After the establishment of breeding goals for dairy cattle in various countries, the breeding goals have been continuously developed, and the composition, definition and weight of traits in the selection index are constantly changing. The selection and breeding of dairy cows started with the focus on milk production performance, and then conformation traits were added. By the end of 20^th century, the focus of selection had moved away from being purely production oriented toward a more balanced breeding goal, and some important functional traits were considered. This shift occurred partly due to increase of health and fertility issues and partly due to welfare concerns. After entering the 21^st century, with the development of society and dairy industry, breeders have pay attention to novel traits, and some have begun to be used in breeding practice. The literatures on novel traits in dairy cattle breeding in the past decade and useful information from the breeding program in various countries are collected in this review. And the novel traits which are being studied and/or have been applied in dairy breeding in dairy developed countries are presented. In this review, the novel traits were summarized as 5 categories, including that related to production efficiency, coping with environmental challenges, improving cattle health and welfare, products and milk processing characteristics, and workability. The background, definition, genetic architecture and genetic selection strategies for these novel traits were introduced in this review. Finally, the workflow of research and application for novel traits in dairy breeding was summarized. This review can provide a reference for the study in dairy breeding and help improve breeding objectives in Chinese dairy population.

Adipose tissues are the important energy metabolism and endocrine organs for animals. Selective fat deposition plays a vital role in the sensory quality, flavor and processing characteristics of animal meat. Therefore, the specific regulatory factors and molecular mechanisms of fat deposition in different parts of animals have attracted the attention of researchers. microRNA (miRNA) was regarded as a family of small non-coding RNA with a length of 22 nt. In recent years, omics technology has been used for high-throughput sequencing of adipose tissues and adipocytes with phenotypic differences, many differentially expressed miRNAs have been screened and found. These miRNAs can perform biological functions by combining with target gene mRNA. The differentially expressed miRNAs in adipose tissues play an important role in the regulation of fat deposition. Here, this review will summarize the function of miRNA involved in subcutaneous adipose tissue and intramuscular adipose tissues of animal, so as to provide theoretical references and new ideas for the further exploration of miRNA regulating on the specific adipose deposition of animal.

The intestinal microbes and their metabolites play an important role in the growth and functional improvement of the animal. When the dynamic balance between the host and the intestinal microbes is broken, the local or systemic inflammation is induced. Butyrate not only provides energy for the body as an energy substance, but also regulates the host's innate immunity and adaptive immune state, promotes the body's immune response. Butyrate affects the host's secretion of antimicrobial peptides(AMPs) to prevent translocation of intestinal pathogenic bacteria and maintain steady state of intestinal microbes. This article will systematically introduce the research progress of the interaction mechanism between the intestinal microbes and the host immune system mediated by butyrate, and clarify their important role in the steady state of intestinal health.

As an important component of bile, bile acid is synthesized by the liver using cholesterol as a raw material. It can be excreted into the digestive tract together with bile under the stimulation of exogenous food and related hormones. Bile acids can emulsify fat, promote intestinal absorption of lipids, and regulate liver and intestine functions, increasing energy consumption, improve insulin sensitivity. The bile acid, generally can be synthesized in two ways, the classical route and the alternative route. The enterohepatic circulation can recycle about 95% of the bile acids synthesized from the beginning, and only 5% which can be refilled through alternative ways to ensure the dynamic balance of the bile acid pool will be lost. Therefore, the enterohepatic circulation plays a vital role in regulating the homeostasis of bile acids. With the deepening of research, the metabolism and transport mechanisms of bile acids have been gradually clarified, and the functions of transporters involved in the enterohepatic circulation have become more clear in recent years. The farnesoid X receptor (FXR), as an important nuclear factor, can regulate the expression of bile acid transporter through small heterodimer receptor (SHP), retinoic acid receptor α (RARα), combined with fibroblast growth factor 15/19 (FGF15/19), and then affects bile acids homeostasis. This article describes the significant transporters involved in the enterohepatic circulation of bile acids and the regulatory mechanism of FXR, which will provide a theoretical basis for further exploration of bile acid functions in the future.

Mastitis is usually an inflammatory reaction of mammary gland caused by microbial infection, and is one of the most common diseases of dairy cows, which can lead to the decline of milk yield and quality, reduce the productive life of dairy cows, and seriously affect the economic benefits of pasture. In recent years, scholars have carried out a lot of researches on the molecular regulation mechanism of dairy cow mastitis, and found that NF-κB and its signaling pathway can participate in regulating the expression of many immune related genes, and play a key role in the process of cellular inflammatory response and immune response, which is also a hotspot in the research of dairy cow mastitis. This paper reviewed the etiology and pathological changes of dairy cow mastitis, as well as the relationship between NF-κB signaling pathway and body immunity, and the latest research progress of the molecular mechanism of regulating dairy cow mastitis by mRNA, non-coding RNA (miRNA, lncRNA and circRNA) and bioactive substances through NF-κB signaling pathway, which will provide the reference for the analysis of molecular regulatory network of mastitis, molecular breeding of anti-mastitis, and the development of bioactive drugs for treatment of dairy cow mastitis.

Bovine torovirus (BToV) belongs to the subgenus Renitovirus, genus Torovirus, family Tobaniviridae, and order Nidovirales. This diarrhea-causing bovine pathogen has dual tissue tropism and implicating this virus is a possible causative agent for bovine respiratory disease. BToV has been reported in 17 countries, such as China, the United States, Japan, with a worldwide geographical distribution. To provide a reference for BToV research, this review describes the latest research progress on BToV, including the biological characteristics, epidemiology, clinical symptoms and pathology, detection methods.

Antibiotic resistance is one of the most severe threats to public health in the 21st century. As an important zoonotic and food-borne pathogen, Clostridium perfringens (C. perfringens) could cause food poisoning, gas gangrene, necrotizing enteritis in humans and animals. The extensive use of antibiotics promotes the resistance of C. perfringens continuously, seriously threaten the public health and the economic benefits of animal husbandry. This review focuses on the occurrence of antimicrobial resistance in the last decade, and the antibacterial mechanism in C. perfringens of common antibiotics, in a bid to provide scientific insight for the prevention and control of antibiotic resistance.

The purpose of this study was to evaluate the inbreeding depression for TNB in Large White pigs with different genetic background after about 8 generations of selection. A total of 1 937 sows with phenotypic and pedigree records were genotyped with GeneSeek GGP Porcine HD chip. Out of them, 1 039 Large White pigs were from the Canadian line and 898 were from the French line. The pedigree of the two lines consisted of 3 086 Large White pigs. Inbreeding coefficients were estimated using 3 different measures based on pedigree data, SNPs and runs of homozygosity (ROH), respectively. Inbreeding depression of TNB was evaluated using an animal model in which the inbreeding coefficient was treated as a covariate. To fine mapping the genomic regions that lead to inbreeding depression of TNB, the inbreeding coefficients of each autosome and significant autosome segments were calculated and their effects were estimated. And then all the significant autosomes were divided into several segments with equal size and these genomic regions were further tested whether they were associated to inbreeding depression of TNB. In Canadian line, the average inbreeding coefficients of F_ROH, F_GRM and F_PED estimated were 0.124, 0.042 and 0.013, respectively. Among pairwise correlations between the different inbreeding coefficients across individuals, the highest correlation was between F_ROH and F_PED and the correlation coefficient was 0.358. In French line, the average inbreeding coefficients of F_ROH, F_GRMand F_PED estimated were 0.123, 0.052 and 0.007, respectively. The highest pairwise correlation was between F_ROH and F_GRM, and the correlation coefficient was 0.371. On the whole genome level, significant inbreeding depression was found for TNB in Canadian line using all 3 measures of inbreeding. Estimates of inbreeding depression were -0.571, -0.341 and -0.823 for TNB per 10% increase in F_ROH, F_GRMand F_PED, respectively. While in French line, only using F_ROH could detect significant inbreeding depression and a 10% increase in F_ROH resulted in a decrease of 0.690 for TNB. On the chromosome level, ROH-based inbreeding coefficient for each chromosome was used to perform inbreeding depression analysis. The results showed that chromosome 6, 7, 8 and 13 in the Canadian line were identified to be significant related to inbreeding depression for TNB, but in French line, no chromosome was associated to inbreeding depression. To narrow down the genomic regions causing inbreeding depression, the 4 significant chromosomes in Canadian lines were further divided into 2, 4, 6, 8 segments with equal size. When these 4 autosomes were divided into 8 segment, the segment lengths ranged from 15.1 to 25.8 Mb. Finally, one, two and three genomic segments on chromosome 6, 7 and 8 were found to be significantly associated with inbreeding depression, respectively. These regions harbored CUL7, MAPK14 and PPARD genes that were associated with placental development, and AREG and EREG genes involved in oocyte maturation. Three calculation measures of inbreeding coefficients were used to evaluate inbreeding depression for TNB in Large White pigs with different genetic background. At the genome level, significant inbreeding depression in Canadian line was detected for TNB using all 3 measures of inbreeding, but only the effect of F_ROH was significant in French lines. At the chromosome level, 4 autosomes showed significant effects. Use of ROH further identified the 4 chromosomes and 6 specific genomic segments that could lead to significant reduction in TNB of Canadian line. These specific genomic segments were annotated candidate genes related to reproduction. The study results not only provided a new sight into revealing the genetic mechanism of inbreeding depression, but also provided a reference for carrying out genomic selection and mating schemes in pigs.

The purpose of this study was to prepare monoclonal antibodies against porcine RNA polymerase Ⅱ and make preliminary applications. The bioinformatics method was used to predict the immunogen, after immunogen was chemically synthesized, 5 female Bal b/c mice aged 4-8 weeks were immunized, and cell fusion experiment was performed on the mice that were positive after immunization, spleen cells of these mice were fused with myeloma cells to obtain hybridoma cells that could stably secrete anti-RNA Pol II antibody. The identification result showed that the heavy chain of RNA PolⅡ monoclonal antibody was IgG 2A type, and the light chain was Kappa type. The hybridoma cells were screened and subcloned by the indirect ELISA method, and 8 hybridoma cell lines that stably secreted RNA PolⅡ monoclonal antibody were obtained. It was preliminary applied to chromatin immunoprecipitation (ChIP-seq) technology, and compared with the enrichment of commercial antibodies, the results showed that the monoclonal antibodies obtained in this study were more enriched. The porcine RNA PolⅡ monoclonal antibody obtained in this study can provide a theoretical basis for epigenetic research and provide important biological materials.

The objective of this study was to investigate the distribution characterization of tandem repeats sequence (TRs) and the evolution of centromeric satellite DNA in bovinae. In this study, based on the genome sequences of 6 bovine subfamily species including Bos taurus, Bos indicus, Bos mutus, Bubalus bubalis, Bison bison and Bos frontalis, the composition, distribution and structural characteristics of TRs among different species were studied, and the evolutionary relationships of centromeric satellite DNA were analyzed. The results showed that:1) The average percentage and length of TRs in the 6 bovine subfamily species were 2.03% and 54.93 Mb, resepctviely. Of which the highest percentage and length was Bos taurus (3.42%, 93.00 Mb), the lowest was Bos indicus (1.42%, 37.88 Mb). 2) Among the 3 types of tandem repeats, microsatellite DNA had the largest number(483 405), accounting for 85.64% of the total TRs, which was higher than minisatellites (43 026, 7.62%) and satellite sequences (38 180, 6.75%). 3) Through the analysis of the abundance and average length of microsatellite DNA, the dinucleo-tide of microsatellite DNA had the highest abundance in bovine subfamily, which was 70.93 loci/Mb and dominated by AC copy types. 4) 1.715 satellite sequence generally present in the genomes of bovinae species by constructing phylogenetic trees of 1.715 and 1.723 satellite sequences, while the 1.723 sequence was lacking in yaks. The two types of satellite sequences had different degrees of differentiation between different species or different chromosomes, which had obvious interspecies specificity. The average proportion of TRs in the 6 species of bovine subfamily was 2.03%. Microsatellite DNA was the main composition of TRs, the abundance of dinucleotide microsatellites was the highest, and the AC copy type was dominant. 1.715 satellite DNA generally existed in the genomes of 6 bovine subfamily species, but there were different degrees of differentiation among species or chromosomes. The results of this study provide an theoretical basis for the study of TRs distribution characteristics and evolutionary relationships among bovinae species.

In order to better protect, develop and utilize Jinnan cattle and ensure the genetic diversity of Jinnan cattle, in this study, gene chip technology was used to detect Jinnan cattle population genetic characteristics and perform genetic evaluation of reserve bulls, and provide theoretical and technical support for molecular-assisted breeding and breeding of Jinnan cattle. Ten blood samples of Holstein, Heshun, Simmental, Yanbian and Limousin cattle with healthy and similar weight of ((350±20) kg) at the age of 18 months were collected, and 25 blood samples of Jinnan reserve bulls were collected, these were divided into 6 groups according to different breeds of cattle, the first 5 groups had 10 repetitions in each group, and Jinnan cattle reserve bulls had 25 repe-titions. The Illumina SNP 50K high-density cattle SNP chip was used to detect their genotypes, the genetic characteristics of Jinnan cattle was analyzed and compared, and the kinship matrix was used to calculate the kinship coefficient of Jinnan cattle reserve bulls, the genetic evaluation was performed by BLUP. The results showed that Jinnan cattle had a farther relationship with Holstein, Heshun, Simmental and Limousin cattle in genetic structure, and was closer to Yanbian cattle, and it was a Chinese local breed group. The Jinnan cattle reserve bulls were genetically evaluated, and the breeding value ranking of the genomic carcass weight variance of the cattle was obtained; the JN23 had the largest carcass weight multiple trait standard deviation, and it could be selected at the genome level as a beef breeding bull. The kinship was analyzed, the Jinnan cattle reserve bull family was classified to avoid inbreeding between groups.The genetic evaluation, inbred family analysis, traditional phenotypic selection and genetic disease detection were carried out on the reserve bulls of Jinnan cattle. Finally, the selected and remained bulls were JN07, JN23, JN05, JN08, JN02, JN13, JN19 and JN14 in Jinnan cattle, the combination of a variety of breeding methods has enhanced the accuracy of bull selection, which laid a foundation for improving the group selection and breeding of Jinnan cattle.

The purpose of this study was to develop Wip1-knockout swine testis (ST) cells using CRISPR/Cas9 system, which laid a foundation for furture exploring the roles of Wip1 gene in the proliferation and apoptosis of ST cells. In this study, two sgRNA (sgWip1-1 and sgWip1-2) targeting the first exon of Wip1 gene were respectively cloned into pX330-GFP and pX330-RFP vectors, and then co-transfected into ST cells (immature Sertoli cell isolated from 80-90 day-old pig embryo testes). The positive ST cells with red and green fluorescence were selected by flow cyto-metry to screen monoclonal cells. The 30 monoclonal cells were performed for genotype identification, and then the PCR products were used to implement T-A clone. In total, 29 monoclonal cells were exhibited fragment deletions, including 5 clones with monoallelic fragment deletions, 8 clones with biallelic homozygous fragment deletions, 16 clones with biallelic heterozygous fragment deletions. The fragment knockout efficiency was 96.7%. In conclusion, we efficiently obtained the Wip1-knockout ST cells. This work not only provides a cell model for subsequent researching the effects of Wip1 gene on the proliferation and apoptosis of ST cells, but also lays a good foundation for exploring the roles of Wip1 gene on the reproductive performance of boars.

RFamide-related peptide receptor(NPFFR1) is a primary affinity receptor of the gonadotropin-inhibitory hormone (GnIH), which plays an important role in controlling animal reproduction. In order to explore the effect of the NPFFR1 on follicle development of geese, nine healthy laying Sichuan white geese of 42-week-old were used as experimental animals. The RT-qPCR technology was used to determine the expression pattern of NPFFR1 in the granulosa cells (GCs) of pre-hierarchical and hierarchical follicles. The NPFFR1 gene expression was upregulated using the overexpression plasmids technique in GCs, and the concentration of reproduction-related hormones (E2, P4 and AMH) was determined by ELISA in the cellular supernatant, the TUNEL assay was employed to detect apoptosis of the remaining anchorage-dependent cell. The RNA sequencing was performed to screen the differentially expressed genes (DEGs) before and after overexpressing NPFFR1 in GCs of 8-10 mm follicles, respectively. The functional cluster analysis of differentially expressed genes was carried out. The results showed that the gene expression of NPFFR1 was extremely significantly different between the pre-hierarchal and hierarchal periods follicular GCs in geese (P<0.01), except F1 follicles. After overexpressed the NPFFR1 gene (72 h), the concentration of E2 in the supernatant of GCs isolated from hierarchal follicles and AMH in the supernatant of GCs isolated from pre-hierarchal follicles were significantly decreased (P<0.05); There were 267 DEGs (119 down-regulated and 148 up-regulated) were selected. The DEGs were mainly enriched in the process of biological rhythm, follicles rhythm. The qPCR results showed that, compared to the control group, the AMH gene was significantly down-regulated (P<0.05), the Clock, FOS, Per and ANTXR2 were extremely significantly (P<0.01) or significantly (P<0.05) up-regulated, respectively. Therefore, it was specu-lated that the NPFFR1 gene might be involved in follicular development by regulating the expression levels of the rhythmic genes, affecting the steroid hormone secretion and apoptosis of follicular granulosa cells of goose.

This experiment was conducted to study the effect of cage density on the production performance, meat quality, welfare index and cecal microorganisms of Xueshan chickens. Totally, 180 6-week-old Xueshan female chickens with similar body weight were randomly selected into high-density, middle-density and low-density rearing system equally with 20 replicates, and 4, 3 or 2 birds in each cage as a replicate. And the production performance, slaughter performance, meat quality, feathers and gait score, and the cecal microflora composition were detected at the market age of 15-week-old. The results showed that:1) The body weight of chickens in the low- and middle-density group were significantly higher than those in the high-density group from the age of 11-to 15-week-old(P<0.05). The abdominal fat weight and abdominal fat percentage of 15-week-old chickens in the low-density group were significantly higher than those in the mid-dle-density group (P<0.05), and the abdominal fat indexes in middle-density were significantly higher than those in high-density group (P<0.05). No significant difference were found in the other slaughter indexes (P>0.05). 2) The feather quality of chickens in the middle- and low-density groups were significantly better than that in the high-density group (P<0.05). There was no significant difference in gait score among three groups (P>0.05). 3) Bacteroidetes, Firmicutes, and Proteobacteria were 3 dominant flora in cecum. And there was no significant diffe-rence in intestinal flora among 3 groups (P>0.05). However, chickens in the middle-density group exhibited less unique OTUs in cecum. Compared with the high density cecal microflora, the middle- and low-density cecal microflora were more similar. The proportion of Firmicutes and Bacterioidetes might be related to body weight. In general, these findings may provide a reliable information for considering the selection of middle-density for female Xueshan broilers can get better production performance.

The purpose of this research is to provide the regulation and mechanism of endoplasmic reticulum stress(ERS) on pyroptosis of THP-1 cells infected by BCG. THP-1 cells were infected by BCG with or without TUDCA treatment. Then, the mRNA expression of GRP78, GSDMD, Caspase1, NLRP3, IL-1β, IL-18, the protein expression of GRP78, Caspase12, GSDMD, Caspase1, NLRP3, the release of IL-1β, IL-18 and the cell survival rate were detected by qRT-PCR, Western blot, ELISA and CCK-8 respectively. The results showed that both the mRNA and the protein expressions of GRP78 were gradually increased with time of BCG infection in THP-1 cells (P<0.01). The protein expression of GSDMD reached the highest level at 24 h post infection. The concentration of IL-1β and IL-18 was increased in a time-dependent manner (P<0.01). Compared with the uninfected group, the mRNA expression of GRP78, GSDMD, Caspase1, NLRP3, IL-1β and IL-18 (P<0.05), and the protein expression of GRP78, Caspase12, GSDMD, Caspase1 and NLRP3 (P<0.001) were both up-regulated, and the concentration of IL-1β and IL-18 was increased (P<0.01), and cells survival rate was reduced at 24 h post infection with BCG (P<0.001). However, with the co-treatment of TUDCA and BCG, the expression of the above molecules decreased and the cell survival rate increased compared with BCG infected group (P<0.01). Our results demonstrate that ERS in THP-1 cells induced by BCG infection regulated pyroptosis.

The aim of this study was to characterize the drug resistance and virulence of pathogens from ducks suspected of cholera in a farm in Guizhou. The pathogenic bacteria were cultured and confirmed by PCR. Capsule genotyping and lipopolysaccharide genotyping, multilocus sequence typing (MLST), ERIC-PCR, animal regression test, drug susceptibility test, whole genome sequencing(WGS) and comparative analysis of local genomes were conducted on those isolated strains. Five Pasteurella multocida isolates (PmCW1-5) were identified as A:L1 ST128 and shared high homology. All of them were resistant to ampicillin, amoxicillin, levofloxacin, and lincomycin. PmCW1 also showed low level resistance to ciprofloxacin. WGS data showed PmCW1 which is highly virulent also harbored genes mediating β-lactams, quinolones, tetracyclines, macrolides and peptides resistance and encoding multi-drug efflux pumps and multi-drug resistance proteins. Meanwhile, PmCW1 carried 201 different virulence-mediating genes including those encoding lipooligosaccharide/lipopolysaccharide (LOS/LPS), capsule, adhesion factor, and etc. In addition, type IV fimbriae genes (ptfA, comE, hofB, hofC, vfr), iron uptake related protein genes (ccmABCEF, hgbBC, fur, hscB), outer membrane protein genes (ompP5) were also detected. PmCW1 has the characteristics of typical A:L1 P. multocida. P. multocida ST128 isolated from ducks in this study has been rarely reported. The drug resistance and virulence analysis on those isolates will provide theoretical basis for clinical medication and vaccine development of duck cholera.

This study was conducted to clarify the role of GE296_RS01450 gene of Riemerella anatipestifer strain RA-LZ01 in mediating its tolerance to heavy metal ions. We constructed GE296_RS01450 gene deletion mutant Δ1450 and complemented strain cΔ1450. The growth curves of these strains, minimum inhibitory concentration (MIC) of antibiotics against them and their tolerance to heavy metal ions were determined, and the transcription levels of GE296_RS01450 gene of these strains treated with specific heavy metal ions were detected. The results showed that the growth ability of the deletion mutant and the MIC values of 11 classes of antibiotics to deletion mutant did not change significantly by comparison with the parent strain. By compared with parent strain and complemented strain, the sensitivity of deletion mutant to Ni, Mn and Co increased significantly. The transcription levels of GE296_RS01450 gene of parent strain treated with Ni and Mn were significantly up-regulated. The above results indicate that the GE296_RS01450 gene does not involve in the growth of the parent strain and the strain's resistance to antibiotics. However, the gene mediates the parent strain's tolerance to Ni, Mn and Co. GE296_RS01450 gene belongs to the GE296_RS01445-GE296_RS01450-GE296_RS01455 RND efflux pump and encodes the outer membrane protein. This protein is named RopM.

The purpose of this study was to analyze the effect of recombinant Lactobacillus reuteri from piglets expressing bovine lactoferrin peptide (pPG-LFCA-E/LR-CO21) as microecological on the growth promotion of piglets and resistance to Salmonella Choleraesuis CVCC79102 infection. In this experiment, the recombinant bacteria were continuously fed to 28-day-old weaned piglets for 21 days, and empty bacteria group, control group and antibiotic group were set up. On the 21st day, weaned piglets were orally infected with Salmonella Choleraesuis for 3 days, and the observation period was set for 7 days, and the serum, intestinal mucus and intestinal tissue of the experimental groups and the control group were collected after infection. The results showed that the average daily gain, feed gain ratio and immune organ index of piglets in the recombinant bacteria group were significantly increased (P<0.05). After being infected with Salmonella Choleraesuis CVCC79102, in the recombinant bacteria group, application of the recombinant bacteria increased the daily gain of piglets and reduced the rate of diarrhea compared with the control group; it significantly increased the amount of IgG in blood and IL-4 and sIgA in intestinal mucus (P<0.01). and significantly reduced the amount of IL-2, IL-12, IL-6 (P<0.05). It significantly increased the gene transcription level of intestinal tight junction protein ZO-1, Claudin-1 and the gene transcription levels of TLR4, Myd88 and MLCK, and significantly decreased the number of Salmonella Choleraesuis in the intestinal tract of piglets (P<0.01). There was no significant difference between the recombinant bacteria group and the antibiotic group. The above results show that pPG-LFCA-E/LR-CO21 can regulate the growth performance, intestinal morphology and intestinal barrier function of weaned piglets, and can protect piglets from Salmonella Choleraesuis infection to a certain extent, indicating that the recombinant bacteria have a certain application prospect feeding as microecological for weaned piglets.

To establish a rapid, sensitive and specific detection method for swine acute diarrhea syndrome coronavirus (SADS-CoV), the conserved region of the SADS-CoV N gene was amplified and cloned into pMD18-T vector. The recombinant plasmid pMD18-T-SADS-qN was used as the positive plasmid standard to establish a SYBR Green real-time PCR. The results showed that the method showed a good linear relationship when the template was among in 3.31×10¹-3.31×10⁷ copies·μL^-1, with a correlation coefficient (R²) of 0.997, and a slope of -3.318. The detection of SADS-CoV was specific, and that porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine delta coronavirus (PDCoV) and porcine reproductive and respiratory syndrome virus (PRRSV) were all tested negative by this method. This detection technology was sensitive and repeatable with the limited detection contend of the standard plasmid being 3.31×10¹ copies·μL^-1 and the intra-group and inter-group coefficients of variation being less than 1%. This method was used to detect the replication of IPI-2I and IPEC-J2 cells infected with SADS-CoV at different time points and different doses. The results showed that the viral load of IPI-2I and IPEC-J2 cells infected with SADS-CoV stayed at a low level at 2 h postinfection; after at 12-36 h postinfection increased rapidly; and at 36 h postinfection the growth rate slowed down, virus RNA maintained at a high level. The results of infection of cells with 0, 0.1, 1 MOI SADS-CoV showed that the mRAN transcription level of the virus showed a dose-dependent increase. When infected with SADS-CoV at an MOI of 1, the viral load in IPI-2I and IPEC-J2 cells was 10^6.7 and 10^5.3 copies·mL^-1, respectively. The established method was further used to detect the clinical samples of piglets that infection with SADS-CoV by oral route. High levels of viral RNA copies were detected in jejunum and ileum, indicating that the virus mainly colonized in jejunum and ileum. In conclusion, the SYBR Green real-time PCR method established in this study can detect SADS-CoV sensitively and specifically, and provide a reliable detection method for the diagnosis of SADS-CoV and virus related basic researches.

The identification of protein components of Haemaphysalis hystricis egg protoplasm illustrated the tick embryo protein profile and laid a foundation for the discovery of new anti-tick strategies. The egg protein extracts of Haemaphysalis hystricis was digested with trypsin according the FASP procedure and the LC/MS/MS method was employed to detect and identify peptides. The MS data were searched against the ovarian, salivary gland and midgut transcriptomic libraries of Haemaphysalis flava, and the 359, 312 and 357 specific peptides were detected, then the 108, 170 and 161 polypeptides were identified. Eventrually, the 103 proteins of them were considered as high confidence proteins (peptides ≥ 2). These high confidence proteins were involved in eight groups including enzymes, protease inhibitors, transporters, cytoskeleton proteins, protein synthesis modifications, secreted proteins and uncharacterized proteins.

The purpose of the experiment is to isolate and culture bovine amniotic fluid mesenchymal stem cells (AF-MSCs), and to study its therapeutic effect on alcoholic liver disease (ALD) mice and its effect on the mRNA expression of hypoxia inducible factor-1 α(HIF-1α), vascular endothelial growth factor (VEGF) and Toll-like receptor 4 (TLR4).AF-MSCs were isolated from amniotic fluid mesenchymal stem cells obtained from 4-to 5-month-old bovine embryos, and were identified by RT-PCR, immunofluorescence and induction of adipogenesis. Forty-eight ICR mice (male) were randomly divided into 4 groups:Blank control group (BC group), Model control group (MC group), AF-MSCs group (MC+AF-MSCs group) and silybin capsule (SC) group (MC+SC group), with 12 mice in each group.Except for the mice in BC group were intragastric administrated with 0.2 mL of normal saline, the rest of the groups were intragastrically administered with 56-degree Hongxing Erguotou (5 g·kg ^-1, 2 times·d ^-1) for 4 consecutive weeks; on the 29th day, the MC+AF-MSCs group was given extrahepatic injection Dil-labeled AF-MSCs (5×10⁶ cells·mL^-1), the MC+SC group was given intragastric SC twice a day for one day, total amount of SC was 2.4 mg (dissolve in 56-degree Hongxing Erguotou); after the experiment, the activities of serum glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST) and triglyceride (TG) were measured. HE staining and immunohistochemical methods were used to observe the modeling. Dil traced the colonization and distribution of AF-MSCs in the liver; qRT-PCR was used to detect the expression of HIF-1α, VEGF and TLR4 genes. RT-PCR and immunofluorescence results showed that bovine AF-MSCs were successfully isolated and had the ability to induce adipogenic differentiation. Serological results showed that the activities of ALT, AST and TG in the MC group were significantly higher than those in the BC group (P<0.01). Histopathological section showed that there were pathological changes such as steatosis, inflammatory cell infiltration and hepatic sinusoidal congestion in the liver tissue of the MC group, indicating that the 56-degree Hongxing Erguotou successfully replicated the ALD mouse model; Compared with the MC+SC group, the ALT and AST activities of the MC+AF-MSCs group were significantly reduced (P<0.01), the fatty change in the liver tissue was mitigated, and the liver sinusoids were not congested, indicating that AF-MSCs improved liver function more effective than SC; The qRT-PCR results showed that the expression of HIF-1α, VEGF and TLR4 genes in the MC+AF-MSCs group was significantly lower than that in the MC+SC group (P<0.01), indicating that AF-MSCs are involved in the regulation of HIF-1α/VEGF signaling pathway, and the effect of inhibiting TLR4 expression is better than SC; Cell tracing proved that AF-MSCs colonized the diseased site and chemotaxis more AF-MSCs migrated to the inflammation site and participated in the repair of liver tissue. In summary, this study successfully isolated bovine AF-MSCs. It can participate in the regulation of oxidative stress, angiogenesis, and inhibit the release of inflammatory factors. And the effect of improving ALD is better than SC.

This study aimed at screening terpenoids with anti-Toxoplasma gondii activity and evaluating their activity in vitro. The activity of 26 terpenoids against Toxoplasma gondii (T. gondii) in vitro was screened. The toxicity and safe concentration of these active compounds to Vero cells were determined by CCK-8 kit. The activity against T. gondii was further identified by Giemsa staining. The activity of the active compounds against T. gondii in vitro was evaluated by cell immunofluorescence, Giemsa staining and biochemical luminescence method. The results showed that among the 26 terpenoids, sabinene had a significant activity against T. gondii. The IC₅₀ of sabinene to RH-2F was 90.76 μg·mL^-1. The results of Giemsa staining and cellular immunofluorescence showed that sabinene had significant anti-proliferative effect on two intracellular strains of T. gondii and had a very significant inhibitory effect on the invasion of T. gondii (P<0.001). It has been found that sabinene had a significant inhibitory effect on the invasion and intracellular proliferation of T. gondii, and can be used as a potential lead compound against T. gondii.

To improve the water solubility and bioavailability of altrenogest (ALT), sulfobutyl ether-β-cyclodextrin (SBE-β-CD) was used to encapsulate ALT. In this study, the freeze-drying method was used to prepare the inclusion complex of ALT-SBE-β-CD, to solve the problems of limited clinical use of ALT due to its water insolubility and low bioavailability, so as to broaden the medicinal way of ALT. The inclusion complexes were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis and microscopy, and the solubility of the inclusion complex was determined by the solubility method. Taking the yield and inclusion rate of inclusion complex as evaluation indexes, the optimal preparation conditions of inclusion complex were selected. The optimal preparation conditions were as follows:at 55℃, the molar ratio of ALT to SBE-β-CD was 1:6, the inclusion time was 5 h, the pH value of the solution was 8, and the amount of solvent was 15 mL (when the dosage of ALT was 0.1 g). The average inclusion rate of ALT was (90.90±1.80)% (n=3), the average drug loading was (4.30±2.30)% (n=3), and the yield of the inclusion complex was (93.19±1.67)%. By Fourier transform infrared spectroscopy, thermogravimetric analysis and microscope imaging method for characterizing the inclusion complex was prepared by, verifiable inclusion complex was successful, solubility method, the test results show that the formation of inclusion complex, its solubility is ALT technical increased 988.86 times, and the inclusion complex has good stability, can meet the requirements of different dosage forms. SBE-β-CD has a good solubilization effect on the drug, which can increase the solubility of the ALT. The preparation method is simple, the conditions are mild, and it is easy for industrial production, which is conducive to the further development and utilization of the drug.

This article aims to prepare florfenicol amorphous solid dispersion (ASD) to improve its solubility and bioavailability. The florfenicol ASD was prepared by antisolvent coprecipitation method with hydroxypropyl methyl cellulose acetate succinate (HPMCAS-MF) as carrier. The ASD was characterized by X-ray diffraction, thermal analysis and scanning electron microscopy. The in vitro and in vivo release of florfenicol was studied by dissolution and pharmacokinetics. When the ratio of florfenicol to carrier was 5:5, a solid dispersion was formed. The results showed that there was no crystal diffraction peak of florfenicol and no specific melting point peak of florfenicol. The SEM results showed that the surface of florfenicol ASD was loose and porous, and increased the surface area, and the saturated solubility of florfenicol increased by 4.8 times, and the relative bioavailability increased by 37.2%, and no drug crystal peak appeared in the solid dispersion within 3 months. The results showed that the new solid dispersion of florfenicol prepared by co-precipitation method has good stability and could effectively improve the solubility and bioavailability of florfenicol.

In this study, we aimed to investigate whether high glucose regulates proinflammatory cytokine IL-1β, IL-6 and TNF-α release from bovine alveolar macrophages (BAMs) through RAGE-TLR4 crosstalk. BAMs were randomly divided into normal glucose group (NG), high glucose group (HG), high glucose + RAGE inhibitor group (H + F), high glucose + TLR4 inhibitor group (H + T) and DMSO group, and the supernatant and lower cells were collected after 12 h of treatment. The mRNA and protein expression of RAGE, TLR4, MyD88 and NF-κB p65 were detected by qRT-PCR and Western blot, and the concentrations of TNF-α, IL-1β and IL-6 in the supernatant were detected by ELISA. The levels of mRNA and protein expression of RAGE, TLR4, MyD88 and NF-κB p65 and the concentrations of IL-1β, IL-6 and TNF-α in the supernatant of HG group were significantly higher than those of NG group, H+F group and H+T group (P<0.01); the levels of mRNA and protein of MyD88, NF-κB p65 and RAGE and the concentrations of three proinflammatory cytokines in the supernatant of H+F group and H+T group were significantly higher than those of NG group (P<0.05, P<0.01). Our results suggest that high glucose can cause the release of proinflammatory cytokines IL-1β, IL-6 and TNF-α from bovine alveolar macrophages via RAGE-TLR4 crosstalk, which further elucidated the molecular mechanism of high glucose promoted the inflammatory response in bovine alveolar macrophages.

In order to investigate the protective effect of melatonin on toxic damage caused by cadmium in cerebral cortex of duck, sixteen GaoYou ducks were randomly allocated into 4 groups:control group, melatonin-treated group, cadmium-treated group, cadmium and melatonin co-treated group. The ducks in the control group were free to eat and drink. The ducks in the melatonin group received drinking water containing 0.2 mg·L^-1 melatonin. The ducks in the cadmium group received feeding mixed with 2 mg·kg^-1 cadmium chloride. The ducks in the cadmium and melatonin co-treated group received drinking water containing 0.2 mg·L^-1 melatonin plus feeding mixed with 2 mg·kg^-1 cadmium chloride. After 60 d, the cerebral cortices of ducks were dissected and collected. The levels of malondialdehyde (MDA) and total antioxidant capacity (T-AOC) in cerebral cortex of duck were measured by colorimetry, the contents of tumor necrosis factor-alpha (TNF-α) and interleukin-1β (IL-1β) were detected by ELISA, the nuclear translocation of Nrf2 was observed by immunohistochemical staining, the protein expression of Nrf2 and HO-1 was detected by Western blot. The results showed that compared with the control group, the Nrf2 nuclear translocation was obvious, the level of T-AOC showed extremely significant decrease (P<0.01), the contents of MDA, TNF-α, IL-1β, the protein expression of Nrf2 and HO-1 showed extremely significant increase (P<0.01) in the cerebral cortex of duck in cadmium-treated group. Compared with the cadmium-treated group, the Nrf2 nuclear translocation was decreased, the level of T-AOC showed significant increase (P<0.05), the contents of MDA, TNF-α, and IL-1β, the protein expression of Nrf2 and HO-1 showed significant or extremely significant decrease (P<0.05 or P<0.01) in the cerebral cortex of duck in cadmium and melatonin co-treated group. The results indicate that melatonin has a protective effect on toxic damage caused by cadmium in cerebral cortex of duck.

The aim of this study was to investigate the optimal dose for establishing the canine type I diabetes model by combined injection of streptozotocin and alloxan. Eighteen healthy mixed-breed dogs aged 9-12 months with an average weight of (5.09±0.30) kg were selected. and randomly divided into 6 groups with 3 replicates per group and 1 per replicate. Group I(20 mg·kg^-1/40 mg·kg^-1), II(30 mg·kg^-1/50 mg·kg^-1)and III(40 mg·kg^-1/60 mg·kg^-1)were given single intravenous injection of streptozotocin and alloxan mixture, respectively; Group IV(10 mg·kg^-1/20 mg·kg^-1), V(15 mg·kg^-1/25 mg·kg^-1)and VI(20 mg·kg^-1/30 mg·kg^-1) were given twice intravenous injection of streptozotocin and alloxan mixture, respectively. The interval between the two injections was 24 h. The diabetes modeling effect was evaluated by the changes of blood glucose, glucose tolerance, blood physiological and biochemical, and pancreatic histomorphology. The results showed as follows:1) The fasting blood glucose in groups I and IV had been at a normal level, but in group II and III both exceeded 15 mmol·L^-1 for 7 consecutive days. Although there was an increase in group V and VI, they did not exceed 10 mmol·L^-1 during the experiment. 2) According to the results of the intravenous glucose tolerance test, the blood glucose in every group rose to its peak within 15 minutes, and then groups I and IV gradually decreased to the pre-injection level, group V decreased but did not reach the pre-injection level, while group II, III and VI continued to maintain high levels. 3) The blood routine indexes of each group were in a normal physiological range. The blood routine indexes of each group were in a normal physiological range. The alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), urea (UREA), creatinine (CREA) of group III were all above the normal physiological range. 4) According to the results of pancreatic histomorphology, the islet structure in group II and III was significantly destroyed. The above results indicate that a single intravenous injection containing 30 mg·kg^-1 streptozotocin and 50 mg·kg^-1 alloxan can effectively establish the canine type I diabetes model, and the blood glucose level can exceed 15 mmol·L^-1 for 7 d consecutively without serious hepatorenal toxicity.

In this present study, in order to investigate the effect of GnIH on the apoptosis and testosterone synthesis of mouse Leydig cells, its overexpression vector was constructed. RNA was extracted from the testis tissues of male mice at 6-8 weeks, reverse transcribed into cDNA. The GnIH gene was amplified and purified by PCR, and connected to the PLVX-IRES-ZsGreen1 vector through T4 ligase. Then, the recombinant plasmid was transformed into the competent cells, and extracted the plasmid after identified by PCR, double enzyme digestion and sequencing. Subsequently, the plasmid was transfected into the TM3 cell line for 72 h. The experiment was divided into two groups:the empty plasmid group and the GnIH overexpression group. Cell fluorescence was observed under a microscope. The mRNA level of GnIH, apoptosis gene and testosterone synthase gene in the TM3 cells were detected by real-time fluorescent quantitative PCR (qRT-PCR), apoptotic rate and testosterone secretion level were detected by flow cytometry and ELISA, respectively. The results showed that the GnIH amplified sequence was consistent with the reference sequence. After transfecting the GnIH overexpression plasmid into the TM3 cell line for 72 h, high-intensity green fluorescence was observed, and the expression levels of GnIH was significantly increased in the overexpression group (P<0.01). These results indicated that GnIH overexpression vector was successfully constructed and highly expressed in the TM3 cell line. After 72 h of transfection, compared with the empty plasmid group, the apoptosis rate in the overexpression group was significantly increased (P<0.01), and the expression ratio of apoptosis genes P53 and Bax/Bcl-2 was also significantly increased (P<0.01). Compared with the empty plasmid group, the concentration of testosterone was extremely significantly cut down in the overexpression group (P<0.01). Meanwhile, the expression of the testosterone synthesis-related enzyme genes, P450 scc mRNA expression was extremely significantly decreased (P<0.01); and the expressions of the StAR, 3β-HSD and P450c17 mRNA were obviously reduced (P<0.05). There was no significant difference of 17β-HSD mRNA expression between the two groups (P>0.05). In summary, overexpression of GnIH in TM3 cells could induce apoptosis of TM3 cells, inhibit testosterone secretion, and decrease the gene expression levels of testosterone synthesis-related enzymes. We can infer that GnIH plays a negative regulatory role in the process of testosterone synthesis in mice. This study can provide a scientific basis for revealing the role of GnIH in the reproductive regulation of male mammals.

Senecavirus A (SVA), a newly emerged causative agent linked to swine idiopathic vesicular disease and epidemic transient neonatal losses, is spreading worldwide and causing economic loss to the swine industry. Since no commercial vaccine or antiviral drug has been licensed, constructing a reporter virus expressing enhanced green fluorescent protein (EGFP) for high-throughput screening antiviral drugs will be undoubtedly conducive to prevent and control of SVA. In this study, the porcine teschovirus-1 2A gene (P2A) was synthesized and cloned into the C-terminal of EGFP gene of pEGFP-C1 to construct pEGFP-P2A vector. Subsequently, the EGFP-P2A gene was inserted into pwtSVA, the DNA-launched infectious clone of wtCH/HeN-2018, at site between 2A and 2B through homologous recombination to create a recombinant plasmid pSVA-EGFP. The pSVA-EGFP was then directly transfected into PK-15 cells and the recombinant virus rCH/HeN-2018-EGFP caused stable cytopathic effect and strong green fluorescence was harvested after twice blind passages. The genetic stability and growth kinetic of rCH/HeN-2018-EGFP was further investigated via RT-PCR and virus infection assays. Results demonstrated that rCH/HeN-2018-EGFP shared similar growth dynamics with that of the parental virus wtCH/HeN-2018 and rCH/HeN-2018. Moreover, no mutation was detected in the inserted EGFP-P2A gene and cells infected by P10 passage of rCH/HeN-2018-EGFP stably expressed green fluorescence. Finally, the rCH/HeN-2018-EGFP was employed to test the antiviral effect of curcumin and baicalin. Data showed that pretreated with both curcumin (20 μmol·L^-1) and baicalin (110 μmol·L^-1) for 2 hours restricted the replication of rCH/HeN-2018-EGFP in PK-15 cells, with green fluorescence intensity lower than that in DMSO control group. Compared to curcumin, however, baicalin exhibited greater anti-SVA effect, which was further confirmed by inhibiting replication of wtCH/HeN-2018 in vitro. Conclusively, the recombinant rCH/HeN-2018-EGFP virus constructed in this study effectively and stably expressed EGFP during infection and provided a robust tool to screen anti-SVA drugs and proteins.

To investigate the endophytic fungi species and population distribution of the poisonous plant, Thermopsis lanceolata, the experimental materials collected from Qinghai Province were disinfected with surface disinfection method and then endophytic fungi were separated. The morphological and 5.8S rDNA-ITS sequence analysis were used to identify these strains, and the MEGA7.0 software was used for phylogenetic analysis. The results showed that twenty-nine endophytic fungi were isolated from the Thermopsis lanceolata, which belong to 7 classes, 9 orders, 11 families and 12 genera. The total relative separation frequency was 85.42%. Endophytic fungi are most commonly isolated from leaves (41.38%), followed by stems (37.93%), seed (13.79%) and roots (6.90%). According to phylogenetic analysis, all endophytic fungi belong to two populations with close genetic relationship. The results indicated that Thermopsis lanceolata had abundant endophytic fungi species and Penicillium sp. was the dominant genus.