Scavenger receptor superfamily is a structurally related transmembrane glycoprotein family. Its characteristic component is a highly conserved scavenger receptor cysteine-rich (SRCR) domain. CD163, which is a type B scavenger receptor and has 9 SRCR domains, is highly expressed in monocyte and macrophages. It was reported that CD163 is an essential receptor of porcine reproductive and respiratory syndrome virus (PRRSV), involved in the uncoating process of PRRSV and possibly participated in the attachment and internalization of African swine fever virus (ASFV). In addition, CD163 plays an irreplaceable role in many important biological processes. This review introduces the basic information, physiological functions and research progress of CD163 in pig disease-resistant breeding on PRRSV, providing reference for disease-resistant animal breeding.

Sow reproductive disorder is one of the important factors that restrict the development of Chinese pig industry. It can cause abnormal estrus, repeated infertility, abortion stillbirth, mummified fetus and weak fetus, which leads to the prolongation of sow breeding cycle, the decrease of live (healthy) litter size and the shortening of service life, and seriously affects the economic benefits. Sow reproductive disorder syndrome (SRDS) is affected by many factors such as genetics, functional disorders, environment, nutrition and disease. This paper has reviewed recent studies on the causes, pathogenesis and prevention measures of SRDS. In order to provide new ideas and reference for prevention and control of sow reproductive disorder.

Chicken is one of important agricultural animals and the eggs they supplied are a high quality and economical source of animal protein. In the layer industry,the early sex identification is indispensable procedure due to the preference for female chicks. Large numbers of male chicks were culled after sex identification,which not only causes a huge loss but also has ethical issues. In recent years,the development of science and technology has made it possible to artificially control the sex of livestock and poultry. The research on sex regulation mechanisms has excavated many important candidate genes,among which the DMRT1 gene is considered to be a key gene in the regulation of gonad formation in roosters. This paper reviewed the role of the DMRT1 gene in chicken sex determination and gonadal differentiation as well as the related pathways and the potential factors affecting its expression. This review provides a reference for in-depth studies on the mechanism of sex determination and development of feasible artificial sex control approaches.

Nanobody is a kind of single-domain antibody derived from Camelidae and sharks. Compared with the traditional antibody, nanobody has the characteristics of small molecular weight, stable structure, easiness of modification and expression, low immunogenicity, high antigen affinity, strong tissue penetration and so on. Its unique molecular characteristics make it promising for application for diagnosis and treatment of various diseases and assays. This review summarized the structure, characteristics, library types, screening and expression strategies of nanobody and research status in the prevention, diagnosis and treatment of animal disease, as well as the detection of foodborne pathogens and drug residues in animal products. The limitations and future development of nanobody are also analyzed.

During the perinatal period, decreased dry matter intake, increased nutritional requirements of the rapid growth fetus in late gestation and the synthesis and secretion of milk in early lactation result in energy metabolism disorder of dairy animals. Energy metabolism disorders can cause a variety of metabolic and inflammatory diseases, which have a significant negative impact on the performance and reproduction of dairy animals. Rumen protected glucose (RPG) is less affected by the rumen microbes during digestion and can be efficiently absorbed within the small intestine, thereby providing a safe and efficient energy source to alleviate energy metabolism disorder of dairy animals during the perinatal period. This paper summarizes the physiological metabolism characteristics during perinatal period, reviews the effects and mechanisms of RPG on energy metabolism, milk yield, milk composition, oxidative stress, immunity and inflammation of perinatal dairy animals, and briefly summarizes the bioavailability of different type of RPG, in order to provide reference for the research and application of RPG in perinatal dairy animals.

Parasitic diseases cause significant economic losses in breeding industry worldwide, and some parasitic diseases pose major public health threats. Thus, it is crucial to make a timely and accurate diagnosis of parasitic diseases for disease prevention and control. Polymerase chain reaction (PCR) technology can effectively improve the accuracy of diagnosis for its advantages of high sensitivity, specificity and convenience, so this technology can provide reliable technical support for the prevention and control of parasitic diseases. In this review, the categories of PCR techniques and their target genes used in the diagnosis of parasitic diseases in the past 10 years (2012-2022) were summerized, in the hope of providing a reference for the diagnosis of parasitic diseases.

Monkeypox virus is a member of orthopxvirus genus in Poxviridae family. Its genome is double-stranded DNA, and it is an important zoonotic pathogen to humans and many other mammals. In May 2022, a large outbreak of monkeypox appeared in non-endemic areas, causing widespread public concern. This paper reviews the current methods of diagnosis, prevention and treatment of monkeypox virus, and summarizes the research and development progress of monkeypox virus vaccine and drugs, in order to provide reference for monkeypox virus diagnosis, disease prevention and control.

Mycoplasma bovis (M.bovis) is an important pathogen that infects cattle, which can lead to a variety of clinical symptoms such as pneumonia, arthritis, mastitis, keratoconjunctivitis and genital tractitis in infected cattle, causing huge economic losses to the cattle industry. M.bovis adhesins play an important role in pathogenesis of M.bovis, including pathogenic infection, cell invasion, immune evasion, and virulence production. So far, more than ten kinds of M.bovis adhesins have been identified. These adhesins primarily bind plasminogen (Plg), fibronectin (FN), heparin sulfate (HS), and amyloid precursor-like protein-2 (APLP2) of host cells. This article will review the current research status of M.bovis adhesins and their host cell target proteins, and provide reference for the identification and application of known and unknown adhesins of M.bovis.

Salmonella is the most common zoonosis pathogen in Enterobacteriaceae, and Salmonella food poisoning is one of the highest proportions and the most harmful bacterial food poisoning, which has important public health significance. The classification of Salmonella is relatively complex with 2 species (Salmonella enterica is divided into 6 subspecies) according to the biological taxonomy, 46 serogroups and more than 2600 serovars according to the difference of surface antigens (some serovars can be further divided into different biovars). The above classification or typing is of great significance for epidemiological and aetiological research. Traditional detection and serotyping have many defects, such as time-consuming, labor-intensive and low sensitivity, which cannot control the epidemic timely and accurately. With the continuous improvement of Salmonella genomic data, more and more nucleic acid targets have been discovered. The molecular detection technology represented by PCR can not only detect Salmonella, but also identify different serovars, with strong specificity, high sensitivity, simple and fast. The specificity of detection target is the key factor for the accuracy of results. Since 1992, when it was first reported that PCR detection method for Salmonella was established with invA as the target, the reports on molecular detection technology of Salmonella are increased, and it has been gradually applied to the rapid detection and typing of Salmonella. This paper introduces the research progress on molecular detection of Salmonella in detail, and summarizes the application from different levels of Salmonella spp., species, serogroup and serovar, in order to provide reference for the promotion and application of molecular detection of Salmonella.

Na⁺/H⁺ exchanger transporters (NHEs)play important roles in various biological processes, including Na⁺ uptake, intracellular pH homeostasis, cell volume regulation, proliferation, and apoptosis. Among them, NHE3 (Na⁺/H⁺ exchanger isoform 3)is highly expressed in the intestine and proximal tubules of the kidney to play a role in neutral NaCl absorption. When pathogens such as bacteria, viruses and toxins infect the intestine, they cause inhibition of the corresponding NHE3 activity on the intestinal epithelium and impairment of Na⁺ transport, which leads to water and electrolyte retention in the intestine, stagnation of nutrient absorption and the development of diarrhea. In recent years, significant progress has been made in the study of the altered NHE3 activity of the gastrointestinal epithelium caused by viral, bacterial, and other microbial infections. This paper reviews the changes of NHE3 activity in diarrhea caused by some pathogenic microbial infections and the mechanisms of NHE3 activity regulation, including the apical to intracellular body circulation of NHE3 from the epithelial membrane, transcriptional and translational regulation, dynamic interactions between proteins, and the regulatory mechanisms of related signaling pathways.

This study aimed to explore the effect of NR2F2 gene on the proliferation and apoptosis of porcine PK15 cells. In this study, the effect of overexpression of NR2F2 gene on cell proliferation was detected by qRT-PCR, Western blot, CCK-8, EdU, flow cytometry, scratch assay and other methods in porcine PK15 cells. CO-IP technology was used to verify the binding ability of NR2F2 to Smad4 protein. The effect of NR2F2 gene on Smad4 gene expression and the expression of downstream gene Cyclin B, CDK1, CDK4, Cyclin D1 were detected by qRT-PCR. The expression of downstream genes and proliferating genes Ki67, PCNA were detected by rescue experiment overexpressed NR2F2 gene and interfered with Smad4 gene. The results showed that overexpression of NR2F2 gene significantly upregulated the expression of Ki67(P<0.01), and significantly upregulated the expression of PCNA(P<0.05), promoted cell cycle progression, inhibited cell apoptosis, and promoted cell proliferation. The CO-IP results proved that NR2F2 and Smad4 proteins were physically bound. After overexpression of NR2F2 gene, the expression of Smad4 gene increased significantly (P<0.01), and the expression of downstream regulated genes by Smad4 gene Cyclin B, CDK1, CDK4 and Cyclin D1 significantly increased (P<0.01). After interfering with NR2F2 gene, the expression of Smad4 gene significantly decreased (P<0.01), and the expression of downstream genes regulated by Smad4 gene was significantly decreased (P<0.01). The rescue experiments showed that after overexpression of NR2F2 gene, interfering with the expression of Smad4 gene could significantly reduce the expression of Cyclin D1, Cyclin B, CDK1, Ki67 and PCNA (P<0.01,P<0.05). In summary, overexpression of NR2F2 gene promoted cell proliferation and inhibited apoptosis, and NR2F2 protein could bind to Smad4 protein and affect the expression of Smad4, which in turn affected the expression of downstream genes.

The purpose of this study was to establish a method to rapidly mine a few SNPs markers that can distinguish the target breeds or strains from other breeds or strains. The goal is to develop a technique for quickly mining the characteristic SNP markers set of chicken breeds or strains. The study used whole genome resequencing data of 19 breeds or strains, including Beijing yellow chicken, Jingxing yellow chicken, 7 Shandong local breeds, 5 Yunnan local breeds, Tibetan chicken, and the fast-growing white feather broiler terminal. After fixation indices analysis,SNP markers with MEAN_FAST ≥ 0.65 were extracted. The SNP markers set was further reduced by extracting independent SNP through linkage disequilibrium analysis. Six breeds or strains were chosen randomly to test this method. The results showed that, after loci screening by once fixation indices analysis and linkage disequilibrium analysis, the fast growing white feather broiler, Jingxing yellow chicken selective line H and Jingxing yellow chicken selective line D2 could be separated from other 18 representative breeds by 114, 220 and 226 SNPs, respectively; Wuding chicken and Piao chicken, which were clustered in one branch by principal component analysis and Neighbour Joining tree, could be separated from other representative breeds by 204 and 178 SNPs, respectively. Obtained the set of 114-226 SNPs markers, and that the target breeds or strains could be distinguished from other representative varieties by principal component analysis. The procedure is straightforward, and the quantity of SNP markers obtained is quite low, which helps for manage detection costs. It can be applied to construct the "molecular identity cards" of local variaties or commercial strairns to assist the protection and identification of germplasm resources.

The aim of this study was to reveal the action mechanism of miR-150 during the differentiation of preadipocytes in Guangling Large-Tailed sheep by investigating the relationship between miR-150 and target gene AOC3. In this study, three 5-day-old Guangling Large-Tailed male sheep with normal development and health were used as the research material. The expression level of miR-150 in subcutaneous, perirenal and tail adipose tissues of Guangling Large-Tailed sheep was detected by qPCR. Tail adipose tissue of Guangling large-tailed sheep was collected to isolate ovine preadipocytes. Cell purity was identified by cell immunofluorescence. Bioinformatics software was used to predict the target relationship between miR-150 and AOC3, and double luciferase reporter system was used to verify this relationship. miR-150 mimics and inhibitors were transfected into preadipocyte and the expression of AOC3 and adipogenic marker genes were detected. The overexpression and interference vector of ovine AOC3 were constructed and transfected into ovine preadipocytes. qPCR and Western blotting were used to detect the expression patterns of miR-150 and AOC3 during the differentiation of preadipocytes in Guangling Large-Tailed sheep. Lipid accumulation ability was detected by Oil Red O staining. Three biological replicates were set up for each of the above tests. The results showed that the expression of miR-150 was the highest in tail fat, and the difference was significant (P<0.001). After overexpression of miR-150, the expression of AOC3 and adipogenic marker genes were significantly down-regulated (P<0.05), lipid droplet accumulation decreased. These results implied that miR-150 inhibits the differentiation of ovine preadipocytes. The results were opposite after inhibition of miR-150. There was a binding site between miR-150 and the 3'-UTR of AOC3, and miR-150 was extremely significant decreased the relative fluorescence activity of wild type AOC3 (P<0.001). After overexpressing AOC3, the expression of adipogenic marker genes were significantly up-regulated (P<0.01) and more lipid droplets were produced, indicating that AOC3 promotes the differentiation of ovine preadipocytes. After interfering with AOC3, the result was reversed. In conclusion, there is a binding site between miR-150 and the 3'-UTR of AOC3. The expression level of miR-150 was negatively correlated with that of AOC3 during the differentiation of ovine preadipocyte. miR-150 inhibits the differentiation of ovine preadipocytes by targeting AOC3. The results provide a theoretical basis for the research of microRNAs (miRNAs) at the molecular level.

The purpose of this study was to clarify the tissue and cell expression pattern of uncoupling protein 3 (UCP3) gene in Sahu sheep, and to clarify the effect of overexpression of UCP3 on revealing the differentiation of preadipocytes in Sahu sheep. This study used real-time quantitative PCR (qPCR) technology to detect the expression distribution of UCP3 gene in the heart, liver, spleen and other tissues of 3 Sahu sheep, as well as its expression levels at different stages of adipogenic differentiation; After overexpression of UCP3, qPCR was used to verify the overexpression efficiency, using Oil Red O staining, lipid extraction analysis, and adipocyte differentiation marker genes testing, clarify the effects of overexpression of UCP3 on lipid production in Sahu sheep preadipocytes from both cellular morphology and biological perspectives.The results showed that the UCP3 gene was widely expressed in various tissues of Sahu sheep, with relatively higher expression levels in the heart and spleen; The temporal expression profile showed that the expression level of UCP3 gene showed an upward trend during the differentiation of preadipocytes in Sahu sheep (P<0.05); After overexpression of UCP3, the number of lipid droplets in the preadipocytes of Sahu sheep increased significantly (P<0.01); In addition, the mRNA levels of PPAR γ, C/EBP α and LEP were extremely significantly up-regulated (P<0.01), and FAS and UCP1 mRNA levels were significantly up-regulated (P<0.05). This study revealed the expression pattern of UCP3 in Sahu sheep and verified that overexpression of UCP3 can promote adipogenic differentiation of Sahu sheep preadipocytes. It is speculated that UCP3 is a positive regulatory factor of Sahu sheep preadipocytes, it possibly regulates the differentiation of preadipocytes through regulating the expression of PPAR γ, C/EBPα and FAS genes.

This experiment was conducted to isolate subcutaneous preadipocytes from yellow cattle and study the effect of leucine on browning white adipocytes and lipid metabolism. Preadipocytes were isolated from subcutaneous adipose tissue of 4 3-year-old healthy male yellow cattle ((351.7±42.5) kg), mixed and added with different concentrations of leucine (0, 0.5, 1, 2, 4 and 8 mmol·L^-1) at the differentiation D 6-D 9 (n=6). RT-PCR was used to detect the expression level of key browning genes to select leucine concentrations that induce browning, and follow-up experiments were executed to set the control group (CON) and optimal concentration leucine addition group (Leu) (n=6). RT-PCR was used to detect the key mitochondrial biosynthesis genes expression, mtDNA copy number and genes expression related to lipid metabolism. The expression of key proteins of browning as well as pathway proteins were detected by Western Blot. Intracellular mitochondrial content and lipid droplet morphology were detected by Mito-tracker Green staining and Oil Red O staining, respectively.The results showed that subcutaneous preadipocytes were isolated and differentiated into mature adipocytes successfully. 4 mmol·L^-1 leucine significantly increased the relative mRNA expression of browning marker factors UCP1, PRDM16 and TMEM26 and key genes of mitochondrial biosynthesis SIRT1, TFAM and NRF1/2, and increased the protein expression of UCP1, CD137 and SIRT1 in adipocytes (P<0.01). Leucine significantly increased the mtDNA copy number and mitochondrial content (P<0.01), decreased the intracellular ATP content (P<0.05). Leucine induced the transformation of lipid droplets from large single-compartment to small multi-compartment and decreased intracellular triglyceride content (P<0.01). Leucine decreased the expression of lipid synthesis genes ACC and FAS, while promoting the expression of lipolysis genes ATGL and HSL and fatty acid β-oxidation genes PPARα, CPT1 and ACOX1 (P<0.01). Leucine also significantly upregulated protein expression levels of p-AMPKα/AMPKα and PGC1α (P<0.01). In conclusion, leucine promotes browning and mitochondrial biosynthesis in preadipocytes, promotes lipolysis and inhibits lipid synthesis, reduces lipid deposition, and upregulates the protein expression of p-AMPKα/AMPKα and PGC1α.

The purpose of this study was to establish a rapid batch determination method for free arginine, histidine and isoleucine in milk by mid-infrared spectroscopy, and to carry out a large number of external verifications. A total of 217 Chinese Holstein milk samples from 4 provinces in North China, Central China and Northwest China were taken as the research objects, using 4 spectral preprocessing algorithms (SG smoothing, difference, multivariate scattering correction, standard normal transformation), 4 feature selection algorithms (known information region, adaptive heavy weighting algorithm, genetic algorithm and minimum angle regression algorithm) and 2 modeling algorithms (partial least squares regression and ridge regression), the MIR spectral quantitative prediction models of free arginine, histidine and isoleucine contents in milk were established. The optimal model was applied to the MIR spectra of 32 559 milk samples collected from 4 690 cows in 9 different dairy farms to explore the effects of lactation stage, pasture, parity and season on the predicted arginine, histidine and isoleucine contents by MIR. The results show that:1) The prediction model of arginine content based on CARS feature selection algorithm, non-spectral pretreatment algorithm and PLSR modeling algorithm was the best, R_P²=0.58, RMSEp=6.89 nmol·mL^-1; The prediction model of histidine content based on CARS feature selection algorithm, SG smoothing (window length is 11, 2-order polynomial) pretreatment and PLSR modeling algorithm was the best, R_P²=0.56, RMSEp=0.88 nmol·mL^-1; Based on 274 characteristic information wave points, SG smoothing (window length is 29, 3-order polynomial) pretreatment and PLSR modeling algorithm, the prediction model of isoleucine content was the best, R_P²=0.49, RMSEp=1.75 nmol·mL^-1; 2) When the optimal model was verified externally across regions, the prediction accuracy was reduced; 3) Applying the established model to the large-scale spectral database of E province (not participating in the establishment of the model), the contents of free arginine, histidine and isoleucine in milk was predicted, it was found that lactation stage, pasture and season had significant effects on the contents of free arginine, histidine and isoleucine in milk (P<0.001), while parity had no significant effect on arginine content, but had significant effect on histidine and isoleucine (P<0.001). The results show that it is feasible to predict the content of free amino acids in milk by MIR, especially, it has certain predictive ability in the trend analysis of milk amino acid content, and the prediction model needs more representative samples to optimize, so as to improve the accuracy and universality of the model.

The experiment investigated the effects of melatonin (MT) on the proliferation, apoptosis, steroid secretion, and the expression of related genes in sheep ovarian granulostic cells, so as to elucidate the pathways in which MT affects sheep reproduction. Fresh ovaries of 20 healthy ewes of small-tailed Han sheep at one-year-old with similar body weight and same feeding conditions were collected. Granulosa cells from follicles of 2-3 mm were collected and randomly divided into 5 groups with 5 replicates in each group, and 0, 10^-9, 10^-8, 10^-7, and 10^-6 mol·L^-1 MT were added, respectively. After 48 h of culture, the proliferation and apoptosis of granulosa cells were detected by CCK8 and Annexin V-FITC, and the culture concentration in vitro was determined to be 10^-7 mol·L^-1. The cells were randomly divided into 2 groups with 3 replicates in each group. The culture medium and lysate supernatant of the experimental group (supplemented with 10^-7 mol·L^-1 MT) and the control group (not supplemented with MT) were collected. The concentrations of progesterone (P₄), estradiol (E₂), and cAMP were detected by ELISA. RT-qPCR and Western blot were used to analyze the effects of MT on the mRNA and protein expression of MT receptor genes, apoptosis-related genes, and steroid secretion-related genes in sheep granulosa cells. The results showed that the proliferative activity of granulosa cells was significantly increased (P<0.05) in experimental groups supplemented with MT, and the proliferative activity of granulosa cells in the 10^-7, 10^-6 and 10^-8 mol·L^-1 groups was significantly higher (P<0.05) than that in the 10^-9 mol·L^-1 group. The cell proliferation activity of the 10^-7 mol·L^-1 MT group was the highest (P<0.05), but there was no significant difference (P>0.05) between the 10^-7, 10^-6 and 10^-8 mol·L^-1 groups. The early and late apoptosis rates of MT granulosa cells added with 10^-7, 10^-6 and 10^-8 mol·L^-1 were significantly lower (P<0.01) than those in the 10^-9 mol·L^-1 and control groups, and the 10^-7 mol·L^-1 group had the lowest (P<0.05) late apoptosis rate. However, there was no significant difference (P>0.05) between the 10^-7 mol·L^-1 and 10^-6, 10^-8 mol·L^-1, while the 10^-9 mol·L^-1 had no significant effect (P>0.05) on cell apoptosis. The addition of 10^-7 mol·L^-1 MT significantly decreased (P<0.05) P₄ secretion of granulosa cells, but had no significant effect (P>0.05) on the levels of E₂ and cAMP. The addition of 10^-7 mol·L^-1MT significantly up-regulated (P<0.05) the mRNA and protein expression of MT1, as well as the mRNA expression of anti-apoptotic gene Bcl-2, and significantly down-regulated (P<0.05) the mRNA and protein expression of FSHR, StAR, CYP11A1, and 3β-HSD, as well as the expression of apoptosis genes BAX, Caspase-3 and BAX/Bcl-2 ratio (P<0.05). There was no significant effect (P>0.05) on the expression of the receptor genes MT2, LHR and CYP19A1. In conclusion, the addition of MT in vitro inhibited the apoptosis of sheep ovarian granulosa cells by up-regulating the expression of anti-apoptotic genes and down-regulating the expression of apoptotic genes. The MT binding to the MT1 receptor inhibited P₄ secretion by down-regulating FSHR, StAR, CYP11A1 and 3β-HSD expression.

The purpose of this study was to explore the role of somatostatin release inhibitory factor (SRIF) in sheep endometrial epithelial cells. The uterine tissue samples of sheep were took from the designated slaughterhouse of cattle and sheep in Shihezi city, Xinjiang. The expression of SRIF in the endometrium of sheep was determined by immunohistochemical staining. Estradiol (E₂), progesterone (P₄) and interferon-tau (IFNT) were used to simulate the hormone changes during embryo implantation in sheep endometrium. RT-qPCR and ELISA were used to detect the expression of progestogen and prostaglandins (PGs), and Western blot was used to detect the expression level of SRIF. SRIF-14 was added to sheep endometrial epithelial cells, and cell proliferation was detected by CCK-8 kit and RT-qPCR. The protein expression levels of Caspase 9, Bax, Bcl-2 and Cyt-c were detected by Western blot. The levels of PGE₂ and PGF_2α secreted by cells were detected by ELISA kit. Immunohistochemical staining results showed that SRIF was widely expressed in sheep endometrial tissues, and cell staining results confirmed that SRIF was expressed in sheep endometrial epithelial cells. The results of hormone combined treatment of sheep endometrial epithelial cells showed that the mRNA expression levels of ISG15, CXCL10, HOXA10 and RSAD2 were significantly increased, the secretion level of PGE₂ was increased, the secretion level of PGF_2α was decreased, and the protein expression level of SRIF was significantly increased after hormone treatment. The results of exogenous addition of SRIF-14 in sheep endometrial epithelial cells showed that SRIF-14 inhibited the proliferation of sheep endometrial epithelial cells and the expression level of CCNA mRNA was significantly decreased. The expression level of Bax, Caspase 9 and Cyt-c proteins were significantly increased, and the expression level of Bcl-2 protein was significantly decreased. The secretion level of PGE₂ increased and the secretion level of PGF_2α decreased. This study confirmed that SRIF is widely expressed in sheep endometrial tissues and epithelial cells. The addition of SRIF-14 to sheep endometrial epithelial cells can inhibit cell proliferation and induce apoptosis.

The study aimed to explore the mechanism of the TGF-β/Smad signaling pathway mediated by TGFβR1 regulating the function of sheep granulosa cells. In this study, sheep ovarian granulosa cells were collected, and pcDNA3.1(+)-TGFβR1 and pcDNA3.1(+)-SMAD4 overexpressed plasmid vectors and shRNA-TGFβR1 and shRNA-SMAD4 interfering plasmid vectors were constructed to overexpress and interfere with TGFβR1 and SMAD4 genes in sheep granulosa cells, respectively. The proliferation, cell cycle and apoptosis of sheep granulosa cells were detected by CCK-8 cell proliferation assay, flow cytometry and Annexin-V FITC/PI double staining. The mRNA levels of TGFβR1 and SMAD4, the protein expression levels of apoptosis-related proteins BAX, BCL2, and Caspase3 and the phosphorylation levels of TGFβR1, TGFβR2 and SMAD2/3 in the TGF-β/Smad signaling pathway were detected by Real-Time PCR and Western blot. The results showed that overexpression of TGFβR1 and SMAD4 extremely significantly promoted the ability of granulosa cells to enter the S phase from the G0/G1 phase (P<0.01), the proliferation of granulosa cells and the expression of anti-apoptotic protein BCL2 (P<0.01), and inhibited the expression of apoptotic protein BAX and Caspase3 (P<0.01). Interference with TGFβR1 and SMAD4 both promoted granulosa cell apoptosis and the expressions of apoptotic protein BAX and Caspase3 (P<0.01) and inhibited the expression of anti-apoptotic protein BCL2 (P<0.01). Further studies showed that overexpression of TGFβR1 could promote the phosphorylation levels of TGFβR1, TGFβR2 and SMAD2/3 in the TGF-β/Smad signaling pathway (P<0.01), and extremely significantly promote the protein expression level of SMAD4 (P<0.01). Interference with TGFβR1 significantly inhibited the phosphorylation levels of TGFβR1, TGFβR2 (P<0.01) and SMAD2/3 (P<0.05), and inhibited the protein expression of SMAD4 (P<0.01). Overexpression of SMAD4 could significantly promote the expression of TGFβR1 protein (P<0.01). The results showed that TGFβR1 promoted the proliferation and cycle of sheep granulosa cells, inhibited granulosa cell apoptosis, positively regulated the proliferation and cycle of sheep granulosa cells, and negatively regulated the apoptosis of sheep granulosa cells by mediating the expression of SMAD2/3 and SMAD4 in TGF-β/Smad signaling pathway.

The aim of this study was to investigate the effect of β-nicotinamide mononucleotide (NMN) on the lipid droplet content and vitrification freezing of bovine oocytes. In this study, bovine oocytes were placed in medium containing 1 μmol·L^-1NMN for in vitro maturation (IVM), vitrification and in vitro fertilization (IVF). The lipid droplet content, reactive oxygen species (ROS) level, apoptosis level and developmental ability of bovine oocytes were examined. The group with the addition of NMN was used as a NMN group; The group without the addition of NMN was used as a fresh control group; The group with the addition of NMN and vitrification was used as a NMN vitrification group; The group without the addition of NMN but with vitrification was used as a vitrification control group. The lipid droplet content, ROS level, and apoptosis level in bovine oocytes of the NMN group were significantly lower than those in the fresh control group (P<0.05), and the cleavage rate ((89.57±7.58)%) and blastocyst rate ((45.63±3.78)%) were significantly higher than those of the fresh control group ((80.77±0.70)%, (36.90±1.20)%) (P<0.05). Meanwhile, the ROS level and apoptosis level of bovine oocytes in the NMN vitrification group were significantly lower than those in the vitrification control group (P<0.05), and the survival rate ((97.25±0.11)%), cleavage rate ((75.47±1.11)%) and blastocyst rate ((33.75±1.43)%) after thawing were significantly higher than those in the vitrification control group ((89.29±1.16)%, (52.00±1.26)%, (15.38±1.73)%, P<0.05). In conclusion, the addition of NMN could effectively reduce the lipid droplet content of bovine oocytes and decrease the ROS level and apoptosis level of vitrified bovine oocytes, thus improving the developmental ability of vitrified bovine oocytes.

The aim of this study was to investigate the effects of overexpression of absent, small, or homeotic 1-like (ASH1L) on the proliferation and apoptosis of bovine cumulus cells (CCs). The cDNA fragment of bovine ASH1L SET domain was amplified by PCR using CCs cDNA as templates and cloned into pcDNA3.1 in order to construct a bovine ASH1L overexpression vector. The bovine CCs transfected with pcDNA3.1+ASH1L vector were used as the overexpression group (ASH1L-OE) and the wild CCs were the control group (Control). The levels of ASH1L protein and H3K36me1/2/3 methylation in CCs of ASH1L-OE and the control group were detected by immunofluorescence staining. The apoptosis and proliferation of CCs in both groups were analyzed by flow cytometry. The mRNA expression levels of apoptosis-related and proliferation-related genes were detected by quantitative RT-PCR. A 2 487 bp DNA fragment of bovine ASH1L SET domain was obtained by PCR amplification using bovine CCs cDNA as template and ligated with pcDNA3.1 vector; the overexpression vector pcDNA3.1+ASH1L was successfully constructed after NheⅠ/NotⅠ double digestion and sequencing. Levels of ASH1L mRNA, protein and H3K36me1/2 methylation were significantly increased in CCs transfected with overexpression vector (P<0.05). Moreover, ASH1L overexpression in CCs significantly increased the percentage of live-cells (91.85±1.25)% vs. (87.39±1.71)% and decreased the apoptosis rate (8.08±1.21)% vs.(12.51±1.72)%. The mRNA expression levels of apoptosis-related genes BAX and CASPASE-3 were significantly downregulated (P<0.05) and expression level of anti-apoptotic gene BCL-2 was upregulated in CCs with ASH1L overexpression (P<0.01). The proliferation rate of cells and the expression levels of proliferation-related genes PCNA and CCND2 in CCs with ASH1L overexpression was significantly increased at 24 and 36 h after transfection (P<0.05). These results suggest that ASH1L overexpression inhibits CCs apoptosis, induces cell proliferation and H3K36me1/2 methylation in bovine CCs. The study provides a technical and theoretical basis for revealing the regulation of ASH1L on CCs growth and follicle development.

Fas-associated factor-1 (FAF1) is a member of the Fas family and contains several multifunctional domains. It can participate in biological processes such as apoptosis, inflammation, necrosis, cell proliferation and protein homeostasis. The purpose of this study was to prepare the polyclonal antibody of yak FAF1, and to explore the biological role of FAF1 in testicular tissues of different ages, and ovaries of different reproductive cycles. The whole sequence of yak FAF1 gene was amplified, and the prokaryotic expression vector pET28a-FAF1 was constructed. The induction conditions were optimized. After purification by Ni-NTA column, the experimental animals were immunized to obtain rabbit FAF1 polyclonal antibody. The testes of male yaks in three age groups of juvenile (1-year-old, 2-year-old), adult (3-year-old, 4-year-old, 6-year-old, 7-year-old) and elderly (11-year-old) were selected, and the ovaries of female yaks in different breeding cycles (follicular phase, luteal phase and pregnancy) were selected. Real-time fluorescence quantitative, Western blot and immunohistochemical(IHC) methods were used to detect the relative expression of FAF1 mRNA, protein and protein expression localization in testes of different ages and ovaries of different breeding cycles. The results showed that the prokaryotic expression vector pET28a-FAF1 was successfully constructed, and the optimal induction conditions were 25℃, IPTG concentration of 0.5mmol ·L^-1 and induction for 5 hours. The recombinant protein was mainly present in the inclusion body. After immunizing experimental animals, rabbit-derived FAF1 polyclonal antibody was obtained, with a antibody titer of 1:1 024 000 and good specificity. Real-time fluorescence quantitative results of testicular tissue showed that the relative expression of FAF1 mRNA in testicular tissue of 3, 4 and 7 years old was significantly higher than that of other ages. The Western blot results of testicular tissue showed that the relative protein expression of FAF1 protein at 11 years old was significantly higher than that at other ages, followed by 6 and 7 years old, and the relative protein expression of FAF1 at 3 and 4 years old was the lowest. Immunohistochemistry showed that FAF1 was mainly expressed in sperm, sperm cells, spermatogonia, primary spermatocytes, Sertoli cells, Leydig cells, peritubular myoid cells and seminiferous tubules. The real-time fluorescence quantitative results of ovarian tissue showed that the relative expression of FAF1 mRNA in the luteal phase was significantly higher than that in the follicular phase and pregnancy, followed by the pregnancy ovary, and the follicular phase ovary was the lowest. The results of ovarian Western blot showed that the relative expression of FAF1 protein in ovarian follicular phase and pregnancy were significantly higher than that in luteal phase, followed by pregnancy, and the lowest expression in luteal phase. Ovarian IHC results showed that FAF1 was mainly expressed in ovarian reproductive epithelial cells, granulosa cells, cumulus cells and luteal cells. The expression of FAF1 mRNA and protein in testis of different ages and ovarian tissues of different reproductive cycles is significantly different. It is speculated that FAF1 may be closely related to the development of testis, spermatogenesis and testosterone secretion of male yaks, as well as the physiological processes of follicular atresia, development, maturation and luteolysis of female yaks. The current study provides the basis for further exploring the reproductive physiological function of FAF1 in yaks.

Pyometra, a common canine disease accompanied by bacterial contamination in the uterus, is a major cause of canine death worldwide. However, the underlying mechanism of pyometra remains poorly understood. The purpose of this study is to identify the gene expression changes associated with canine pyometra, and to screen differentially expressed genes, regulatory pathways and hub genes to further understand the molecular mechanism of pyometra. RNA sequencing (RNA-seq) was uesd to study the gene expression profile of canines with pyometra compared with those with benign uterus. The results showed a discrepancy of 11 812 gene expressions, among which 7 086 genes were up-regulated while 4 726 genes were down-regulated in the uteri of diseased animals. GO analysis showed that differentially expressed genes were mainly involved in biological processes such as immune system processes, cytokine activity, chemokine activity, and cytokine receptor binding. KEGG-Pathway analysis found that differentially expressed genes were involved in the signaling pathways of inflammation and immune response activation, such as NF-κB signaling pathway, PI3K-Akt signaling pathway, B cell receptor signaling pathway, phagosome and natural killer cell-mediated cytotoxicity, revealing that inflammation and immune response activation are the main pathological stages of canine pyometra. Eleven key hub genes were identified by STRING database network, such as CDK1, ESR1 and SHC1, which can be used as biomarkers for early diagnosis of pyometra. In conclusion, the current study presents a comprehensive landscape of the gene expression in pyometra canines and contribute to a better understanding of the molecular mechanisms of canine pyometra.

This experiment aimed to explore the effects of different exercise amounts on rumen microflora of Tan sheep. Sixty Tan sheep weaned lambs (body weight (20±0.5) kg) with good body condition were randomly divided into three groups:1) Control group (NC group), whole barn feeding; 2) 5 kilometers group (5 km group), daily exercise along the ecological pasture fence 5 km; 3) 10 kilometers group (10 km group), daily exercise along the ecological pasture fence 10 km. The are 5 replicates per group and 4 sheep in each replicate. The experiment lasted for 130 days, including 10 days of pre-experiment and 120 days of formal experiment. At the end of the experiment, rumen contents were collected for metagenomic sequencing to determine the diversity of rumen microflora. Results were showed as follows:1) The Shannon, Simpson and Chao1 indexes of 5 km and 10 km groups were higher than those in NC group (P>0.05); 2) Taxonomic annotation showed that Bacteroidetes and Firmicutes accounted for the highest proportion, whereas no significant differences in relative abundance of the two bacteria phyla were observed among the experimental groups and the NC group (P>0.05). The relative abundance of Ruminococcus and Lactobacillus were significantly higher in the experimental groups than those in the NC group (P<0.05). In summary, under the conditions of this experiment, increasing the amount of exercise can increase the abundance and diversity of rumen microorganisms of Tan sheep, change the relative abundance of some bacteria, but have no significant impact on the uniformity of rumen microorganisms.

Porcine primary alveolar macrophages (PAMs) were the main target cells of African swine fever virus (ASFV), and it was difficult for ASFV to grow and multiply continuously in other in vitro cell lines except host monocytes/macrophages. The aim of this study was to investigate the factors affecting ASFV in in vitro passaging cultures. By comparing the genome-wide transcriptional profile differences between ASFV inoculated susceptible cells PAMs and non-susceptible cell lines 3D4/21 and PK15 cells; Using small molecule drugs to alter cell metabolism or cycle, physical methods to alter cell adhesion; detecting virus proliferation in 3D4/21 and PK15 cell lines by fluorescence quantitative PCR, Western blot, red cell adsorption. The results showed that:ASFV inoculation of both 3D4/21 and PK15 cells and PAMs cells were significantly enriched in genes common to both cell adhesion, cell cycle and cell metabolism; The ability of ASFV to infect PK15 and 3D4/21 cells was not significantly affected by treatment of cells with various small molecule drugs that regulate cell cycle and cell metabolism; The ability of ASFV to infect PK15 and 3D4/21 cells was significantly affected by suspension culture to regulate The ability of ASFV to infect PK15 and 3D4/21 cells was significantly improved after cell adhesion was regulated by suspension culture, but the replication ability of the virus gradually decreased with the increase in the number of passages. In conclusion, cell adhesion may be one of the important factors affecting the in vitro infection ability of ASFV, but altering cell adhesion did not maintain the replication ability of ASFV. This study provides a preliminary reference for the establishment of ASFV in vitro cell lines.

This study is aimed to prepare monoclonal antibodies against African swine fever virus (ASFV) p30 protein. Five 6-8 weeks female BALB/c mice were immunized with prokaryotic recombinant p30 protein as immunogen. Spleen cells were isolated and fused with SP2/0 cells after 3 times of immunization. Two hybridoma cell lines, named 2G10-2 and 6D2-1, which could stably secrete monoclonal antibodies against ASFV p30 protein were obtained. The antibody titers were 1:12 800 and 1:3 200, respectively. The results of subtype identification showed that the heavy chain of the two monoclonal antibodies belonged to IgG, and the light chain was κ type. The results of Western blot and indirect immunofluorescence assay showed that both monoclonal antibodies had good binding activity to p30 protein. Western blot analysis showed that the two monoclonal antibodies could effectively bind the 170-194 amino acids of the recombinant p30 protein, which proved that the antigenic recognition region was the 170-194 amino acids. In this study, two monoclonal antibodies against p30 protein were prepared and their epitopes were identified, which laid a foundation for the study of the structure and function of ASFV p30 protein and the development of serological diagnostic reagents.

This experiment was conducted to obtain monoclonal antibodies against the GP3 protein of porcine reproductive and respiratory syndrome virus (PRRSV) and analyze its antigenic epitopes. The prokaryotic expression plasmid of PRRSV glycoprotein 3(GP3) from NADC30-like strain FJ1402 was constructed and the recombinant GP3 protein was obtained. Positive hybridoma clones were obtained through BALB/c mouse immunization and indirect ELISA. The specificity of monoclonal antibodies was further identified by indirect immunofluorescence assay and Western blot. The recognizing epitope regions of monoclonal antibodies on GP3 protein were identified by constructing truncated GP3 protein and antigenic epitope ELISA. The results showed that seven hybridoma cell lines stably secreting antibodies against the GP3 protein were successfully selected,Moreover, all monoclonal antibodies can specifically react with PRRSV FJ1402 strain. And four antigenic epitopes were identified as following,⁵⁵PLCPTRQAAAEILE⁶⁸,⁶⁹PGKSFWCRI⁷⁷, ⁷⁸GHDRCSESDH⁸⁷, and ⁸⁸DELGFMVVPPGLSS¹⁰⁰. And 2E12 mAb can also react with PRRSV BB0907 and S1 strains by using IFA and Western blot, but 4H9、9F3 and 6B3 cannot. This study successfully obtained monoclonal antibodies against GP3 protein of PRRSV, and identified four GP3 protein epitopes, laying an important foundation for the diagnosis of PRRSV and the study of the function of the viral GP3 protein.

This experiment analyzed the variations of key antigen genes and virulence genes of infectious laryngotracheitis virus (ILTV) WF03 strain isolated from chickens that failed to be immunized with live vaccine. And also, the virulence of WF 03 was tested in SPF chickens. The phylogenetic tree of key immunogenic genes and virulence related genes of clinical ILTV isolate WF03 was performed, and the pathogenicity of this strain infected 28 and 56 days-old SPF chickens was tested. The results showed that the key immunogenic genes and virulence related genes of the WF03 strain were in the similarity evolutionary branch with the early isolates from China and some commercialized vaccine strains. The pathogenicity experiment showed that when the 28 days-old SPF chicken were challenged with WF03 strain with the doses of 10^4.5, 10^4.0, 10^3.5 and 10^3.0 EID₅₀, respectively, the morbidity was 100%, 100%, 95% and 85%, respectively, and the mortality was 45%, 30%, 35% and 10%, respectively. The morbidity and mortality to 56 days-old SPF chickens challenged with 10^4.0 EID₅₀ dose were 100% and 27.3%, respectively. The main clinical symptoms were hemoptysis, dyspnea, mouth breathing with same as typical symptoms of laryngotracheitis. The results showed that the clinical strain WF03 of ILTV was a high virulent strain and can be used as a challenge strain for vaccine efficacy, and its genetic characteristics were not significantly different from those of early isolates and vaccine strains in China. The results suggested that high virulent strains were prevalent in Chinese flocks, which will enrich the epidemiological database of current ILTV strains in China.

In order to determine the infection situation of Escherichia coli (E.coli), virulence genes and drug resistance of the E.coli strains in goat and sheep farms with diarrhea in Eastern China, the intestinal tract and fecal samples of sick and dead goat and sheep with diarrhea were examined by means of E.coli isolation and identification, phylogenetic grouping, virulence gene detection and drug sensitivity test. The results showed that a total 163 E.coli strains were isolated from 187 samples, of which the majority were classified into group B1 and group A, with detection rates of 59.51% and 28.83%, respectively. The detection rates of B2 and D groups were 9.82% and 1.84%, respectively. The results from 31 virulence genes detection showed that the detection rates of yijp, crlA, mat, ompA, fimC and ibeB were all greater than 87%. Virulence genes afa/draB and LT were not detected in all strains. fimC, mat, crlA, ibeB, yijp and ompA were widely present in all evolutionary groups. According to the results of virulence gene test, 72 strains were Shiga toxin-producing E.coli (STEC), which mainly carried virulence gene stx2. The detection rate of the other marker virulence gene eae was 61.11%. Drug sensitivity test results showed that the resistance rate of 4 and 13 out of the 16 antibacterial drugs tested was greater than 80% and 50%, respectively. Among the 163 strains, 150 were multi-drug resistant (MDR) ones, accounting for 92.02%, and most of them were resistant to five or more drug categories (84.05%). In conclusion, the results of this study indicated that the clinically prevalent E.coli strains from goat and sheep in eastern China were highly virulent, common and strong drug resistance. It provided a scientific basis for the prevention and control of E.coli in the farms in this region.

This experiment was conducted to understand the microbial diversity of the nasal cavity of sheep with respiratory disease and the surface deposits of their house, and analyze the relationship between the respiratory diseases of sheep and the microbial community composition of their environment. Our study is expected to provide references for prevention and treatment of sheep respiratory diseases. We collected the nasal swabs of sheep and the surface deposits of their house from a sheep farm in Yinchuan, Ningxia. Then, control with nasal swabs and surface deposits of the healthy sheep, 16S rRNA gene sequencing were performed, and the microbial community composition of the nasal cavity and the environment of healthy sheep with respiratory disease were analyzed by bioinformatics. The results showed that the diversity and richness of microbial flora in the nasal cavity and their environment of sheep with respiratory diseases were reduced compared with those healthy sheep. The microbial community composition of the nasal cavity in healthy sheep was significantly different from those sheep with respiratory diseases (P<0.01). Stenotrophomonas is the main genus in the nasal cavity of healthy sheep. Moraxella, Bergeyella and Filobacterium are the predominant genus in the nasal cavity of sheep suffering from respiratory diseases. The microbial community composition in the surface deposits of house between the healthy and the sheep with respiratory diseases also had a significant difference (P<0.05). Firmicutes was the most prominent phyla in diseased sheep. It is concluded that the microbial community composition and relative abundance were changed in the nasal cavity and their environment of sheep with respiratory diseases, but the environment microbial community is not directly related to the respiratory disease of sheep. The imbalance of microbial flora and the increased relative abundance of bacterial pathogens such as Moraxella, Bergeyella, and Filobacterium in diseased sheep may be important factors leading to respiratory diseases in sheep.

This study aimed to explore the effect of gut microbiota disturbance on susceptibility to BVDV by establishing a mouse model of gut microbiota disturbance. The experiment was divided into two groups. Gut microbiota disturbance mice were treated with amphotericin-B, neomycin trisulfate salt hydrate, ampicillin, metronidazole, and vancomycin hydrochloride by gavage, while mice in control group were given an equal volume of physiological saline.After 13 consecutive days, fecal samples of mice were collected to extract bacterial DNA and 16S rRNA sequencing was performed to detect the changes in the diversity and abundance of gut microbiota in mice.The mice exploring the impact of gut microbiota disturbance on susceptibility to BVDV were divided into 2 groups. Mice in antibiotic treated and untreated groups were intraperitoneally injected with 10⁵ TCID₅₀ CP BVDV. The blood and duodenum of mice at day 7 of post-infection were collected, and viral loads were detected by qRT-PCR and Western blot. The pathological changes of duodenum were detected by hematoxylin and eosin staining (HE staining). The experiment of fecal microbiota transplantation (FMT) was divided into two groups. Mice in the experiment group were treated with FMT while mice in control group were given the same volume normal saline. Aimed to further verify the effect of restoring gut microbiota on BVDV infection. The results showed that antibiotic treatment significantly reduced the α-diversity of gut microbiota, Venn diagram analysis also showed that antibiotic treatment reduced the number of OTUs of gut microbiota. The β-diversity combined with the histogram analysis further showed that the composition of gut microbiota was significantly different between the antibiotic treated group and the untreated group. The relative abundance of Firmicutes and Bacteroidetes decreased significantly in the antibiotic treated group, while the relative abundance of Proteobacteria increased significantly. Exploring the susceptibility to BVDV showed that viral loads in blood and duodenum of mice with gut microbiota disturbance were significantly higher than that of mice with normal microbiota. The disturbance of gut microbiota increased the expression of BVDV E0 protein in duodenum of mice, and aggravated duodenal pathological damage were detected by Western blot and histopathological analysis. However, FMT treatment significantly reduced viral loads and improved duodenal pathological damage. The above results indicated that the antibiotic induced mouse model of gut microbiota disturbance was successfully constructed. On the basis of this model, it was further confirmed that the disturbance of gut microbiota increased the susceptibility to BVDV, and FMT supplementation showed the effect of inhibiting BVDV infection. These studies lay a foundation for developing probiotics for the prevention and control of BVDV infection and the screening of antiviral drugs.

The purpose of this study was to analyze the expression profile of circRNA in protoscolex, hydatid cyst wall and adult of Echinococcus granulosus, and to provide theoretical basis for further elucidating the role of circRNA in the growth and development of Echinococcus granulosus.Total RNA was extracted from Echinococcus granulosus protoscoleces, hydatid cyst wall and adult worms, and the circRNA, miRNA and mRNA libraries were constructed.The differentially expressed circRNA were screened by bioinformatics analysis and functional prediction, and then were analyzed by the GO, KEGG and ceRNA regulatory networks. The differentially expressed circRNA were validated by real-time fluorescent quantitative PCR (qRT-PCR).In this study, 636 circRNA molecules were identified from protoscolex, cyst wall and adult of Echinococcus Granulosus, of which 140 were differentially expressed.The specific expression of circRNA was 44, 2 and 0 in protoscolex, cyst wall and adult worms, respectively.The results of the GO analysis showed that compared with protoscolices and cyst walls, the differentially expressed circRNA molecules in the adult worms were mainly concentrated in the biological processes of sexual reproduction, spermatogenesis, male gametogenesis, axogenesis, neurogenesis and differentiation.The results of KEGG analysis showed that the differentially expressed circRNA was significantly enriched in Notch, tricarboxylic acid cycle, phosphatidylinositol, riboflavin metabolic pathway and fatty acid synthesis and degradation pathway. The differentially expressed circRNA in cyst wall and protoscolices were significantly enriched in dorsal-ventral axis formation, Notch, wnt and FoxO signaling pathway.The differentially expressed circRNA in cyst wall and adult worms were significantly enriched in Notch, carbon metabolism, tricarboxylic acid cycle and glycolysis/gluconeogenesis pathway.Targeted prediction of circRNA-miRNA-mRNA regulatory networks showed that 164 up-down-up circRNA-miRNA-mRNA regulatory networks were formed by 24 differentially expressed circRNAs, 14 miRNAs and 54 mRNAs, 36 down-up-down circRNA-miRNA-mRNA regulatory networks were composed of 13 differentially expressed circRNAs, 7 miRNAs and 16 mRNAs.The results of qRT-PCR for some circRNA molecules showed that the mRNA transcription level was consistent with the RNA-sequencing results.In conclusion, this study is the first to systematically analyze the differential expression profiles of circRNA in protoscolex, cyst wall and adult Echinococcus granulosus, the potential regulatory network of circRNA-miRNA-mRNA of differentially expressed circRNA was predicted, the high expression and specific expression circRNA molecules in protoscolex, cyst wall and adult worms were also screened, this study lays a foundation for further research on the role of circRNA in the growth and development of Echinococcus granulosus.

To investigate the effect of anthocyanins from Lycium ruthenicum Murray of Qaidam, Qinghai, on hypoxia-induced apoptosis of H9c2 rat cardiomyocytes, the H9c2 rat cardiomyocytes were selected for the study, including normoxia group, hypoxia group, hypoxia+anthocyanin group and hypoxia+salidroside group, LDH content was detected by enzyme label, cell morphological changes were observed by microscope, the content of ROS was explored by fluorescent enzyme labeling instrument, cell apoptosis was detected by Hoechst33342/PI double staining and flow cytometry. In addition, the expression of apoptosis related factors (Bcl2, Bax) at mRNA and protein levels were tested by qRT-PCR and Western blot, respectively. The results showed that compared with the normoxia group, hypoxia significantly increased the contents of LDH, ROS and the rate of apoptosis (P<0.01), and down-regulated the expression of Bcl2 and Bcl-2/Bax (P<0.01), the mRNA and protein expression of Bax were significantly up-regulated (P<0.01). Under hypoxic condition, anthocyanin significantly reduced the contents of LDH and ROS (P<0.05), significantly reduced the rate of apoptosis (P<0.01), significantly up-regulated the expression of Bcl2 and Bcl2/Bax ratio mRNA and protein (P<0.05), the protein expression of Bax was significantly down-regulated (P<0.01). In conclusion, under hypoxic conditions, Lycium ruthenicum Murray anthocyanin can effectively delay the apoptosis of H9c2 rat cardiomyocytes induced by hypoxia by reducing the content of LDH and ROS in cells, up regulating the expression of Bcl2 and Bcl2/Bax ratio at mRNA level and protein level, and down regulating the expression of Bax at protein level.

This study aims at establishing a method for the determination of related substances in aspirin eugenol ester(AEE) granules. Ultra performance liquid chromatography(UPLC) was used to detect the related substances of AEE granules, with the column was Phenomenex luna C18 (150 mm×4.6 mm, 5 μm), mobile phase was 0.5% phosphoric acid squeous solution and acetonitrile, flow rate was 1.0 mL·min^-1, wavelength was 279 nm, column temperature was 35℃, injection volume was 10 μL. Under the selected chromatographic conditions, the separation of the main peak of AEE and the impurity peak was well. Impurity A had a good linear relationship with the peak area in the concentration range of 0.25-60.0 μg·mL^-1, R²=0.999 2 (n=8). The material recoveries ranged from 98.34% to 100.15% (n=9, RSD=0.68%), and the robustness of the method under different conditions was well, the RSD value was less than 3.33%. The method is simple and specific, which suitable for the determination of related substances in AEE granules after methodological verification.

In order to explore the histopathological characteristics of Salmonella infection in female rabbits, 6 dead female rabbits with depressed, shortness of breath and aborted from a large-scale rabbit farm in Henan province were examined. Endometrial bleeding, stillbirth, and hyperemia and congestion on the surface of heart, liver, spleen, lung and kidney were found by anatomical examination. The isolated pathogen was identified as Salmonella by isolation culture, morphological observation, serological typing, PCR identification, sequencing analysis and pathogenicity test. It was found that the myocardial fibers were bent or arranged disordered, liver cell granular degeneration or steatosis, lung extensive congestion, suppurative hysteritis, endometritis and other organs hyperemia by histopathological observation. The results of drug sensitivity test showed that the pathogens were sensitive to chloramphenicol, cephalosporins, penicillins, quinolones, tetracyclic, polypeptide, β-lactam and sulfa antibiotics, and had certain resistance to aminoglycoside (butamicana). In this study, Salmonella was successfully isolated from rabbits, the histopathological and drug sensitivity analysis of the infected female rabbits were carried out, which provides a basis for better prevention and control of rabbit salmonellosis.

The aim of this research was to study the antagonistic mechanism of Lycium barbarum polysaccharide (LBP) on cyclophosphamide induced immunosuppression in chicks based on RNA-Seq technology, and to analyze the changes of immune-related differentially expressed genes in spleen, so as to provide reference for the antagonistic mechanism of LBP on immunosuppression of chicks. One hundred and twenty 7 days old Hy-line brown laying hens were randomly divided into three groups:blank group (NC), cyclophosphamide group (CY) and Lycium barbarum polysaccharide group (CYLbGp). The immunosuppressive chick model was established by injecting 80 mg·kg^-1 cyclophosphamide everyday into the pectoralis muscle for three consecutive days, and the same volume of normal saline was injected to the hens in NC group. After the establishment of the model, the chicks in the CYLbGp group were supplemented with Lycium barbarum polysaccharide (LBP) 5 mg·kg^-1 by drinking water everyday for 30 consecutive days. At the end of administration, spleens were collected from 6 chicks in each group, and the differentially expressed genes (DEGs) in the three groups were detected by RNA-Seq. KEGG pathway enrichment analysis and protein-protein interaction (PPI) network analysis were used to screen key differentially expressed genes and pathways. The results showed that after LBP intervention a total of 178 DEGs significantly reduced, and were significantly enriched in mitochondrial autophagy-animals, chemokine signaling pathways, C-type lectin receptors signaling pathways, B cell receptor signaling pathways, platelet activation, natural killer cell mediated cytotoxicity and Th1 and Th2 cells differentiation and other related immune pathways. PPI analysis showed that PIK3CD, JAK3, GATA3, HIF1A, NKX2-5 and INPPL1 were the key genes. The results of RT-qPCR were consistent with the trend of transcriptomic data, which further indicated the high reliability of transcriptomic data. It can be concluded that Lycium barbarum polysaccharide can act on key targets such as PIK3CD, JAK3, GATA3, HIF1A and INPPL1. Lycium barbarum polysaccharide is involved in the regulation of mitochondrial autophagy, chemokine signaling pathway, C-type lectin receptor signaling pathway, B-cell receptor signaling pathway, platelet activation, natural killer cell-mediated cytotoxicity, Th1 and Th2 cell differentiation and other signaling pathways, and thus it can relieve the immune suppression of chicks.

The aim of this experiment was to investigate the effect of the combination of adipose derived mesenchymal stem cells (ADSCs) and methylprednisolone (MP) on immune rejection and wound healing in allogeneic skin grafts from miniature pigs. An allogeneic skin graft model was established for 12 minipigs, and the minipigs were randomly and equally divided into four groups according to different postoperative interventions on allogeneic skin graft skin pieces:allogeneic skin graft group (ASG), methylprednisolone group (MP), adipose mesenchymal stem cells (ADSCs), and combined adipose mesenchymal stem cells and methylprednisolone (ADSCs+MP) group.Blood and tissue samples were collected preoperatively and at 7 and 14 d postoperatively, and clinical changes of the grafted epidermis were photographed and recorded immediately, at 7, 14 and 21 d postoperatively. The effects of the four different interventions on the immune rejection and wound healing of allogeneic skin grafts in minipigs were evaluated by the phenological examination, pathological histological examination, blood and serum biochemistry, and the indexes of oxidative stress, inflammation and regeneration of the grafted skin tissues. From the skin slice phenotypic and pathological analysis, it was observed that the survival time of the grafted skin slice was around 7 d in the ASG group, 7-14 d in the MP group, and 14-21 d in the ADSCs and ADSCs+MP groups. and the expression of GSH and SOD was significantly higher in the ADSCs+MP group at 7 and 14 d. In terms of immune rejection, the ADSCs+MP group had significantly higher CD4⁺ T cells, protein and gene expression of NF-κB, and expression of IL-2, IFN-γ, TNF-α, IL-1β, and IL-6 pro-inflammatory factors were significantly lower in the ADSCs+MP group relative to the ASG group.In terms of wound healing, the ADSCs+MP group significantly increased the protein expression of VEGF and TGF-β, and the expression of SDF-1 and CXCR4 relative to the ASG group. the ADSCs+MP group could significantly inhibit the protein and gene expression of PI3K, AKT, and 11β-HSD1. In conclusion, the ADSCs+MP group could prolong the survival time of grafted skin pieces relative to the ASG and MP groups. And ADSCs+MP could effectively inhibit oxidative stress by promoting the expression of GSH and SOD. In terms of rejection, ADSCs+MP can inhibit the expression of inflammatory factors such as IL-2, IFN-γ, IL-6 and TNF-α by suppressing the expression of NF-κB, thus suppressing the immune rejection reaction after transplantation. Moreover, ADSCs+MP could promote wound healing by promoting the expression of healing factors such as VEGF and TGF-β. Further study revealed that ADSCs may affect the expression of 11β-HSD1 by inhibiting PI3K/AKT, which in turn suppresses skin cortisol content and promotes the expression of growth factors such as TGF-β and VEGF to promote wound granulation tissue regeneration and vascular migration.

We attempted to investigate the role of endoplasmic reticulum stress (ERS) in lipopolysaccharide (LPS)-induced inflammatory responses in goat endometrial epithelial cells (gEECs) and provide a preliminary basis for elucidating the mechanism of ERS in ruminant endometritis. In this study, gEECs were treated with ERS activator tunicamycin (TM) and inhibitor 4-phenylbutyric acid (4-PBA) separately to activate and inhibit ERS, and RT-qPCR and Western blot techniques were used to detect the effect of ERS on inflammation-related genes expression. Next, gEECs were pretreated with TM and 4-PBA, respectively, and then treated with E.coli LPS to induce inflammatory responses in gEECs, the effects and mechanisms of ERS on the inflammatory responses induced by LPS in gEECs were detected by RT-qPCR and Western blot. The results showed that activation of ERS by TM treatment significantly inhibited the expression of inflammatory cytokines IL-6 and TNF-α mRNA in gEECs (P<0.05). The mRNA expression levels of classical inflammatory pathway-related genes TLR4, NF-κB P65, and NLRP3 was also significantly suppressed (P<0.01). Meanwhile, it also significantly inhibited the phosphorylation of NF-κB P65 protein and the expression of NLRP3 protein (P<0.05). However, 4-PBA treatment significantly promoted the expression of inflammation-related genes (IL-6, TNF-α, TLR4, NF-κB P65, and NLRP3) in gEECs (P<0.05). Further study revealed that TM pretreatment of gEECs significantly inhibited the expression of LPS-induced inflammatory cytokine IL-6 mRNA (P<0.05). It also significantly inhibited LPS-induced TLR4, NF-κB P65, and NLRP3 mRNA expression as well as phosphorylated NF-κB P65 protein and NLRP3 protein (P<0.05). 4-PBA pretreatment of gEECs then significantly promoted the LPS-induced inflammatory response in gEECs (P<0.05). The above results suggest that ERS alleviates LPS-induced inflammatory response in gEECs by inhibiting the activation of NF-κB pathway and NLRP3 inflammatory vesicle pathway. These results provide a pre-theoretical basis for further elucidation of the development of goat endometritis.

The objective of this study was to preliminaries verify the potential target and mechanism of berberine (BBR) in the intervention of enteritis in broilers by using network pharmacology, molecular docking and molecular dynamics simulation combined with Salmonella challenge test. Firstly, PharMapper database and Swisstargets database were used to predict drug targets. The disease targets were collected through the GeneCards database, and the intersection of drugs and disease targets was screened. The obtained intersection targets were used to construct protein interaction network and GO and KEGG enrichment analysis. Molecular docking verification and kinetic simulation were carried out for the selected core targets. Subsequently, the intestinal inflammation model of broilers was established to observe the morphology changes of intestinal tissue, and the effect of BBR on the mRNA expression of target protein was verified by qPCR test. Finally, 374 potential BBR targets were screened, and 174 overlapped with enteritis. GO and KEGG enrichment analysis obtained 197 items and 73 items, respectively, which mainly involved signal transduction, protein phosphorylation process, PI3K-Akt, chemokine and Ras signaling pathway. The results of molecular docking and kinetic simulation showed that BBR could bind well to the core target, mainly through hydrophobic force and hydrogen bond. The results of tissue section showed that BBR could significantly alleviate intestinal tissue injury. The results of RT-qPCR showed that the expression of HSP90AA1 was significantly decreased (P<0.001), and the expression of PIK3CA was significantly increased (P<0.01) in cecal tissue of the inflammatory model of broilers. Through network pharmacology and experimental verification, it was found that BBR may regulate the occurrence and development of intestinal inflammation by regulating the expression of HSP90AA1 and PIK3CA, which may involve PI3K-Akt, cAMP, AMPK and other signaling pathways, providing new ideas and methods for further research on the mechanism.

The network pharmacology and related molecular biology experiments were used to explore the protective mechanism of Codonopsis pilosula on acute lung injury (ALI). The main chemical components of Codonopsis radix and related targets were mined through the network pharmacology database. The related targets of ALI were obtained through disease-related databases. protein interaction (PPI) network was constructed,and GO analysis and KEGG pathway enrichment analysis were performed on the mined data. The results showed that 8 compounds in Codonopsis pilosula could act on 303 targets, 1 522 targets related to ALI, 119 intersecting targets involved in ALI, 103 targets in PPI network including MAPK, NF-κB and TLR4. GO functional enrichment analysis showed 133 molecular functions, 79 biological processes and 523 cellular components, and KEGG showed 132 related pathways. The results were verified by Western blotting, ELISA and immunofluorescence. The results showed that Codonopsis pilosula polysaccharides pretreatment could down-regulate MAPK and NF-κB inflammatory signal pathways activation and TNF α and IL-1 β secretion in E.coli-induced macrophages. Furthermore, Codonopsis pilosula polysaccharide could down-regulate TNF α and IL-1β production, and down-regulate HMGB1 expression to reduce the lung injury in the E.coli-infected lungs of mice. In conclusion, Codonopsis pilosula could protect the lung by regulating disease-related targets of ALI through multiple pathways and biological processes.

T-2 toxin is the most toxic trichothecene mycotoxin and can be present in many human foods and animal feeds. Currently, most of the studies related to the toxicology of T-2 toxin based on animal models are acute attack tests, but the contamination level of T-2 toxin in feed materials and livestock and poultry compound feeds is mostly low-level contamination. In this study, C57BL/6J mice were treated with 1 mg·kg^-1 BW T-2 toxin three times a week to assess its effects on the colonic barrier and the colonic flora. 100 mg·kg^-1 BW tannic acid (TA) was applied every day to ameliorate the toxicity of the T-2 toxin. T-2 toxin did not cause significant morphological damage and marked inflammatory responses in colonic tissue but significantly decreased the number of goblet cells in colon tissue (P<0.01). T-2 significantly downregulated the expression of mucoprotein Muc1 and the tight junction proteins Occludin, Claudin1, and ZO-1 (P<0.01). T-2 toxin changes the diversity of colonic flora in mice, the intervention of TA can make harmful bacteria in the colon of T2 group mice, such as Patescibacteria, Saccharimonadales, Candidatus-Saccharimonas, Odoribacter, Bilophila, and unclassified_f__Ruminoccaceae decreased significantly (P<0.05) or extremely significantly (P<0.01), while the abundance of beneficial microorganisms norank_f__Oscillospiraceae and Oscillibacter increased significantly (P<0.05) and extremely significantly (P<0.01) respectively. In summary, a low-dose T-2 toxin exposure will lead to the impaired colonic barrier and dysbiosis of colonic flora in mice, and TA treatment can alleviate T-2 toxin-induced colonic mucosal damage and improve intestinal health in mice.