly decreased, while the protein expression levels of P53 and CASP3 did not change significantly. In the m-3M3FBS group, the protein expression levels of PLC-γ1 and BCL6 significantly increased, the protein expression levels of BAX, P53 and CASP3 significantly decreased, while the protein expression levels of BAK and CASP8 did not change significantly. In conclusion, this study suggests that PLC-γ1 plays an important role in in vitro maturation culture of sheep oocytes and regulates oocyte maturation and early embryo development.

This study aimed to explore the mechanism of ADP-ribosyl transferase 3 (ART3) regulating spermatogenesis, and to provide a theoretical basis for improving semen quality and livestock reproductive performance. In this experiment, three kinds of concentrations of ART3 inhibitor 3-methoxybenzamide (3-MBA) (0.302, 0.906 and 1.510 mg·mL^-1) were injected into testis of mice in 3 groups aged 6-8 weeks respectively, the testis and epididymis tissues of each mouse were collected at 3 h, 6 h, 12 h, 1 d, 2 d, 3 d and 5 d after injection. The testis tissues were made into paraffin section for HE staining, and the dynamics and morphology of sperm in epididymis caudal were analyzed. The results showed that the area of vacuoles in the seminiferous tubules of mouse testis was maximized, the spermatogenic cells decreased, and arranged dispersedly and irregularly on 3 d after injection in the treatment of 0.302 mg·mL^-1 3-MBA; The area of vacuoles decreased, the spermatogenic cells tended to recover on 5 d after injection; While the area of vacuoles was maximized at 5 d after injection in the treatments of 0.906 and 1.510 mg·mL^-1 3-MBA, and the empty seminiferous tubules also appeared. In the 3 concentration groups, the density, motility and progressive of sperms in epididymis caudal was decreased in a time-dependent manner, and the sperms with abnormal morphology in epididymis caudal also gradually increased, and the abnormal morphology of sperm tails such as breakage, bending and curling appeared. In summary, ART3 may be involved in the regulation of spermatogenesis, such as proliferation, differentiation, migration of spermatogenic cells and formation of spermatogenic tail.

The aim of this study was to investigate the effect of an alternative splicing (WNT4-β) of WNT4 on the follicular granulosa cells proliferation of goat. In this study, 20 healthy ewes from 4 to 6 months of age were selected, goat ovarian tissues were collected, follicular granulosa cells were isolated in vitro and cultured. The expression location of WNT4-β was determined by immunofluorescence staining. Overexpression or interference of WNT4-β in goat granulosa cells, RT-qPCR and Western blot were used to detect the expression of key marker factors (ROA1 and RHOA) in the WNT signaling pathway, and granulosa cell proliferation marker genes cyclin-D2 and CDK4. CCK-8 was used to detect the granulosa cells proliferation. ELISA was used to analyze the changes of reproductive hormone levels in granulosa cells. Immunofluorescence staining result showed that WNT4 was expressed only in follicular granulosa cells of goat, but not in oocyte cells. WNT4-β and granulosa cell proliferation factors cyclin-D2 and CDK4 mRNA significantly increased (P<0.01) after overexpression of WNT4-β, and significant increase in protein expression levels (P<0.05). The expression levels of WNT signaling pathway marker factors ROA1 and RHOA mRNA significantly increased (P<0.05), and β-catenin protein levels significantly increased (P<0.05). The mRNA expression of WNT4-β, cyclin-D2, CDK4, ROA1 and RHOA significantly reduced after interfering with WNT4-β (P<0.05). WNT4-β, cyclin-D2, CDK4 and β-catenin proteins expression significantly reduced (P<0.05). The CCK-8 result showed that overexpression of WNT4-β promoted proliferation of granulosa cells (P<0.05). ELISA results showed that estradiol (E₂) levels significantly increased (P<0.05) and progesterone (P₄) levels increased but not significantly (P>0.05) in granulosa cells after overexpression of WNT4-β. The opposite result was got after interfering with WNT4-β, the granulosa cell proliferation was inhibited (P<0.05) and the levels of E₂ and P₄ significantly reduced (P<0.05). In conclusion, the WNT4 alternative splicing WNT4-β promotes follicular granulosa cell proliferation and ovulation-related steroid hormone secretion in goats by regulating the WNT signaling pathway, and this study provides a theoretical basis for revealing the molecular function of WNT4 in reproductive traits in goats.

There are few reports on the effects of different feeding modes on the intestinal flora of Kazakh horses. This study compared the composition and biomarker of the fecal flora of Kazakh horses under free grazing and captive rearing, thus to reveal the fluctuation of the intestinal flora of Kazakh horses under different feeding modes. Fresh feces were collected from 5 Kazakh horses under free grazing and 5 Kazakh horses in captivity. The composition and functional pathways of the intestinal flora of Kazakh horses were analyzed by high-throughput sequencing technology. The results showed that the diversity of intestinal flora in the grazing group was significantly higher than that in the captive group (Shannon index, P<0.05; Simpson index, P<0.01); The biomarker species of the grazing group were significantly more than that of the captive group. At the phylum level, the abundance of Firmicutes (76.10%; 78.08%) and Bacteroidetes (17.16%; 15.72%) in grazing group and captive group was higher, which accounting for more than 93% of the total flora. At the family level, cellulose decomposing/fermenting bacteria (Ruminococcaceae, 30.44%; Lachnospiraceae, 21.96%;[Mogibacteriaceae], 4.86%) were dominant in the grazing group. While the cellulose decomposition/fermentation bacteria Ruminococcaceae (28.73%) and Lachnospiraceae (14.51%), as well as the starchcarbohydrates digesting bacteria Streptococcaceae (15.08%) were dominant in the captive group. In addition, the abundance of Streptococcaceae significantly higher than that of the grazing group (P<0.05). Annotation for the functional gene sequences showed that, 77.44% of the sequences were annotated as "metabolism". The results indicated that, the colony diversity of the free grazing group was higher than that of the captive group, and the digestion type of flora was more unified. There were overall differences in the composition of the intestinal flora between the two groups, which laid a theoretical foundation for the further study of the intestinal flora of Kazakh horses.

The aim of this study was to investigate the effects of aspartic acid supplementation in low-protein diets on growth performance, blood biochemical indexes and free amino acids, apparent digestibility of nutrients and meat quality traits in pigs from weaning to fattening. Twenty healthy three-way cross (Duroc×Landrace×Yorkshire) weaned piglets at 45 days old with similar body weight ((10.93±0.79) kg) were selected and randomly divide into two groups with 10 replicates in each group, 1 pig per replicate, single piglet pen rearing. The experiment lasted for 84 days. The control group was fed with low-protein corn-soybean meal basal diet, and the test group was fed with basal diet supplemented with 0.5% aspartic acid. The results were showed as follows:1) The addition of aspartic acid significantly increased the average daily gain (P<0.05) and extremely significantly reduced the feed to gain ratio (P<0.01) of pigs at the weaning stage, but had no significant effect on the growth performance in the fattening and growth stages (P>0.05);2) The addition of aspartic acid significantly increased the serum ALB level and ALP activity in the weaning stage (P<0.05), but there was no significant effect on the blood biochemical indexes in the growth and fattening stages (P>0.05); 3) The addition of aspartic acid significantly reduced the content of Pro, Met, Thr, Asp, His, Tyr in the serum (P<0.05) and increased the level of Lys (P<0.01) at the weaning stage, the content of Asp and Lys in the serum during the growth stage and the content of Asp and Thr in the fattening stage were significantly reduced (P<0.05), while there were no significant effect on the content of other amino acids (P>0.05); 4) Aspartic acid treatment extremely significantly increased the apparent digestibility of total energy, crude protein, crude fat, crude ash and crude fiber of fattening pigs (P<0.01); 5) Aspartic acid treatment effectively increased the cooked meat rate of pigs (P<0.01) and the mRNA expression level of MyHC1 in the longissimus dorsi muscle (P<0.05). The above results showed that the addition of 0.5% aspartic acid to low-protein diets throughout the breeding process could effectively improve the growth performance of pigs at the weaning stage, and beneficially promote the development of muscle fibers in the later stage so as to improve meat quality traits.

The purpose of this study was to gain a cell protein profile that interact with the porcine circovirus type 2 (PCV2) replication initiation region (Ori) in PK-15 cells and the regulation of PARP1 protein on PCV2 replication was preliminarily investigated. To gain a protein profile that interact with the PCV2 Ori in PK-15 cells, we performed a biotinylated DNA-protein pull down assay in conjunction with LC-MS/MS analysis; GO analysis and KEGG pathway enrichment analysis were performed and the protein interaction network was mapped; subsequently, we constructed eukaryotic plasmids of PARP1 and viral protein Rep, and the interaction between PARP1 protein and Ori region was verified by DNA pull down and IP experiments; Co-IP assay was used to detect the binding of PARP1 protein to Rep proteins; finally, the effect of over-expression or silencing of PARP1 protein on virus replication was detected. A total of 130 host proteins were identified, in which 45 were related to DNA function, mainly involved in host DNA damage repair and replication function; the interaction between PARP1 protein and PCV2 genome Ori or Rep protein was verified; after siRNA silenced the expression of PARP1 protein, the copy number of PCV2 genome was significantly decreased, and PARP1 protein was overexpressed instantly, and the copy number of PCV2 genome was significantly increased. In conclusion, the replication initiation of PCV2 in host cells involves the repair of host DNA damage repair and replication function. PARP1 protein could bind to Ori and Rep proteins of PCV2 and promote the replication of PCV2, which lays a foundation for the study of drug targets of PCV2 and the replication initiation of PCV2.

This study aimed to analyse the pathogenic characteristics and the epidemiological situation of canine respiratory coronavirus (CRCoV) in Beijing. From December 2015 to March 2017, Pharynx nasal swabs from 487 dogs were collected and reverse transcriptional polymerase chain reactions was used to detect CRCoV. Meanwhile, some of the samples were also used to detected canine parainfluenza virus (CPIV), canine adenovirus type 2 (CAV-2) and canine distemper virus (CDV) for exploring the situation of mixed infection. The results showed that:1) The positive rates of CRCoV was 21.36% (104/487). The mixed infection rates of CRCoV and CPIV, CAV-2, CDV were 3.43% (11/321), 0% (0/156) and 3.85% (6/156) respectively. 2) Among all 455 cases which had respiratory symptom records, dogs which had non-respiratory symptom accounting for 27.19% of all dogs without respiratory symptoms, dogs which had mild respiratory symptoms (coughing and nasal discharging) accounting for 19.73% of all dogs with mild respiratory symptoms, dogs which had moderate to severe respiratory symptoms (pneumonia) accounting for 14.28% of all dogs with moderate to severe respiratory symptoms. 3) From November 2016 to March 2017, the mixed infection between CRCoV and CPIV, CAV-2, CDV in 146 cases was explored. The mixed infection rates of the dogs which were tested CRCoV positive and had non-respiratory symptom was 12.50%. The mixed infection rates of the dogs which were tested CRCoV positive and had mild respiratory symptoms was 41.18%. The mixed infection rates of the dogs which were tested CRCoV positive and had moderate to severe respiratory symptoms was 100.00%. 4) The infection rates of CRCoV varied from 15.56% to 22.97% in different age groups among 454 samples. Except for the high positive rates in July, the positive rates in cold season is higher than warm season. These results indicated that the pathogenicity of CRCoV is rather weak. Dogs suffer pure infection of this virus often show non-respiratory symptom or mild respiratory symptoms. Dogs which are CRCoV positive suffer mixed infection often show mild respiratory symptoms or moderate to severe respiratory symptoms. This virus is more likely to be prevalent in winter and spring. The infection rates has no significant relationship with age.

In order to understand VP1 genes and pathogenicity of feline calicivirus (FCV), 13 FCV strains were obtained after isolation and identification. The VP1 genes of these FCV strains were obtained by RT-PCR and sequencing. Then, phylogenetic analyses based on the VP1 sequences were performed with reference strains. Cat pathogenicity experiments were carried out with 2 FCV strains. The results showed that the nucleotide sequence similarity among these 13 FCV VP1 genes was 74.3%-99.8%, and the homology of the nucleotide and amino acid was low with reference strains. The characteristics of variation were consistent with FCV. The analysis of phylogenetic tree showed that the major strains in Shanghai were from northern China, and other strains were from foreign countries. Seven VS-FCV strains were obtained by clinical symptoms, phylogenetic tree of VP1 and VS-FCV characteristic amino acid sites analysis. Cat pathogenicity experiments showed that the strains of FCV-SH202101 and FCV-SH202113 could cause the disease and death of cats, and the incubation period was about 1 to 2 days. The FCV strains would be detected on the 3rd day after inoculation. The durations of FCV in different age groups were different, kittens would be 5 days, and adult cats would be 11 to 18 days. So the FCV-SH202101 strain and FCV-SH202113 strain had the same characteristics with VS-FCV strains in pathogenicity. This study enriched the molecular epidemiological data of FCV in China, provided strong evidence for the screening of VS-FCV strains, and also provided technical support for the research of FCV vaccine.

The purpose of this study was to identify the avian metapneumovirus (aMPV) as the pathogen of head shaking, mental depression and swollen head syndrome in some poultry farms in China. In this study, nasal turbinate and lung samples were collected from chicken farms in Shandong, Fujian and Heilongjiang. Firstly, the clinical samples were detected by aMPV specific RT-PCR method, and then the positive samples were inoculated into Vero cells for virus isolation. Secondly, the subtypes of the viruses were identified by G and F gene sequence analysis and indirect immunofluorescence assay (IFA). Finally, the pathogenicity of the isolate to SPF chickens was analyzed. Among the 220 samples, the RT-PCR results indicated that three nasal turbinate samples had specific bands at about 228 bp. After the positive samples were inoculated into Vero cells for 5 consecutive generations, the cells showed aMPV characteristic cytopathy (CPE) such as rounding, aggregation and fusion. These results showed that three isolates of aMPV were successfully isolated and named SD2001, SD2002 and HLJ2101, respectively. Phylogenetic analysis showed that the G and F genes of layer-originated SD2001, SD2002 and broiler-originated HLJ2101 isolates had high nucleotide and amino acid sequence homologies with other subtype B aMPV strains, and the nucleotide similarities were 93.4%-98.6% and 95.6%-100.0%, respectively, and the amino acid similarities were 88.7%-97.8% and 97.6%-100.0%, respectively. However, the homologies with the G and F genes of subtypes A, C and D aMPV strains were low, and the nucleotide similarities were 27.1%-61.8% and 66.8%-74.8%, respectively, and the amino acids similarities were 16.1%-36.7% and 72.5%-86.5%, respectively. These results indicated that SD2001, SD2002 and HLJ2101 isolates belonged to subtype B aMPV. Further, the specific positive serum of subtype B aMPV was used for IFA test. The specific green fluorescence signal could be observed in Vero cells inoculated with SD2001, SD2002 and HLJ2101, respectively, which furtherly confirmed that the three isolates belonged to subtype B aMPV. Next, the pathogenicity of the isolate to SPF chickens was tested. SPF chickens infected with SD2001 isolate showed clinical symptoms such as depression, head shaking and sneezing 3-6th day after infection. Histopathological analysis showed that there were pathological lesions in the turbinate, trachea and lung of infected SPF chickens. The incidence rate was 90%(18/20). In this study, three strains of subtype B aMPV were successfully isolated. Our results not only identified the pathogen of swollen head syndrome in some poultry farms in China, but also confirmed that the epidemic strain of subtype B aMPV had obvious pathogenicity to chickens. These results provided an important theoretical basis for the diagnosis and effective prevention of aMPV infection in China.

This study attempted to isolate a novel C. perfringens (Cp) phage from poultry. Biological characteristics and genomic information have been analyzed, which can provide new strategy for Cp infection and control. A Cp phage was isolated and purified from broiler intestinal tissue samples by double-layer and single-layer plate methods. The morphology of the phage was observed by transmission electron microscope, and the MOI, one-step growth curve, pH and temperature tolerance characteristics, host range and antibacterial activity in vitro were analyzed. The genomic information was analyzed by genome sequencing, and finally the effect of phage on reducing the pollution of Cp was evaluated in a space simulated environment. The results showed that the electron microscope showed that the phage was a long tailed phage, the latent period was less than 20 min, the burst size was 28.5 PFU·cell^-1, and the best MOI was 0.01. It could specifically inhibit avian Cp strains, and the host range was as high as 83.72%. Genome analysis showed that the phage was a double stranded DNA phage with a total length of 41 590 bp and 59 open reading frames (ORF), of which 33 ORFs had certain homology with known functional proteins. The phage was named as vB_CpeS_SD72. The phylogenetic tree analysis of terminase small subunit showed that the phage had high homology with Clostridium phage vB CpeS-CP51. In the environmental control test, it was observed that the phage could inhibit 98.36% Cp at 30 min and nearly completely inhibited within 2 h. A novel Cp phage with strong lytic activity can effectively inhibit Cp contamination in the environment, which provide a powerful strategy candidate source for Cp pollution and control in food, animal breeding and livestock and poultry products.

The purpose of this study is to investigate the pathogenicity and drug resistance of Clostridium perfringens (CP) derived from large-scale chicken farms, and to evaluate the preventive effect of avilamycin and tylosin phosphate premixture on Necrotizing enteritis (NE) caused by CP infection, which can guide the prevention and control of chicken NE. Feces were randomly collected in large-scale chicken farms with a history of NE caused by CP in Hebei and Shanxi Province. CPs were isolated and their toxin types were determined by multiplex PCR. Three strains of type A CPs were selected from three farms to infect 14-day-old SPF chicken at dose of 10⁹CFU for 5 days. Diarrhea and intestinal lesions were observed and the CPs' pathogenicity were evaluated. Minimum inhibitory concentration (MIC) of avilamycin, lincomycin and tylosin were measured using the microbroth dilution method. According to the MIC results, avilamycin premixture and tylosin premixture were selected to prevent chickens with no clinical symptoms from NE disease caused by CP by observing mental appetite, diarrhea symptoms and intestinal lesions, counting the CP detection rate and NE incidence, thus evaluate the prophylactic effect of the two drugs. Results were as follows:91 CP were isolated from 753 chicken stool samples, and all toxin types were type A. The results of the challenge test showed that both the incidence of NE lesions and intestinal lesion score were significantly higher in the A, B, C challenged group than D unchallenged group (P<0.05). Compared with the unchallenged group, the challenged group showed obvious diarrhoea symptoms and intestinal lesions, with significant differences (P<0.05), and all three CP strains could cause chicken NE. Results of drug sensitivity test:The MIC range of avilamycin, lincomycin and tylosin were 0.25-4, 0.125-128 and 0.25-32 μg·mL^-1, respectively. Results of drug efficacy test:Feeding avilamycin premixed for 21 consecutive days could decrease the NE symptoms or prevent the lesion deterioration, significantly reduce the incidence of NE and NE lesion score (P<0.05), and significantly reduce the CP detection rate (P<0.05). The effect of feeding tylosin premixture for 7 consecutive days was comparable to that of avilamycin premixture, but a few of the chickens showed NE symptoms after the drug withdrawal. At present, most of the CPs detected from large-scale poultry farms in Hebei, Shanxi Province are type A which can cause NE. Meanwhile, type A CP is still sensitive to avilamycin and tylosin. Their premix are highly effective in chicken NE prevention.

The present study aimed to explore the toxicity mechanism of inflammatory injury and immune regulation disorders in organisms, and lay a foundation for further in-depth exploration of the pathogenic mechanism of Clostridium perfringens disease. The cultured supernatant of C. perfringens type C was intraperitoneally injected into BALB/c mice, and the small intestine samples were collected for transcriptome sequencing. Based on the differentially expressed genes (DEGs), GO functional annotation and KEGG pathway enrichment analysis were performed. The results showed that in total, there were 40.99 Gb effective bases and 795 DEGs were obtained, among which 229 were up-regulated and 566 were down-regulated. The ten randomly selected DEGs were verified by real-time fluorescent quantitative PCR that their relative expression was consistent with the transcriptional expression profile. GO functional annotation mainly involved G-protein coupled nucleotide receptor activity, and G-protein coupled purine nucleotide receptor activity. KEGG pathway enrichment analysis found that it was mainly concentrated in TNF signaling pathway, IL-17 signaling pathway, p53 signaling pathway, FOXO signaling pathway, Toll-like receptor signaling pathway, and NF-κB signaling pathway. When C. perfringens exotoxin invades the body, inflammatory signaling pathways such as TNF will be activated, resulting in inflammatory damage and even necrosis of the intestine.

hyaD is a gene involved in capsular polysaccharide synthesis of Pasteurella multocida type A. To investigate the effect of hyaD gene on P. multocida virulence and immune protection characteristics, an hyaD mutant (ΔhyaD) derived from bovine P. multocida type A strain CQ2 (PmCQ2) was constructed by homologous recombination. The results showed that, compared to the wild type strain, the capsule production and virulence of ΔhyaD were significantly reduced, and the bacterial loads in the mice organs infected with ΔhyaD were also decreased. The mutant strains were easier to adhere to mice peritoneal macrophage and were easily been swallowed, leading to the up-regulation of the expression of related inflammatory factors in macrophage. The hyaD gene deletion significantly down-regulated the expression of genes involved in capsule synthesis, LPS synthesis and transport, and iron transport. While significantly up-regulated the expression of protective antigen genes. The inactivated bacterin of wild type PmCQ2 and ΔhyaD strains were prepared by conventional methods, and the mice were immunized with both bacterin, respectively (one booster following the primary immunization). On the day 21 after first immunization, the immunized mice were challenged with homologous and heterologous serotypes P. multocida, the lungs of mice immunized with ΔhyaD had none or slight histopathological lesion at 24 h after challenged. Immunization of mice with ΔhyaD conferred significant protection against challenge from bovine P. multocida type A, type B and type F strains, as well as rabbit, porcine and avian P. multocida type A strains, with protection rate at 100%, 100%, 80%, 90%, 100%, and 100%, respectively; While immunization with similar doses of killed wild-type was failed to confer protections against heterologous and homologous serotypes excepting bovine P. multocida type A strains challenge, with protection rate above 80%. Taken together, the results show that the hyaD gene can affect the virulence of P. multocida by regulating capsule production and expression of virulence-related factors, and the hyaD gene deletion endows mutant cross-protection characteristics by up-regulating the expression of cross-protective antigens, and the mutant can be used as a candidate for the development of P. multocida vaccine. This study lays the foundation for the development of a universal vaccine against P. multocida infection.

The aim of this study was to investigate the role of four hypothetical MIC-related proteins in the tachyzoite stage of Toxoplasma gondii and the effect of gene deletion on virulence. The sgRNA sequences of TGME49_222975, TGME49_275798, TGME49_238220 and TGME49_238210 were designed online at the website, respectively, and the sgUPRT on the pSAG1::CAS9-U6::sgUPRT template was mutated to sgRNA by PCR to obtain the target gene of the CRISPR plasmid. The obtained target gene plasmids and DHFR fragments were subsequently mixed and electrotransferred to T. gondii tachyzoites, and the target gene deletion strains were obtained by successful PCR identification after ethidium monoclonal screening. Subsequently, the target gene deletion strains and the RH wild strain were used for replication, egress, plaques formed and virulence in mouse assays, and the results were compared to evaluate the effect of the target genes on the function and virulence of T. gondii tachyzoites. There was no significant difference in the number of vacuoles containing 1, 2, 4, 8 and 16 tachyzoites formed by of the deletion and wild strains within the same period of time (P>0.05), and all the tachyzoites in the vacuoles could egress within 2 min under the stimulation of calcium ions, plaques in the size and area of plaques in 12-well plates infected with the same number of tachyzoites for the same period of time (P>0.05). The differences in the time from the beginning of the death to the total death were not significant (P>0.05). In this study, four deletion strains of T. gondii MIC-related genes (RHΔTGME49_222975, RHΔTGME49_275798, RHΔTGME49_238200 and RHΔTGME49_238210) were successfully constructed, but the virulence of the deletion strains was not significantly different from that of the wild strain, indicating that the four genes are not important independent virulence factors of T. gondii. But the four genes may play other important biological functions. This study preliminarily analyzed the basic biological functions of four T. gondii MIC-related proteins, and provided data for the later comprehensive analysis of T. gondii protein functions.

To investigate the correlation of high mobility group box 1 (HMGB1) with liver inflammatory fibrosis induced by Schistosoma japonicum infection, in the present study, BALB/c mice were randomly divided into control group (n=10), 4 weeks' infection group, 6 weeks' infection group and HMGB1 inhibited group (n=5). Blood routine tests and biochemical tests were performed as well as liver pathology observation and Masson staining. qRT-PCR was used to detect the transcription levels of HMGB1,IL-1β,TNF-α,IL-6,IFN-γ,α-SMA,Col1α1,TGF-β1,Smad2 and Smad3 in the liver. Results showed that 4 weeks after infection, serum alanine aminotransferase (ALT) and the transcription levels of HMGB1, α-SMA, TGF-β1, IL-6 and TNF-α were increased significantly (P<0.05 or P<0.01); 6 weeks after infection, WBC, Neu, Mon, Eos, aspartate aminotransferase and ALT were significantly increased (P<0.01), the transcription levels of HMGB1, IL-1β, TNF-α, IL-6, IFN-γ, α-SMA, Col1α1, TGF-β1, Smad2 and Smad3 were significantly increased as well (P<0.05 or P<0.01); 6 weeks after infection, large number of eggs were deposited in the liver accompanied with neutrophils infiltration and collagen fiber accumulation. The HMGB1 inhibitor could significantly reduce the ALT level and transcription levels of HMGB1, α-SMA, TGF-β1, IL-6 and TNF-α in the liver (P<0.05). In conclusion, there was a correlation between HMGB1 and liver inflammatory fibrosis induced by S. japonicum infection.

Interferon stimulated gene 15 (ISG15), a gene stimulated by interferon, plays an important role during host-viral interaction. To study the effect of ISG15 on porcine pseudorabies virus (PRV) replication, in this study, ISG15 gene knockout cell line was constructed by CRISPR/Cas9 technology. Next, the cell viability of PK15-ISG15^-/- cells was monitored by CCK-8 assay. Then, the following indexes were used to comprehensively evaluate the effect of ISG15 knockout on PRV replication:fluorescence intensity of PRV was measured by indirect immunofluorescence assay (IFA); mRNA level of PRV-EP0, PRV-gE, PRV-VP16 and IFN-β were detected by RT-qPCR; protein expression levels of PRV-gE and ISG15 were evaluated by Western blot; PRV titer was detected by virus plaque assay. Results were as follows:Firstly, ISG15 gene was successfully knocked out in PK-15 cell by sgRNA1 and sgRNA2; ISG15 gene knockout had no effect on cell viability; IFA detection showed that PRV fluorescence intensity of PK15-ISG15^-/- cells was higher than that of PK-15 cells after PRV infection. Moreover, RT-qPCR and Western blot results showed that PK15-ISG15^-/- cells could up-regulate PRV mRNA transcription and protein translation. Viral plaque assay further showed that knockout ISG15 could promote PRV replication. Besides, RT-qPCR results showed that up-regulation transcription of IFN-β induced by PRV infection was resisted in PK15-ISG15^-/- cells. In conclusion, the above results indicate that ISG15 inhibits PRV replication in PK-15 cells, which might be linked to the IFN signaling pathway.

Escherichia coli type III secretion system 2 (ETT2) is involved in the pathogenesis of avian pathogenic Escherichia coli (APEC). This study investigated the biological characteristics and pathogenic effects of ETT2 structural gene epaPQR on APEC in order to provide the basis for further understanding of the pathogenic effects of ETT2. The epaPQR mutant strain and complemented strain were constructed based on CRISPR-Cas9 gene editing technology. The effects of epaPQR gene on the biological characteristics of APEC were analyzed by detection of growth curve, motility and biofilm formation ability, etc. The effect of epaPQR gene on the pathogenicity of APEC was analyzed by serum bactericidal and tissue bacterial load tests. The results showed that the deletion and complemented strains of epaPQR gene of ETT2 were successfully constructed. Deletion of epaPQR gene did not significantly influence the growth and biofilm formation ability (P>0.05). After deletion of epaPQR gene, the motility was significantly reduced (P<0.05), and the number of flagella observed by transmission electron microscopy was significantly reduced. RT-qPCR showed that the transcription levels of flagellar T3SS structural genes and flagellar output protein genes were significantly down-regulated (P<0.05). After the deletion of epaPQR gene, its antiserum bactericidal ability was significantly increased (P<0.05), and the colonization ability of AE81ΔepaPQR strain in different organs of chicks was significantly decreased. The results indicated that epaPQR was involved in the regulation of APEC flagellar formation, and affected antiserum bactericidal ability of APEC, as well as the ability to colonize tissues and organs in vivo, suggested that epaPQR played an important role in pathogenesis of APEC. This study provides a reference for the further understanding of the function of ETT2 and the pathogenesis of APEC.

The objective of this study was to investigate the protective effect of yeast mannatide (MTY) on mice infected with pathogenic Escherichia coli (PEC). Firstly,40 SPF male KM mice were randomly divided into 4 groups:control group, model group, positive drug group and MTY group (10 replicates per group and 1 mouse per replicate). Control group and model group were given normal saline, while the positive drug group and the MTY group were given antibiotic amoxicillin and MTY, respectively. In this paper, the Kunming mice were gavaged MTY for 4 weeks before the challenge experiments with PEC. By analysing enteric microorganism of mice that before challenge test, mortality of mice after PEC infection, organ index, intestinal structure, organs histopathological analysis, and at the same time, observing the physical change of mice in vivo testing, the protective effect of MTY on PEC infected mice was comprehensively evaluated. The results showed that MTY could inhibit PEC proliferation. Compared with blank group, MTY group had significant difference in intestinal microflora (P<0.05), which mainly showed that beneficial bacteria increased and bacterial diversity increased. Compared with model group, MTY treatment can effectively reduce the mortality of mice infection with PEC. MTY significantly increased thymus index (P<0.05), decreased spleen index (P>0.05) and extremely significantly decreased liver index (P<0.01). MTY inhibited the decrease of goblet cells in jejunum of mice (P>0.05). MTY inhibited the down-regulation of MUC2 and ZO-1 expression in intestinal of mice (P<0.05). Therefore, MTY has a good protective effect on intestinal, liver, spleen and lung organs of mice infected with PEC, which has a wide application future in animal feed.In conclusion, oral administration of MTY can reduce the damage of PEC to the body and then play a protective role in mice.

This experiment aimed to clarify the role of C5a-C5aR axis in the liver injury of ducklings caused by Riemerella anatipestifer (R. anatipestifer) infection. The preserved R. anatipestifer was resuscitated and duck anticoagulant blood was also prepared. The whole blood experiments were conducted by mixing R. anatipestifer with duck anticoagulant blood and then incubating with anti-C5a antibody and anti-C5aR antibody, respectively. ELISA was used to detect the expression levels of C5a and various inflammatory factors. Then the ducklings were infected with R. anatipestifer and treated with anti-C5a antibody and anti-C5aR antibody, respectively to conduct the animal experiments. The main tissues of ducklings were collected to conduct the histopathological examination. Peripheral blood and liver tissues of ducklings were collected for bacterial counts. Peripheral blood was collected to separate the serum for serum enzyme activity measurement. ELISA was used to detect the expression levels of C5a and inflammatory factors in the serum. The liver tissues were collected to detect the mRNA transcriptional levels of main inflammatory factors by fluorescent quantitative PCR. The results showed that in the whole blood experiments and animal experiments, compared with R. anatipestifer group, the expression levels of C5a, TNF-α, IL-6 and IL-1β in R. anatipestifer + anti-C5a group and R. anatipestifer + anti-C5aR group were significantly decreased (P<0.05). Blocking the C5a-C5aR axis could significantly reduce the morbidity and mortality of ducklings infected with R. anatipestifer, and meanwhile alleviated the tissue damage of the heart, liver and spleen. Blocking the C5a-C5aR axis could significantly reduce the number of bacteria in the peripheral blood and liver tissues, and decreased the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) of the ducklings infected with R. anatipestifer. The results of fluorescence quantitative PCR showed that compared with R. anatipestifer group, the mRNA transcriptional levels of C5aR, Sphk1, FGL2, TNF-α and IL-1β in R. anatipestifer + anti-C5a and R. anatipestifer + anti-C5aR groups were significantly decreased (P<0.05). The study successfully confirmed that the activation of C5a-C5aR axis could help R. anatipestifer infect ducklings, and blocking the C5a-C5aR axis could significantly alleviate the tissue damages and inflammatory responses of ducklings, and provided new ideas for further research on anti-R. anatipestifer infection drugs.

The aim of this research was to determine the dose and injection rate of contrast medium and the optimal delay time for multi-phase contrast-enhanced scanning of liver in dogs. Different iohexol doses (500, 575 and 650 mg·kg^-1, calculated based on I content) and injection rates (2 and 3 mL·s^-1) were selected for dynamic scanning modality in dog. The CT enhancement values of aorta, portal vein and liver parenchyma were respectively calculated to determine the optimal dose and injection rate of contrast medium. Then the dogs of different sizes were scanned using the optimal contrast medium dose and injection rate. The time-density curves of aorta, portal vein and liver parenchyma were drawn after dynamic scanning. The peak arrival times of aorta, portal vein and liver parenchyma were counted, and the difference between the peak arrival time and injection time (Δt_AO/Δt_SP/Δt_L) were calculated to determine the optimal scanning delay time of each phase. The results showed that the CT enhancement values of aorta, portal vein and liver parenchyma were higher with a dose of 575 mg·kg^-1 at an injection rate of 3 mL·s^-1. Perfect contrast enhancement effect could be obtained in these situations. The Δt_AO values of small, medium and large size dogs were 7, 9 and 4 s, Δt_SP were 21, 23 and 17 s, and Δt_L were 41, 44 and 34 s, according to the time density curves. The optimal scan delay times in each phase could be calculated by the equation "injection time+Δt_ROI-1/2 scan time". It has been verified in clinical cases that the dose of contrast medium (I, 575 mg·kg^-1), injection rate (3 mL·s^-1) and delay times ("injection time +Δt_AO/Δt_SP/Δt_L-1/2 scan time") used in this research had better clinical contrast enhancement effects on liver image and can be applied to contrast-enhancement CT diagnosis of liver diseases in dogs.

The disorder of calcium and phosphorus metabolism in pigs is one of the long-term problems affecting the economic efficiency of pig farms. Calcium deficiency of pregnant sows leads to a decline in the production performance of the pigs, and the fattening effect of piglets is relatively poor, further resulting in the loss of production profit. The effects of calcium loss in pregnant sows on lipid metabolism in fetal pigs were studied in this paper. In this paper, the effect of calcium loss in pregnant sows on lipid metabolism in fetal pigs was studied. Twenty healthy sows with 7-8 gestational age were randomly divided into control group (CON, n=10) and low-calcium group (LCa, n=10). The sows were continuously fed according to different Ca/P ratio from the 85th day of gestation to the 114th day of delivery. The blood, liver and kidney tissue samples were collected from piglets immediately after birth. Metabolomics was used to analyze the changes of metabolic pathways in piglets. Lipids and lipid metabolism in serum were detected by biochemical apparatus and ELISA, respectively. The expression of lipid metabolism-related genes was detected by qRT-PCR. The results showed that low calcium stress mainly affected glycerol phospholipid metabolism pathway of fetal pigs, and also interfered fatty acid metabolism and palmitic acid synthesis; while, there was no significant change in serum lipid content of piglets (P>0.05). Low calcium stress significantly affected the secretion of FAS, Visfatin and Resistin in fetal pigs (P<0.05). The mRNA expressions of SCD, LDLR, SCD, FABP4, HSD17B12 and ABCC4 in liver and kidney of piglets were significantly decreased (P<0.05) in low calcium group. These results indicated that calcium deficiency in pregnant sows can affect the lipid metabolism of fetal pigs, which may be detrimental to their growth and development.

This study was conducted to evaluate the clinical outcome and complications of radius and ulna fractures in toy-breed dogs treated with conventional bone plate fixation. Medical records of toy-breed dogs with radius and ulna fractures repaired with open reduction and internal fixation utilizing conventional bone plate (round hole plate or veterinary cuttable plate) in Veterinary Teaching Hospital of China Agricultural University were reviewed, and these cases were followed up. The inclusion criteria were as follows:Body weight no more than 7 kg; follow-up time more than 12 months; case information record was complete. Results were as follows:64 cases of radius and ulna fractures in 63 dogs were included, 49 cases (76.6%) had no lameness, 7 cases (10.9%) had barely visible lameness, 6 cases (9.3%) had mild lameness and 2 cases (3.1%) had moderate lameness, with a major complication rate of 6.3% and a minor complication rate of 27%. Conventional bone plate internal fixation of radius and ulna fractures in toy-breed are effective means of fixation that has good clinical effect with low incidence of major complications.

This study was conducted to investigate the effects of Radix Salviae Miltiorrhizae on the intestinal flora of mice injected with Escherichia coli(E. coli) based on network pharmacology and 16S rDNA high-throughput sequencing technology. The active components and corresponding targets of Radix Salviae Miltiorrhizae were screened by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform, and the E. coli targets were searched by Genecards database. Protein interaction analysis of the intersection of the target genes was performed through SRTING database to construct the protein-protein interaction(PPI) network, and the "Radix Salviae Miltiorrhizae active ingredient-potential action target" network was constructed by Cytoscape 3.8.2 software. DAVID online tools were used to analyze the function of gene ontology (GO), Metascape online tools were used to analyze the enrichment of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway. One hundred and ten male Kunming mice were randomly divided into control group, E. coli group, and low-, medium- and high-dose Radix Salviae Miltiorrhizae injected with E. coli group, with 22 mice in each group. E.coli O₁₀₁ was injected intraperitoneally after continuously gavaged with Radix Salviae Miltiorrhizae solution for 28 days, while sterile normal saline was injected into the control group. After observation for 24 h, feces were collected from each group. The 16S rDNA high-throughput sequencing technology was used to analyze the intestinal flora of mice in each group. The results were showed as follows:1) The network pharmacological analysis showed that the active components of Radix Salviae Miltiorrhizae were 36, and the number of action targets was 669. The Genecards database searched for 8 902 E. coli targets, and the intersection between the two was selected and isolated points were removed, finally, 51 potential targets for E. coli treatment with Radix Salviae Miltiorrhizae were obtained. Through GO and KEGG analysis, 18 key targets and 16 key pathways were identified, including 174, 76 and 45 biological processes (BP), molecular functions (MF) and cellular components (CC), respectively; 2) A total of 950 855 valid sequences and 2 537 operational classification units (OTUs) were obtained by Illumina Miseq sequencing; 3) Alpha diversity analysis results showed that the intestinal flora diversity of Radix Salviae Miltiorrhizae groups was significantly different from that of the control group, and the intestinal flora diversity of the E. coli group was significantly lower than that of the control group (P<0.05); 4) Compared with E. coli infection group, at the phylum level, the abundance of Bacteroides was extremely significant increased(P<0.01) and Firmicutes was significantly increased(P<0.05) and Proteobacteria was extremely significant decreased (P<0.01) in the low-, medium- and high-dose Radix Salviae Miltiorrhizae injected with E. coli groups, while at genus level, the abundance of Bacteroides was extremely significant increased (P<0.01); 5) Based on LEfSe analysis, a total of 14 significantly different microflora were found in the control group, E. coli infection group, and high-dose Radix Salviae Miltiorrhizae injected with E. coli group. 6) PICRUSt functional prediction analysis showed that the main functional pathways of Radix Salviae Miltiorrhizae for intestinal flora of mice infected with E. coli were amino acids, carbohydrate transport and metabolism; Translation, ribosomal structure and biotransformation; Cell wall/membrane/biosynthesis, etc. In summary, network pharmacology combines with 16S rDNA high-throughput sequencing technology has revealed that Radix Salviae Miltiorrhizae regulates the relative microflora biological abundance through multi-component, multi-target, and multi-pathway, which alleviates the intestinal flora damage in mice with flora imbalance caused by E. coli infection, and provides theoretical basis for clinical application of Radix Salviae Miltiorrhizae in the treatment of E. coli infection.