Hox genes encode conserved transcription factors that control skeletal patterning in the developing embryo. They are expressed in restricted domains and function to regulate the morphology of specific vertebral elements. It has been more than 40 years since the gene family was first discovered in drosophila. The updated studies allow an emerging understanding of Hox genes function in patterning the vertebrate axial skeleton. This review summarizes genetic and embryologic findings regarding the role of Hox genes in establishing axial morphology and how these combined results impact the current understanding for the vertebrate Hox control, which will be of great help in studying vertebrate morphology and quantitative variation of animals.

Silent estrus has been the bottleneck of estrus identification, which seriously impedes the development of dairy farming industry. The research on pheromone provides a new idea for promoting the technical innovation of cow estrus detection through the way of bionics. The observation of mating between male and female cattle shows that there is an obvious flehmen between estrous cows before mating, which indicates that there is stronger pheromone communication between male and female animals in addition to sensory stimulation of sexual organs, which has been confirmed in bees and silkworm moths. Therefore, this paper reviews the study on pheromones from bovine feces, urine, vaginal mucus, milk, saliva and so on, hoping to provide an important reference for further revealing the pheromone secretion rule of estrous cows, so as to promote the research and development of timely and accurate estrous identification technology of cows in non-invasive manner.

Exosomes are nano-level extracellular vesicles, which are widely present in the tissues and body fluids of a variety of animals. In various types of extracellular vesicles, exosomes can carry multiple compounds with biological activity. At the same time, the sorting of compounds in exosomes is a regulated and non-random process. In recent years, studies have shown that milk-derived exosomes can promote development of the intestinal and immune system of young animals after being absorbed in digestive tract, and they are also involved in some disease processes. This article reviews the production and function of exosomes, the extraction and identification of milk-derived exosomes, as well as their application of milk exosomes, in order to provide the theoretical foundation for further research on milk-derived exosomes.

Bovine adenovirus type 3 (BAdV-3), a member of the genus Mastadenovirus in the family Adenoviridae, is an unenveloped linear double-stranded DNA virus. It is one of the important pathogens causing respiratory diseases in cattle. This paper reviews the progress of research on the biological characteristics, prevalence, clinical symptoms and pathological changes, and detection techniques of BAdV-3 in order to provide a reference for subsequent scientific research and effective prevention and control of the disease.

The aim of this study was to use the YE1-BE3-FNLS vector to carry out a single-base mutation of C>T in the porcine CD163 gene to form the stop codon TAA, which would lead to the premature termination of the translation of the CD163 gene. The website was used to design high-efficiency sgRNA sequence at the 7th exon of porcine CD163 gene and make it conform to C5 feature, and the following two bases of C5 were AA, and the number of bases between CAA and the start codon ATG was the integer multiple of 3. The CD163-sgRNA and APOBEC-Cas9n-UGI fragment in the YE1-BE3-FNLS vector was amplified by PCR, and CD163-sgRNA and APOBEC-Cas9n-UGI mRNA were produced by in vitro transcription. Porcine oocytes were cultured in vitro and undergone parthenogenetic activation. The CD163-sgRNA and APOBEC-Cas9n-UGI mRNA were injected into parthenogenetic embryos by micromanipulator. After the embryo developed to the stage of morula and blastocyst, the genomic DNA of the embryo was extracted and its single-base editing region was subjected to PCR amplification and sequencing. The results showed that a CD163-sgRNA was successfully screened among 49 candidate sgRNAs. The CD163-sgRNA and APOBEC-Cas9n-UGI fragments were successfully amplified by PCR, and the porcine parthenogenetic embryos developed to the 2-4 cell stage after injection of CD163-sgRNA and APOBEC-Cas9n-UGI mRNA. After PCR amplification and product sequencing of selected injected 20 embryos at 2-4 cell stage, there were 12 embryos with the expected site C(num1479)>T of the 7th exon of the CD163 gene, and the single-base editing efficiency was about 60%. It was found that the CD163-sgRNA had 5 off-target regions in the case of a mismatch of 3 bases based on the off-target analysis. After analyzing these 5 regions, it was found that there was no C>T mutation using PCR amplification and sanger sequencing. The pig CD163 gene was suffered with base editing by YE1-BE3-FNLS tool at the embryo level in this study, and the 7th exon expected site (num1479) C was successfully changed to T, which can terminate the translation of CD163 gene in advance by making the codon CAA to TAA.

The aim of this study was to investigate the differences in genetic patterns between Hetian and Qira Black sheep, to screen superior genes and their molecular markers, and to promote the development and utilization of genetic resources of sheep breeds in southern Xinjiang. In this study, 84 healthy, adult Hetian ewes and 41 Qira Black sheep were selected, respectively, and ear tissues were collected and put into 75% alcohol. DNA was extracted based on phenol-chloroform method, and Illumina Ovine SNP50 was prepared by electrophoresis and nucleic acid instrumentation. For genomic data, quality control was performed with Plink1.07, and quality control criteria: exclusion of SNPs with detection rate less than 90%, minimum allele frequency (MAF)<5%, and Hardy-Weinberg equilibrium P<1×10^-6. PCA populations of both breeds were classified, and HapFLK analysis was performed to take the SNPs corresponding to the top 1% of P values, and after sheep genome Oar_v4.0 annotation of genes, the GO and KEGG analyses were performed on the candidate genes. The results showed that: 1) Hetian sheep and Qira Black sheep differed in genetic structure after different degrees of selection and breeding, but they were not highly differentiated compared with the Tibetan sheep population. 2) A total of 167 candidate genes were obtained, and based on the differences in production performance between Hetian sheep and Qira Black sheep, the genetic patterns of the two breeds were resolved at the genomic level, and genes related to sheep wool growth were found to be KRT82 and KRT84, etc., wool color-related genes KIT, etc., growth and development-related genes AMHR2, SP1, OARDACVR1B, ADIPOQ, etc., and environmental adaptation-related genes PDGFRA, ADIPOQ, etc. Genome-level differences between Hetian and Qira Black sheep in different production traits were resolved by genomic selective sweeping, and it was found that the two breeds differed in wool quality traits, coat color, and tail type, while the differences in reproduction-related traits were small. In conclusion, the sheep genome microarrays was used to compare the differences in genetic structure of Hetian and Qira Black sheep and to search for the related superior genes, which would provide reference for the conservation of sheep germplasm resources and molecular selection and breeding in Southern Xinjiang region.

The purpose of this study was to clone the sequence of goat RPL26 gene and explore its expression in tissues and its regulation on adipocyte differentiation of goat. The one-year old male Jianzhou da’er goat (with good healthy growth status, weight of 50 kg, n=3) was selected as the experimental object, the RPL26 sequence was cloned by RT-PCR, and the sequence of gene and protein were analyzed by bioinformatics; Goat tissue cDNA was used as template to construct tissue expression profiles by qPCR; The overexpression vector of RPL26 was constructed by double enzyme digestion method. RPL26 was overexpressed in goat intramuscular adipocytes by liposome transfection. The morphological changes were observed by Oil red O staining and Bodipy staining after induction. The overexpression efficiency and expression of adipogenesis related genes were detected by qPCR to clarify its effect on intramuscular adipocyte differentiation. The results showed that the cloned goat RPL26 sequence was 544 bp, the CDS region was 438 bp, and the coding protein was negatively charged unstable hydrophilic protein, which was mainly located in the nucleus. RPL26 was widely expressed in goat tissues, and highly expressed in abdominal subcutaneous fat and back subcutaneous fat, which were extremely significantly higher than that in other tissues (P<0.01). RPL26 was overexpressed in goat intramuscular adipocytes, and the lipid droplets in intramuscular adipocytes increased significantly, the OD value detected by Oil red O staining showed the same result; qPCR results showed that the expression level of Pref1 decreased significantly(P<0.01), the expression levels of adipocytes differentiation marker genes such as PPARγ, and lipid metabolism marker gene such as LPL showed an extremely significantly upregulated expression(P<0.01). The cloned goat RPL26 sequence is 544 bp, and the CDS region is 438 bp, its highest expression was in abdominal subcutaneous fat. RPL26 has a positive regulatory effect on the differentiation of goat intramuscular preadipocytes.

The aim of this research was to identify non-coding RNA, circNMT1, clarify its tissue and cells expression patterns, and explore the effect of circNMT1 overexpression on adipocyte differentiation. In the study, the heart, liver, spleen, lung, kidney, longissimus dorsi, dorsal subcutaneous adipose tissue and preadipocytes of 30-month-old Chinese swamp buffalo (Xinyang buffalo, n=3) and 3T3-L1 cells were used as experimental materials. Semiquantitative PCR and real-time quantitative PCR (qRT-PCR) were used to identify circNMT1, explore its location in the nucleus or cytoplasm of buffalo adipocytes. Relative expression level of circNMT1 in different tissues and preadipocytes of buffalo at different differentiation stages were detected by qRT-PCR. circNMT1 was overexpressed in 3T3-L1 cells and buffalo preadipocytes respectively, and the accumulation of lipid droplet was detected by morphology and quantitative method. Meanwhile, the relative expression level of adipogenic differentiation marker genes was detected by qRT-PCR. The results showed that circNMT1 was stably expressed circRNA, which was expressed in both nucleus and cytoplasm of buffalo preadipocytes. And circNMT1 was highly expressed in adipose tissue and maturation adipocytes (P<0.001). Gain-of-function experiments demonstrated that the overexpression of circNMT1 significantly promoted the accumulation of lipid droplets in 3T3-L1 cells and buffalo adipocytes, and significantly upregulated the relative expression level of adipogenic marker genes PPARG, C/EBPα and FABP4 (P<0.01). These results indicated that circNMT1 might be a positive regulator for buffalo adipocytes differentiation, which would provide new insights into the regulatory roles of circNMT1 in buffalo adipocytes.

The study aimed to explore the relationship between lysozyme activity in donkey milk and lactation stages and treatment temperature. The lysozyme activities in fresh milk of cow, sheep and donkey were compared. To investigate the effects of lactation stages and lactation level on lysozyme activity in milk，female donkeys aged 4-9 years were divided into 3 groups according to the average daily milk yield: low (below 400 g), medium (400-1 000 g) and high (above 1 000 g) groups，3 animals in each group, and 9 animals in total were used to observe the lysozyme activity in the milk of female donkeys from the 3rd to 10th lactation month regular continuous sampling every month for 7 days. Finally, mixed donkey milk obtained on 5 sampling days was divided into 6 aliquots, one of which was not treated, and the others were heated at 62 ℃ for 30 min, 72, 77, 82 and 87 ℃ for 15 s, respectively. Lysozyme activity was detected after cooling to study the effect of heat treatment condition on lysozyme activity in milk. The results showed that the lysozyme activity in donkey milk (12 367 U·mL^-1) was significantly higher than that in cow milk (334 U·mL^-1) and sheep milk (233 U·mL^-1) (P<0.05). Lysozyme activity in milk secreted by female donkeys at the 8th lactation month after parturition was the highest (14 383 U·mL^-1) (P<0.05). During the processing of raw milk, sterilization at 62 ℃ for 30 min had no significant effect on lysozyme activity (P>0.05), while sterilization at 72-87 ℃ for 15 s significantly decreased lysozyme activity (P<0.05). Compared with cow and sheep milk, donkey milk has higher lysozyme activity, and long-term sterilization at low temperature does not affect the lysozyme activity.

The purpose of this study was to explore the regulatory effect of MEI1 gene with alternative splicing event on spermatogenesis in Mongolian horses, improve sperm quantity and semen quality of horses by using molecular regulation techniques, effectively improve reproductive efficiency, reduce production costs, accelerate genetic progress, and promote population reproduction. The testicular sertoli cells(SC) of two-year-old Mongolian horses were used as the subjects, the MEI1-1-pIRES2-EGFP recombinant plasmids without exon skip (ES) events and MEI1-2-pIRES2-Dsred recombinant plasmids with ES events were constructed and transfected into testicular sertoli cells, respectively. There were 3 repeats in each group. The proliferation and activity of transfected cells were detected by CCK-8 at 0, 24, 48, 72 h of transfection, the transfected cells were screened by G418. The transfected cells were collected and total RNA was extracted, and the differential expression of meiosis related genes in the two cells was detected by qRT-PCR after reverse transcription. The MEI1-1-pIRES2-EGFP and MEI1-2-pIRES2-Dsred vectors were successfully constructed. Agarose gel electrophoresis and sequencing experiments showed that the vectors construction was successful, and the recombinant plasmid mass spectrum was drawn. CCK-8 assay showed that the cells transfected with two plasmids had the highest cell activity and proliferation level at 48 h after transfection, and cells transfected with MEI1-2-pIRES2-Dsred had the highest cell activity. The transfection efficiency of transfected cells increased by G418 screening; The test results showed that some genes in meiosis pathway (BUB1, CUL1, CCNB1, PPP2R5C, CCNB2, etc.) and some factors regulating sperm meiosis in sertoli cell signaling pathway (ARID4A, FSH, GDNF, Stra8, NRG1, etc.) had significant differences in expression in the two types of cells, and all had higher expressions in SC transfected MEI1-2-pIRES2-Dsred plasmids. The MEI1 gene with ES events occurence is more conducive to regulating the meiosis and spermatogenesis, which has laid a theoretical foundation for exploring the mechanism of alternative splicing events affecting spermatogenesis in Mongolian horses.

The study aimed to investigate the proteins expression in peripheral blood at early pregnancy, and to discover potential protein biomarkers for early pregnancy diagnosis in sows. In this study, the peripheral blood from 8 healthy, normal production performance and similar physical condition of pregnant and non-pregnant Landrace×Large White multiparous sows was collected on the 15th day after mating. Proteins were isolated, then iTRAQ technique and bioinformatics software were employed to screen differentially expressed proteins related to embryo attachment. ELISA was used to verify its expression and ROC curve was analyzed. A total of 24 differentially expressed proteins were screened in the peripheral blood of pregnant and non-pregnant sows(19 were up-regulated and 5 were down-regulated in pregnant sows), which mainly enriched in PI3K-Akt, p53, MAPK, Estrogen, mTOR and other important signaling pathways. ELISA test verified their expression trend were consistent with iTRAQ results, which proved the accuracy of iTRAQ results. ROC curve analysis showed that FLNC, ADIPOQ, HP, TERF2, FLNC-ADIPOQ and ADIPOQ-HP could be used as a potential biomarker for early pregnancy of sows, with good accuracy and specificity. These results suggest that the differential proteins FLNC, ADIPOQ, HP and TERF2 can be used as potential biomarkers for early pregnancy diagnosis in sows, and provide a new view for the study of the mechanism of embryos implantation in sows.

This study aimed to investigate the effect of TIMP2 protein derived from porcine uterine fluid exosomes on embryo implantation during early pregnancy. Exosomes were isolated from the uterine fluid of Yorkshire pigs at day 9 (n=3) and 12 (n=3) of pregnancy by ultracentrifugation. The relative abundance of TIMP2 protein in exosomes was determined using Western blot (WB)， and the expression and location of TIMP2 in uterus was detected by immunohistochemistry. Subsequently, co-culture experiments of exosomes and porcine trophoblast cells (PTr2) were used to investigate the targeted transport of exosomes for TIMP2 protein. Finally, the TIMP2 gene was interfered or overexpressed in PTr2, and the effects of TIMP2 on proliferation and migration of PTr2 cells were determined using CCK-8, EdU and wound healing assay. The exosomes were successfully isolated from uterine fluid by ultracentrifugation, and their particle sizes were mainly distributed from 30 to 150 nm. WB results showed that the TIMP2 protein abundance in exosomes of pig uterine fluid at day 12 of pregnancy was significantly increased (P<0.05). While the co-culture experiments of exosomes and trophoblast cells showed that TIMP2 protein could be targeted transport to trophoblast cells through exosomes. Immunohistochemical results showed that TIMP2 protein was found in both the embryo and endometrium, and the abundance level in endometrium at day 12 of pregnancy was significantly higher than that at day 9 of pregnancy (P<0.01). In addition, in vitro cell culture experiments found that interfering with TIMP2 gene in PTr2 significantly enhanced cell proliferation (P<0.01) and migration ability (P<0.05)， while the overexpression of TIMP2 impaired the proliferation (P<0.01) and migration ability (P<0.05). These results suggest that TIMP2 protein in uterine fluid exosomes may be secreted by the maternal endometrium, and could be targeted into the trophoblast cells through exosomes to regulate their proliferation and migration, thereby affecting embryo implantation.

The aim of this study was to investigate the effect of RFRP-3 on in vitro maturation of porcine oocyte. In this study, GV stage oocytes from the ovaries of healthy sows collected from slaughterhouses were randomly divided into 3 groups. The maturation rate of porcine oocytes in each group was calculated after incubating with 0, 10^-6 and 10^-8 mol·L^-1 RFRP-3 for 44 h, respectively. In the subsequent experiment, the collected oocytes were randomly divided into 2 groups. Porcine oocytes were cultured in medium with 0 and 10^-8 mol·L^-1 RFRP-3 for 44 h, respectively. The cumulus expansion of porcine oocytes was observed and the cumulus expansion index and maturation rate of oocytes in each group were calculated. The expression of cumulus expansion factors (PTGS2, HAS2, PTX3), oocyte secretory factors (GDF9, BMP15) and cyclin related genes (CCNB1 and CDK1) were detected by qRT-PCR. The contents of MPF and cAMP in oocytes were detected by ELISA kit. The concentrations of progesterone and estrogen were measured by radioimmunoassay. Oocytes in each group were no less than 100, and each test was repeated 3 times. Compared with the control group, the maturation rate of porcine oocytes cultured with 10^-8mol·L^-1 RFRP-3 was extremely significantly reduced (P<0.01). No significant change in cumulus expansion rate and cumulus expansion index were observed(P>0.05), but the expression of cumulus expansion factors (PTGS2, HAS2, PTX3) was extremely significantly decreased (P<0.01). RFRP-3 could extremely significantly reduce MPF content in porcine oocytes (P<0.01), and had no significant effect on cAMP content (P>0.05). After the addition of RFRP-3, the expression levels of GDF9 (P<0.01) and BMP15 (P<0.05) were promoted, while the expression levels of CCNB1 (P<0.05) and CDK1(P<0.05) were inhibited; meanwhile, the concentrations of progesterone and estrogen in medium of experimental groups were extremely significantly decreased (P<0.01). In conclusion, RFRP-3 inhibits in vitro maturation of porcine oocytes by regulating the expression of maturation related factors and cumulus expansion factors and the secretion of steroid hormones. This study will lay a theoretical foundation for elucidating the regulatory effect of RFRP-3 on mammalian oocytes.

High-energy diet is one of the important causes of obesity, and intestinal microorganisms play an important role in it. The aim of this study is to explore the dynamic changes of intestinal microbial composition in the process of high-fat and high-sugar diet and screen the intestinal microorganisms which closely related to the development of obesity. In this study, Guangxi Bama mini-pigs were randomly divided into normal diet group (CN group) and high-fat and high-sugar diet group (HFD group) for 30 d of dietary intervention. Fecal samples were collected at 0, 7, 15 and 30 d after intervention, respectively. The effects of high-fat and high-sugar diet on intestinal microbial composition, structure and function of Guangxi Bama mini-pigs were analyzed by 16S rRNA gene sequencing. The results showed that the diversity of intestinal microbiota in HFD group was generally lower than that of CN group, and high-fat and high-sugar diet changed the structure of intestinal microflora in mini-pigs. With the increase of intervention time of high-fat and high-sugar diet, the overall change trend of intestinal microbiota was observed. At the phylum level, the abundance of Firmicutes increased while Bacteroidetes decreased gradually in the pigs of HFD group. At the genus level, the abundance of Lactobacillus decreased gradually while the abundance of Ruminococcus increased continuously in the HFD group. Twelve bacterial genuses, including g__Lactobacillus, g__norank_f__p_251_o5 and g__unclassified_c__Bacilli, were significantly enriched in the CN group, while 8 genuses including g__Peptococcus, g__Collinsella and g__Senegalimassilia were significantly enriched in the HFD group, which might be potential microorganisms related to obesity. Microbial function prediction analysis found no significant difference in microbial function between the two groups, which were mainly enriched in amino acid biosynthesis, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, glycolysis and gluconeogenesis. This study indicated that the high-fat and high-sugar diet reduced the diversity of intestinal microorganisms, changed the microbial structure, resulting in a certain imbalance of gut microbiota, and some potential microorganisms which were closely related to development of obesity were found.

The study aimed to investigate the main genes and pathways of healthy tribeneficial bacteria regulating lipid metabolism in broilers, and to elucidate the mechanism of their effects on lipid metabolism. A total of 200 one-day-old male Jute broilers were randomly divided into 2 groups with 5 replicates per group and 20 broilers per replicate. The individuals in control group drank water normally, and the individuals in probiotic group drank water added with 1% healthy tribeneficial bacteria compound preparation (Lactobacillus casei∶Lactobacillus acidophilus∶Bifidobacterium=1∶1∶2). The experiment lasted for 42 days. The meat quality of broilers were measured and ileal transcriptome sequencing was performed. The results showed that, compared with the control group, the abdominal fat percentage and shear force of broilers supplemented with healthy tribeneficial bacteria decreased, and the breast muscle percentage and intramuscular fat increased (P>0.05). Transcriptome sequencing showed that there were 12 significantly differentially expressed genes related to lipid metabolism. Compared with the control group, the expressions levels of LPL, PLIN1, GOS2, FABP4, RBP7, THRSP, FABP3, SCD, THEM4 and CYP2C18 in probiotic group were significantly up-regulated (P<0.05), and the expressions levels of CYP7B1 and GGT5 were significantly down-regulated (P<0.05). These genes were mainly enriched in GO items such as response to fatty acids, chylomicron remodeling, response to lipid, white fat cell differentiation and long-chain fatty acid binding, as well as important pathways related to steroid hormone biosynthesis, PPAR signaling pathway, arachidonic acid metabolism and regulation of lipolysis in adipocytes. The results showed that healthy tribeneficial bacteria can affect lipid metabolism, reduce abdominal fat content and promote intramuscular fat deposition of broilers by regulating the related pathways and the expression of genes related to lipid deposition, transport and decomposition.

Antigenic drift on hemagglutinin (HA) protein of H9N2 subtype avian influenza virus (AIV) happens frequently, however, the key antigenic sites for identification of antigenic changes of H9N2 subtype AIV in China remain unclear. Two monoclonal antibodies(mAbs)2E4 and 2D6 with high hemagglutination inhibition (HI) titers against H9N2 subtype AIV were used, and antigenic sites were identified by selection of mAbs escape mutants with H9N2 AIV strain A/Chicken/Shanghai/F/1998. The antigenic profile of representative strains of H9N2 subtype AIV in 2017-2019 was investigated by HI assay using mAbs 2E4 or 2D6. The relationship between antigenic sites and genotype and variation characteristic of antigenic sites for H9N2 AIV in China were also analyzed. Two antigen sites 145 and 153 on the globe of HA protein of H9N2 AIV were identified by mAbs 2E4 or 2D6. H9N2 AIVs isolated in Eastern China in 2017-2019 were divided into two major branches:Group 1 and Group 2, according to the genetic distance of the HA gene. The representative viruses in Group 1 had good reactoin with two mAbs. In contrast, the viruses in Group 2 had poor reaction with two mAbs. Sequence analysis showed that S145D (16/16) and D153G (15/16) mutation happened in the viruses of Group 2. Natural mutation rate analysis showed that the rates of H9N2 subtype AIV strains isolated in China in 1996-2021 with 145D or 153G have risen to 68% or 66%, respectively, which become prevailing strains. The mAb 2E4 may be a tool for antigenic typing of the prevailing H9N2 subtype AIV, and antigenic site 145 may be one of molecular target to identify the difference between Group 1 and Group 2.

The purpose of this study is to establish a blocking ELISA antibodies detection method for porcine epidemic diarrhea virus (PEDV). The purified N protein was used as the coating antigen, and the ELISA reaction conditions were optimized by the chess rboard titration. A blocking ELISA method for detecting PEDV antibodies was established, and its specificity, sensitivity and repeatability tests were carried out. One hundred and forty clinical serum samples were tested, and the results were compared with commercially IDvet PEDV indirect ELISA antibodies detection kit. The results showed that the best antigen coating concentration was 625 ng·mL^-1, and the best dilution ratio of serum was 1:1; The best dilution of the HRP-conjugated antibody working solution was 1:5 000; There was no cross-reaction with healthy pig serum and the positive sera of common pig disease pathogens, such as classical swine fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2), and transmissible gastroenteritis virus (TGEV). The sensitivity of PEDV positive serum was 1:16, which was equivalent to that of IDvet ELISA kit (titer 1∶32). The coefficient of variation of within-run and between-run repeatability test is less than 10%, so it showed that the blocking ELISA established in this study had good repeatability and stability; the kappa value of detected 140 clinical porcine serum using this method was 0.87 when compared with IDvet ELISA. The above results indicated that the established blocking ELISA method for detecting PEDV antibodies in this study could be applied to the prevention and control of PEDV, epidemiological investigation and the monitoring of antibody levels after vaccine immunization.

The antigenicity of the p72 protein of African swine fever virus (ASFV) was compared with that of a truncated p72 protein (p72s) to provide experimental basis for ASFV specific antibody detection. The p72 and p72s proteins were expressed by Escherichia coli expression system, and the expressed protein was identified by Western blotting and Liquid chromatography mass spectrometer (LC-MS/MS), and the epitopes of the protein were analyzed. Mice were immunized with the purified intact p72 protein or p72s protein, respectively, and the immunogenicity of p72 and p72s proteins was compared. The immunoreactivity of the p72 and p72s proteins with ASFV-positive serum was evaluated by ELISA and Western blotting.Both recombinant p72 and p72s proteins were expressed as inclusion bodies with molecular weights of 76 and 42 kDa, respectively, which were consistent with the expected molecular weight. The coverage of predicted amino acid sequences of p72 and p72s by the hydrolysate peptide of the expression proteins was 89.3% and 88.39%, respectively, which included 27 and 17 epitopes. After mice were immunized with p72 or p72s for three times, the serum specific antibody titers were in the range of 1∶200 to 1∶25 600 and 1∶12 800 to 1∶409 600, respectively. Among 108 swine sera, 78 (72.2%) and 85 (78.7%) positive sera were detected by ELISA based on p72 and p72s proteins with a good consistency (kappa=0.826, P=0.016). Seven of the 10 ASFV positive sera detected by Western blotting reacted with p72 protein, and all of them reacted with the p72s protein. The truncated p72 protein has better antigenicity than the intact p72 protein, and it is more suitable for ASFV antibody detection.

In order to study the effect of Brucella bovis type IV secretion system (T4SS) on the endoplasmic reticulum stress and apoptosis of macrophages, the molecular pathogenic mechanism of Brucella was deeply explored. In this study, bovine Brucella vaccine strain A19 and its T4SS promoter deletion strain A19ΔVirB were used to infect mouse macrophages. RNA and total protein were collected at 6, 12 and 24 hours after infection, transcription level and protein expression level of endoplasmic reticulum stress marker molecules GRP78 and CHOP, apoptosis related molecules BAX and BCL-2 were detected by qRT-PCR and Western blot, apoptosis at different time points was detected by flow cytometry. The results showed that at 6, 12 and 24 h after infection, the transcription level and protein expression level of GRP78 in deletion strain A19ΔVirB group were significantly lower than those in parent A19 group (P<0.05); at 6 h after infection, the transcription level and protein expression level of CHOP in the deletion strain group were also significantly lower than those in the parent strain (P<0.05), but at 24 h, it was significantly higher than that of the parent strain (P<0.05); at 24 h after infection,transcription level and protein expression level of BAX in deletion strain A19ΔVirB group were significantly higher than those in parental strains (P<0.05); at 24 h after infection,transcription level and protein expression level of BCL-2 in deletion strain A19ΔVirB group were significantly lower than those in parental strain (P<0.05); the results of flow cytometry further showed that the apoptosis rate of the deletion strain group was significantly higher than that of the parent strain (P<0.05). The results showed that deletion of T4SS would weaken the ability of Brucella bovis to induce endoplasmic reticulum stress and enhance its ability to induce apoptosis.This study laid a foundation for further understanding the pathogenic mechanism of Brucella, and also provided a scientific basis for the functional research of Brucella T4SS in the future.

This experiment was conducted to establish a TaqMan multifluorescent quantitative PCR method for simultaneous detection of Escherichia coli O157∶H7, Salmonella and Listeria monocytogenes. Specific primers and TaqMan probes were designed according to the conservative sequences of E. coli O157∶H7 rfbE gene, Salmonella invA gene and L. monocytogenes hlyA gene, respectively, and a multiplex qPCR reaction system was established to detect their specificity, sensitivity and repeatability. The established method was used to detect pathogenic bacteria in fresh food and compared with the national standard method. The multiplex qPCR method established in the experiment has good specificity, only three kinds of target bacteria can be amplified, the lowest sensitivity detection value is 10⁴ copies·μL^-1, and the repeatability is good, the intra-assay coefficient of variation is 0.09%-2.97%, and the inter-assay coefficient of variation is 0.23%-7.82%. One hundred and twenty fresh meat samples were detected by the multiplex qPCR method and compared with the national standard method, 2 samples of E. coli O157∶H7 and 21 samples of L. monocytogenes were detected by the two methods, and 14 samples of Salmonella were detected by the multiple qPCR method, one more than the national standard method. All contaminated samples were detected by the established method. The results confirmed that the established multiple qPCR detection method can quickly and accurately detect the three foodborne pathogens, E. coli O157∶H7, Salmonella and L. monocytogenes, and provide technical support for food safety.

The present study was designed to evaluate the effect of dietary supplementation with Enterococcus faecium on growth performance, survival rate, caecal lesion, fecal oocyst excretion and intestinal barrier in broiler chickens challenged with E. tenella infection. Total of 180 one-day-old broiler chickens (Ross 308) with similar body weight were randomly distributed into 5 groups, including control group (NC), positive group (PC), salinomycin group (SAL), E. faecium group (EA and EB). Chickens in NC and PC groups were fed with basal diet, while SAL, EA, EB groups were fed with the diet supplemented with 70 mg·kg^-1 salinomycin, 1.0×10⁸ CFU·kg^-1 E.faecium and 2.5×10⁹ CFU·kg^-1 E.faecium, respectively, until the end of the experiment. At 15 days of age, the chickens in all groups except NC were orally infected with 1.0×10⁵ sporulated E. tenella oocysts. Then, the anticoccidial efficacy of dietary supplementation with E. faecium was evaluated by the average daily gain (ADG), oocyst per gram faeces (OPG), caecal lesion score, histopathological examinations and gene expression levels of cecal barrier related molecules (iNOS, MUC2, Occludin, JAM2, ZO-1). The results showed that: 1) Compared with NC group, ADG was significantly increased in EA and EB groups before treated with E. tenella (P<0.05). The ADG of SAL and EB groups were higher than that in PC group after infected with E.tenella (P<0.05). 2) Compared with PC group, the OPG in all pre-treated groups (SAL, EA, and EB) were significantly reduced (P<0.05). Besides, the obvious pathological damage with immune cell infiltration and villi blunting was observed in the cecum in PC group compared with pre-treated groups, while no significant difference was found in all pre-treated groups regard to the lesion score (P>0.05). 3) During the whole test period, the survival rates of NC, PC, SAL, EA and EB groups were 100%, 58.33%, 83.33%, 58.33% and 75.00%, respectively. 4) Compared with NC group, the expression of iNOS, MUC2, Occludin, JAM2, ZO-1 in PC group was significantly decreased (P<0.05). The expression of iNOS in all pre-treated groups and MUC2, Occludin, JAM2 in EB group showed a remarkable increase compared with PC group (P<0.05). In conclusion, the results of this study are promising and indicate many beneficial effects of E. faecium-supplementation in poultry diets to reduce the negative consequences of E. tenella disease by regulating gene expression of intestinal barrier related molecules.

To develop DNA vaccine against Blomia tropicalis allergy, we constructed eukaryotic expression vector pVAX1-PADRE-Blo t 5-21-CpG of major allergen fusion gene of Blomia tropicalis by codon optimization of Blo t 5 and Blo t 21 gene sequences. To verify the immune effect of the eukaryotic expression vector, BALB/c mice were immunized with 100 μg/100 μL of pVAX1-PADRE-Blo t 5-21-CpG by subcutaneous injection (n=6)，and the serum levels of specific IgG, IgG1 and IgG2a were detected by ELISA. The changes of regulatory T cells in spleen lymphocytes of mice were detected by flow cytometry. The results showed that the eukaryotic expression plasmid pVAX1-PADRE- Blo t 5-21-CpG could induce the increase of specific IgG and IgG2a levels in mice, and the increase of IgG2a/IgG1 ratio suggested a bias towards a Th1 type immune response in mice after immunization. In addition, the plasmid pVAX1-PADRE-Blo t 5-21-CpG can induce higher levels of CD4⁺CD25⁺Foxp3⁺ T cells in mice, suggesting that the eukaryotic expression plasmid may induce immunosuppression.

The study aimed at investigating the effect and mechanism of exogenous neuropeptide cocaine-and amphetamine-regulated transcription factor (CART) against oxidative stress in hippocampal neurons. The primary culture of hippocampal neurons was carried out in rats within 24 hours of birth. Following the purity test, 200 μmol·mL^-1 hydrogen peroxide were used to induce oxidative stress. On this basis, the effects of CART on the neuronal viability, ATP content, mitochondrial membrane potential (MMP)and the expression levels of apoptosis-related genes and trkB gene were investigated. Also the correlation between trkB gene and apoptosis-related genes was analyzed. The results showed that compared with the control group, the neuron viability and ATP content in H₂O₂ group were extremely significantly decreased(P<0.01), and the MMP was significantly decreased (P<0.05); CART with different concentrations significantly increased the neuronal viability, ATP content and MMP(P<0.05). Compared with the control group, the mRNA levels of bax and caspase-3 were extremely significantly increased in H₂O₂ group(P<0.01), and the mRNA levels of bcl-2 were extremely significantly decreased (P<0.01) and trkB were significantly decreased (P<0.05) in H₂O₂ group. Compaced with the H₂O₂ group, the mRNA levels of bcl-2 (P<0.01) were extremely significantly increased and trkB (P<0.05) were significantly increased in CART + H₂O₂ group. While the mRNA levels of bax were significantly decreased (P<0.05) and the mRNA levels of caspase-3 was extremely significantly decreased (P<0.01) in CART+H₂O₂ group compared with H₂O₂ group. The results of Western blot showed that the protein levels of bax and caspase-3 in H₂O₂ group were significantly higher than those in control group (P<0.05), and the protein levels of bcl-2 and trkB were significantly lower than those in control group (P<0.05). The protein levels of bcl-2 and trkB were significantly increased in CART+H₂O₂ group (P<0.05), while the protein levels of bax and caspase-3 were significantly decreased (P<0.05) compared with H₂O₂ group. The correlation analysis showed that, trkB gene was negatively correlated with the expression level of bax and caspase-3 genes, while positively correlated with the expression level of bcl-2 gene. The results indicate that CART can resist oxidative stress in hippocampal neurons by inhibiting apoptotic signaling pathway while increasing the expression level of trkB gene.

The objective of this study is to investigate the correlation between the expression pattern of genes in swainsonine biosynthesis gene cluster (SWN) and swainsonine production in Alternaria oxytropis, fungal endophyte from locoweed. A. oxytropis UA003 was treated with Ethyl methanesulfonate (EMS). The SW content of each mutated strain was determined to screen strains with significant changes in SW yield. The expression levels of AATL, swnR, swnN, swnH1, swnH2, swnK (A, KS, SDRe1, SDR) in SWN gene cluster of some strains including A. oxytropis UA003, EMS mutagenesis strains and A. gansuense were analyzed. The swnK gene sequences of the above-mentioned strains were further analyzed to mutation sites. The results showed as follows: the yield of SW in E02, E23 and E25 strains induced by EMS was significantly increased, and the gene expressions of AATL, swnR, swnN, swnH1, swnH2 were significantly down-regulated (P<0.05 or P<0.01). The gene expressions of A, KS, SDR and SDRe1 were significantly up-regulated (P<0.05 or P<0.01 in all strains). E02 strain mutated by EMS had 13 nucleotide mutation sites of swnK gene and 6 amino mutation acid sites, respectively. Compared with UA003, EA strain had 163 different nucleotide sites and 67 different amino acid sites. The amino acid sequence MLTPAVSLKNLTKPK deletion was absent in the initial segment of the transcription product, and an amino acid sequence of TEVDGVP was inserted in the position 1135 and 1143 of the AT gene transcription product. In conclusion, EMS mutagenesis could significantly change the biosynthesis of swainsonine and gene expression of SWN gene cluster in A. oxytropis. The expression patterns of AATL, swnR, swnN, swnH1 and swnH2 genes in the SWN gene cluster were negatively correlated with the biosynthesis of swainsonine in E02, E23 and E25 strains. However, the expression patterns of A, KS, SDR and SDRe1 were positively correlated with the biosynthesis of swainsonine in above-mentioned strains. The biosynthesis changes of SW in endophytic fungi may be related to the structural and functional changes of related genes in SWN gene cluster.

The aim of this study was to investigate the expression and clinical significance of NLRP3 inflammasome and downstream inflammatory genes in canine mammary tumors(CMTs). The mRNA expression levels of NLRP3, Caspase-1, IL-1 and IL-18 in 45 CMTs (15 benign tumors and 30 malignant tumors) and 16 tumor adjacent normal mammary gland tissues were detected by real-time quantitative polymer chain reaction. The relationship between NLRP3 and clinicopathological features (tumor size, tumor subtype, histological malignant grade and prognosis) was also analyzed. The results showed that the mRNA expression of NLRP3 and downstream inflammatory factors were up-regulated in tumor tissues compared with normal mammary tissues, and the relative mRNA expression of NLRP3 (P=0.006) and Caspase-1 (P=0.048) in MCMTs was significantly higher than that in BCMTs. Besides，the expression of NLRP3 mRNA in MCMTs was statistically different in tumor type (P=0.029) and malignant grade (P=0.049). This study suggests that up-regulation of NLRP3 expression may promote canine mammary tumorigenesis, and could be a diagnostic predictor for CMTs and its malignant features.

The aim of this study was to optimize the classification of canine endometritis in order to improve its clinical diagnostic rate and provide theoretical basis for further exploration of the standard classification of canine endometritis. Forty bitches were hystero-oophorectomy, and canine endometritis was classified more deeply by observing the presence or absence of clinical symptoms, histological dilatation of endometrial structures, endometrial to Myo thickness (Endo/Myo) ratio, stromal components, and inflammatory cell infiltration of dogs. The results showed that endometritis could be roughly divided into two groups in histopathology, the group without clinical symptoms and the group with clinical symptoms. The group with clinical symptoms could be further divided into four classifications: no inflammation, mild inflammatory infiltration, severe inflammatory infiltration (hyperplasia) and severe inflammatory infiltration (atrophy), and the group without clinical symptoms included healthy and cystic endometrial hyperplasia (CEH). This study provides a more objective method for determining the standard classification of canine endometritis.

This experiment aims to investigate the mechanism of zearalenone (ZEA) on autophagy block of PC12 cells. Taking PC12 cells (rat renal pheochromocytoma) for research materials, treated with different concentration of ZEA for 24 h, Cell Counting Kit-8 (CCK-8) was used to detect the cell survival rate. The optimal concentration of ZEA was screened. PC12 cells were divided into control group, 15 μmol·L^-1 ZEA group and 30 μmol·L^-1 ZEA group. The two ZEA groups were treated with 15, 30 μmol·L^-1 ZEA for 24 h, respectively, and the control group was administered with the same amount of 1640 medium. Western blot was used to detect the expression levels of autophagy associated proteins Atg5, LC3Ⅱ, p62, Beclin1, lysosomal associated membrane proteins LAMP1, cathepsin B(CTSB) and cathepsin D (CTSD), TFEB proteins in cytoplasm and nucleus, autophagosomal and lysosomal fusion associated proteins STX-17 and SNAP29. The immunofluorescence assay was used to detect LC3 fluorescence dots and TFEB nuclear translocation. The mRFP-GFP-LC3 was used to detect autophagy flow. AO staining was used to observe the changes of intracellular acidic environment. The results showed that compared with the control group, the expression levels of Atg5, LC3Ⅱ, p62 and Beclin1 in all ZEA treatment groups were significantly increased (P<0.05). The immunofluorescence showed that LC3 fluorescence dots increased gradually. The mRFP-GFP-LC3 assay showed autophagy flow was blocked. The expression levels of cytoplasmic protein CTSD and CTSB in ZEA-treated group were significantly increased (P<0.0), while the expression level of LAMP1 decreased significantly when ZEA was 30 μmol·L^-1(P<0.05). The result of AO staining showed that ZEA could damage the lysosome of PC12 cells. The expression level of p-ERK and TFEB protein in nucleus was significantly decreased (P<0.05). However, there was no significant change in the expression levels of TFEB protein in cytoplasm. The expression level of SNAP29 and STX-17 protein decreased significantly (P<0.05).In conclusion, ZEA can increase the autophagy level and increase the number of autophagosomes in PC12 cells, accompanied by autophagy flow block. The mechanism was related to ZEA lysosomal damage, inhibition of TFEB entry into the nucleus, and reduction of autophagosomal-lysosomal fusion related protein expression.

This study aims at studying the effects of the total flavonoids of Drynaria Rhizoma (TFDR) on osteogenic differentiation potential of canine bone marrow mesenchymal stem cells (BMSCs) in hypoxic. TFDR was used to intervene canine BMSCs in a low oxygen concentration (10%) environment for 4 weeks to induce osteogenic differentiation. Observation of calcium nodule formation in BMSCs stained with Alizarin Red by an inverted microscope. The alkaline phosphatase (ALP) activity level was detected by colorimetric method. The cell mitochondrial membrane potential was detected by flow cytometry. The expression of Runx2 and Osterix, the key genes for osteogenic differentiation were detected by RT-PCR and confocal laser microscope. Results were as follows: Compared with the control group, 10% oxygen concentration inhibited the formation of calcium nodules and ALP activity in canine BMSCs (P<0.01), Mitochondrial membrane potential level dropped (P<0.05), and the expression of Runx2 and Osterix decreased (P<0.05 or P<0.01). Compared with the hypoxic group, TFDR can significantly promoted the formation of calcium nodules and ALP activity of BMSCs in Hypoxic (P<0.05), the level of cell mitochondrial membrane potential increased (P<0.05), and Runx2 and Osterix expression increased (P<0.05 or P<0.01). TFDR can promote the osteogenic differentiation of canine BMSCs in hypoxic.

The goals of this study were to study the anesthetic effect of xylazine combined with midazolam on goats, and to detect the effects of combined anesthesia on central neurotransmitters and NO/cGMP signal transduction system, so as to provide a new method for goat anesthesia and to clarify the mechanism of general anesthesia of combined anesthesia. Goats received 1.31 mL·kg^―1 combined anesthetics with intramuscular injection, and the control group received normal saline. The behavioral changes of the animals were observed, and the physiological indexes and anesthesia effects were monitored. The contents of neurotransmitters and NO/cGMP pathway in the brain and hippocampus were detected by HPLC during induction, anesthesia, recovery I and recovery Ⅱ, respectively. Results were as follows: 1) xylazine-midazolam compound could induce anesthesia rapidly in goats, and the induction time was (5.63±2.35) min.Anesthesia maintenance time (56.72±5.38) min. The compound exhibited well sedation, analgesia and muscle relaxation effects, and has little effect on basic physiological parameters of goats; 2) During anesthesia, compound drugs inhibit the expression of norepinephrine (NE), dopamine (DA), aspartic acid (Asp) and glutamic acid (Glu) both in the brain and hippocampus, and increase the contents of serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA), glycine (Gly) in hippocampus and γ -aminobutyric acid (GABA) in the brain; 3) During the anesthetic period, the compound inhibited the expression of NO, NOS and cGMP in the brain and hippocampus. We can conclude that the xylazine-midazolam compound has a good anesthetic effect on goats, and exerted the central anesthesia mechanism by affecting the NO/cGMP signal transduction system and the expression of various neurotransmitters in the brain and hippocampus.

This study aimed to understand the molecular characteristics of infectious laryngotracheitis virus (ILTV) in China. TK, ICP4, gB, UL32 gene sequences of ILTV BT strain isolated from domestic farms was determined, and its pathogenicity to 2-week-old SPF chickens was studied. Phylogenetic analysis results showed that the nucleotide sequence of BT strain has a consistency with other epidemic strains in China was more than 97%, which were consisted within one branch and there was no significant variation. However, it is quite different from American virulent strain 1874C5 and domestic standard strain WG, which mainly focuses on ICP4 gene. There is a deletion of 4 amino acids at position 87-90 of (AAQD), which is also found in other domestic isolates. The deletion of (QPQEPQ), with 6 amino acids at position 862-868 was consistent with attenuated vaccine strains such as K317, Serva, Laeyngo and LTBlen. When the strain was inoculated back into the larynx and trachea, no chicken death was observed, only a few chickens showed conjunctival secretion. Autopsy showed that the larynx trachea was normal. It is suggested that domestic ILTV epidemic strains are prevalent in chicken flocks in a mild form and show unique genetic characteristics.

In order to investigate the prevalence and genetic variation of feline parvovirus (FPV) in domestic cats in Chengdu region. From 2017 to 2021, 168 diarrheal fecal samples were collected from domestic cats in Chengdu region of Sichuan province. FPV was detected in 168 samples, and 13 positive samples’nucleotide sequence of VP2 genes were obtained. The homology and variation of FPV nucleotide and amino acid (aa) sequences was analyzed by MegAlign software, and the phylogenetic tree was constructed by neighbor-joining method through MEGA 7.0. Of the 168 samples examined, 70.2% (118/168, 95% CI=62.7%-77.0%) samples were positive for FPV. Sequence homology analysis showed that the 13 full-length VP2 genes shared 97.1% to 100% homology to reference strains. The aa sequence of SMU-D18 and SMU-D14 strains were consistent with new CPV-2a from cats. Interestingly, the SMU-D46 strain showed a unique substitution from Ser (S) to Phe (F) at both 293aa and 297aa. Furthermore, phylogenetic analysis showed that the FPV strains from China and other countries clustered into three subgroups (G1, G2 and G3). The FPV isolates obtained in this study were located in G1 and G3, respectively. It was worth noting that the commercial FPV vaccine strain was located in G2, which had distantly related to the FPV strains circulating in China. The results demonstrated a very high infectious rate of FPV among cats in Chengdu region. The protective efficiency of the existing commercial vaccines should be re-evaluated as soon as possible.

Nebovirus (NeV) is an emerge diarrhea-causing virus in calves, the aim of this study is to establish an insulated isothermal RT-PCR(iiRT-PCR) for detecting NeV on field. Based on the RdRp sequences of NeV in GenBank database, a pair of primers and a fluorescent TaqMan probe were designed and synthesized. After optimizing the react system and condition, the iiRT-PCR method for detection of NeV was established. The iiRT-PCR assay could amplify specific fragment of NeV, without amplification of irrelevant pathogens, including bovine coronavirus, bovine norovirus, bovine rotavirus, bovine viral diarrhea virus, bovine torovirus, bovine Cryptosporidium parvum, bovine Eimeria. The intra- and inter-coefficients of variation were 3.07%-3.12% and 2.45%-3.01%, respectively, and the detection limit of viral nucleic acid of the assay was 5.38 copies·μL^-1. One hundred and one calf diarrhea samples, collected from Hongyuan County, Ruoergai Prefecture, Xichang City, Liangshan Yi Autonomous Prefecture and Langzhong City in Sichuan Province during 2020-2021, were used to detect NeV, and 64.36% samples were detected as NeV positive. The study established an iiRT-PCR method for NeV detection with good specificity and reproducible as well as high sensitivity. Moreover, combined with the premixed detection reagent and PetNAD nucleic acid extraction kit, this assay could be used to NeV detect on-site, and only 1 hour from nucleic acid extraction to result report,which contribute to the fast detection for NeV.

Compare the effect of surgical treatment and conservative treatment of cat idiopathic chylothorax under thoracoscopy. Cats with idiopathic chylothorax are treated in two ways. Conservative treatment is to place a thoracic catheter for daily suction of pleural effusion, while taking rutin drugs and adopting a low-fat diet. Surgical treatment adopts thoracic duct ligation and pericardectomy under thoracoscopy. The recovery degree of the cat can be judged by checking the physical condition of the cat, blood examination, cytology examination, and imaging examination. Seven days after conservative treatment, the body weight of the cat was still decreasing, the production of chylo fluid did not decrease, and the cytological examination revealed that the neutrophil nucleus shifted to the left, indicating that the cat’s pleural effusion condition has not been significantly improved. The fluid was significantly reduced, the weight gradually recovered, and the postoperative recovery was good without infection. Conservative medical treatment is not satisfactory in the efficacy of this case, while thoracoscopic thoracic duct ligation and pericardectomy are safe and effective procedures for the treatment of cat idiopathic chylothorax, which can effectively reduce pain, speed up recovery, and reduce Postoperative complications, etc.