CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) is a novel editing system, which is originally developed from the bacteria defense immune mechanism against viruses infection and plasmid transfer. As a simple, accurate, economical and efficient tool, this technology has been extensively used in the gene-editing and related research fields. The progress and application of CRISPR/Cas9 technology in pig genome editing in the past few years are summarized in this review, and also discusses its future perspectives in pig breeding and clinical trials.

In most female mammals, the migration of primordial germ cells, germ cells meiosis, and breakdown of germ cell nests are regarded as the 3 critical processes for the formation of early primordial follicles. In addition, there are some special factors and signaling pathways regulating the formation and development of primordial follicles. However, the reproductive ability of a female is determined by the size and reserve capacity of her primordial follicle pool. This review summarizes the factors and signaling pathways involved in the formation and development of primordial follicles, with the aim of understanding the cellular and molecular mechanisms and providing research ideas for maintaining the primordial follicle pool and promoting primordial follicle activation.

Busulfan has high toxic effect on the spermatogenesis and can cause male sterility. It is an ideal agent for the preparation of transplant recipients of spermatogonial stem cells (SSCs). Orchitis also has an important effect on the spermatogenic function and can even lead to male sterility. However, the mechanism by which busulfan damages the blood-testis barrier (BTB) and affects spermatogenesis is unclear. It is unknown whether it will cause non-infectious testicular inflammation, affect cytokine secretion, and impair fertility. Therefore, the damage effect of busulfan on the BTB, its effects on testicular cell-related functional proteins, and the relief methods of busulfan-damaged testis are summerized in this review. It deeply reveals the toxic effect of busulfan on the function and structure of testicular cells and BTB. Furthermore, we provide theoretical guidance for the efficient and safe preparation of SSCs transplantation recipients, and protection and recovery of male spermatozoa under the action of therapeutic and environmental toxins.

Newbury-1 virus, usually designated by Nebovirus (NeV), is the only member of the genus Nebovirus in the family Caliciviridae at present. NeV is a single-stranded positive-sense RNA virus with a genome length of 7 453-7 460 bp and is an important diarrhea-causing virus in calves. The detection of NeV mainly relies on RT-PCR and Real-time RT-PCR assays. To date, NeV has been detected in 13 countries including the USA, the UK, Brazil, Turkey, etc., suggesting that NeV has been widely distributed around the world. Recently, our studies suggested that the virus has been widely circulating among dairy cows and yak in China, presenting a unique evolution trend, and new genotypes have been determined. To provide a reference for NeV research, this review describes the latest research progress on NeV, including the biological characteristics, epidemiology, clinical symptoms and pathology, detection methods, prevention and control.

The study was conducted to clone the complete coding sequence (CDS) and transcript variants of porcine hnRNPUL1, and to analyze its tissue expression and subcellular localization. In this study, three 3-month-old Min pigs were used. The CDS and transcript variants of porcine hnRNPUL1 were cloned using RT-PCR method, and the expression was detected in tissues including heart, liver, spleen, lung, kidney, stomach, large intestine, small intestine, back muscle and leg muscle by qRT-PCR. At the same time, the subcellular localization was analyzed by inserting the fragments of CDS into pEGFP-N1 vector on the basis of bioinformatics prediction. It was found that the CDS of porcine hnRNPUL1 gene (GenBank accession No.:MN399154) was 2 580 bp of length, and the predicted polypeptide contained SAP, SPRY, and AAA domains, the characteristic domains of hnRNPs family. Porcine hnRNPUL1 was ubiquitously expressed in all detected tissues, with the highest level in the spleen but the lowest in leg muscle. The nuclear localization sequence(NLS) was located at 133-430 aa of the polypeptide chain, which was different from the result predicted by the bioinformatics methods. In addition, 2 splice variants and 11 alternative splicing fragments were identified. The results have provided the basis for further revealing the function and transcriptional regulation mechanism of porcine hnRNPUL1.

In this study, the long non-coding RNAs (lncRNAs)of sheep embryonic skeletal muscle were identified and analyzed to clarify its regulatory mechanism in myofiber switching and hypertrophy. Chinese Merino ewes with similar body weight were selected for simultaneous estrus and artificial insemination, and the fetal longissimus dorsi at the 85th(D85N), 105th (D105N), and 135th day (D135N) of pregnancy were sampled and sequenced by whole transcriptome sequencing technology. Three comparison groups (D85N vs D105N, D105N vs D135N, and D85N vs D135N) were set up, and the significantly differentially expressed lncRNA and mRNA were selected by comparison. Co-expression modules were constructed using the weighted gene co-expression network analysis (WGCNA) method. GO and KEGG enrichment analysis were performed using DAVID online tools and R-package to find modules related to muscle development. Finally, lncRNAs and mRNAs with high connectivity were selected from the target modules, and the lncRNA-miRNA-mRNA co-expression network was established through the predicted targeting relationship between mRNAs and miRNAs. A total of 25 modules were obtained according to the WGCNA analysis. The core genes in the related functional modules were mainly enriched in cell adhesion, Wnt, tight junction, mTOR, AMPK, and ECM-receptor signaling pathways. lncRNAs and mRNAs with high connectivity in modules were selected to establish a sub-network in which some genes such as TNNI2, PIP5K1A, PDK4, etc., and 10 lncRNAs (MSTRG.3903, MSTRG.10154, MSTRG.1629, MSTRG.10496, MSTRG.9559, MSTRG.10178, MSTRG.10521, MSTRG.3911, MSTRG.4586, MSTRG.7232) related to muscle development, muscle disease, and cell proliferation were found successfully. The lncRNA-miRNA-mRNA co-expression network related to myofiber development in Merino sheep was constructed, and several potential candidate genes related to embryonic skeletal muscle development in late pregnancy were found. All above results provided the solid basis for studying the regulatory mechanism of lncRNA during Merino sheep embryo development and references for the same study in other livestocks.

The aim of this study was to explore the relationship between goat HOX transcript antisense RNA (HOTAIR) and its partial family genes and skin pigmentation in goats as well as its expression pattern in the melanocytes. qRT-PCR was used to detect the mRNA expression level of HOTAIR, HOXD10, HOXC11 and HOXC12 genes in skin tissues of adult female Youzhou Dark-skin goats ((19.16±1.44) kg) and Banjiao goats ((23.27±3.24) kg). Then the expression pattern of these genes in mouse skin melanoma cells (B16-F10) at different proliferation and differentiation stages (1, 3, 5 and 7 d cultured) were also detected by qRT-PCR, and the relationship in expression level between HOTAIR and the other 3 genes was analyzed. The results showed that expression of HOTAIR and HOXC12 in the skin tissue of Youzhou Dark-skin goats were significantly higher than those of Banjiao goats (P<0.01), however, expression of HOXD10 in the skin tissue of Youzhou Dark-skin goats was significantly lower than that of Banjiao goats (P<0.05). Pearson correlation analysis indicated that expression level of HOTAIR was significantly positively correlated with HOXC12 (P<0.05), but significantly negatively correlated with HOXD10 (P<0.05). In the proliferation stage of B16-F10 cells, expression of HOTAIR was not significantly different (P>0.05) among different proliferation stages; The expression of HOXC11, HOXC12 and HOXD10 showed a pattern of "high-low-high", and their mRNA levels on 1 d cultured were higher than those on 3 and 5 d cultured (P<0.01); The expression level of HOTAIR was positively correlated with HOXC11 (P<0.01). At the differentiation stage of B16-F10 cells, expression of HOTAIR kept increasing during cell differentiation; Instead, expression of HOXD10 kept decreasing during this stage; Expression of HOXC11 was not significantly different (P>0.05) among different differentiation stages; Expression of HOXC12 on 1 and 5 d cultured were very significantly higher than those on 3 and 7 d cultured (P<0.01); Expression level of HOTAIR was significantly negatively correlated with HOXD10 (P<0.01). HOTAIR shows high expression level in the dark skin of goats, and its expression level remains increasing during differentiation in B16-F10 cells. HOTAIR has a negative regulatory effect on HOXD10. The interaction between HOTAIR and HOXD10 may affect skin pigmentation by regulating the differentiation of skin melanocytes in goats.

The aim of this study was to clone goat Smad3, to clarify its tissue and cell expression profiles, and finally to elucidate the effect of interfering Smad3 gene on the differentiation of goat intramuscular and subcutaneous adipocytes. In this study, 5 one-week-old Jianzhou Big-ear goats in good physical condition were selected, slaughtered after fasted for 24 h, and the corresponding tissues and cells were sampled for testing. The cDNA sequence of goat Smad3 gene was cloned by RT-PCR and analyzed by bioinformatics. Real-time quantitative PCR (qPCR) was used to detect the tissue and cell temporal expression of Smad3 gene. siRNA targeting to Smad3 was designed and synthesized. Oil red O staining was used to detect the effect of interfering Smad3 on adipogenic differentiation of goat preadipocytes, and qPCR was used to detect the effect of interfering Smad3 on the expression levels of adipocyte differentiation marker genes (C/EBPα, C/EBPβ, LPL, SREBP1, AP2, PPARγ, Pref-1, KLF3, KLF4, KLF6, KLF7, KLF8, KLF9, KLF10 and KLF15), Smads-related genes (Smad2, Smad4, Smad7) and TGF-β1 mRNA, with exploring the possible mechanism. The results showed that the goat Smad3 gene was 1 449 bp in length, the CDS region sequence was 1 278 bp, encoding 425 amino acids. Smad3 was widely expressed in goat tissues with the highest expression level in the kidney (P<0.01); Smad3 expression was lowest in goat intramuscular and subcutaneous adipocytes at 36 h of induced differentiation, which was significantly lower than that in undifferentiated precursor adipocytes (P<0.01). The interference of Smad3 significantly promoted the accumulation of lipid droplets in intramuscular and subcutaneous adipocytes of goats, the expression level of adipocyte differentiation marker genes, KLF3, KLF4, KLF8, KLF9, KLF10 and KLF15 were significantly increased (P<0.05), and the relative expression level of Pref-1 was extremely significantly decreased (P<0.01). At the same time, the interference of Smad3 gene down-regulated the relative expression levels of Smad2, Smad4 and Smad7 genes (P<0.05). The results indicate that interference of Smad3 promotes the differentiation of goat adipocytes by regulating the expression of adipocyte differentiation marker genes LPL, SREBP1, AP2, C/EBPα, C/EBPβ, Pref-1, KLF3, KLF4, KLF8, KLF9, KLF10 and KLF15 cooperating with Smad2, Smad4 and Smad7.

The aim of this study was to enhance the understanding of mammals' adaptability to high altitude hypoxia environment at the circRNA level and explore the differentially expressed circRNA and related regulatory networks in brain of Zang cattle and Sanjiang cattle, which would lay the theoretical foundation for further researching the molecular regulation mechanism of circRNA in hypoxia adaptation in Zang cattle. In this study, the brain tissues of three 4.5-year-old female healthy Zang cattles and Sanjiang cattles were sampled to construct the cDNA libraries for high-throughput sequencing. Bioinformatics methods were used to analyze host genes of circRNA by GO and KEGG, and predict the target genes of differentially expressed circRNA. At the same time, the circRNA-miRNA and circRNA-miRNA-mRNA visualization regulatory networks were constructed. Moreover, the reliability of sequence data was verified by RNaseR enzyme resistance assay and real-time quantitative PCR (qRT-PCR). The results showed that 858 significantly differentially expressed circRNAs were filtered in 6 samples of Zang cattle and Sanjiang cattle, included 394 up-regulated circRNAs and 464 down-regulated circRNAs. The host genes of these differentially expressed circRNAs were involved in 49 functional subclasses, involved in 31 significantly enriched signaling pathways (P<0.05), mainly included MAPK signaling pathway, glutamatergic synapse, Rap1 signaling pathway, phospholipase D signaling pathway, cGMP-PKG signaling pathway, etc.. The result of target gene prediction revealed that 350 up-regulated and 364 down-regulated differentially expressed circRNAs were able to target 492 and 508 miRNAs, respectively. Among them, the novel_circ_017825 (235) and novel_circ_046762 (231) had the most target points to miRNAs, indicated that novel_circ_017825 and novel_circ_046762 might play an important role in the function regulation of Zang cattle brain. The circRNA-bta-miR-2284z-mRNA regulatory network showed the targeting relationship between bta-miR-2284z and circRNA, mRNA. It was speculated that the above circRNAs might indirectly affect the related targeted mRNA expression levels in Zang cattle brain by acting as bta-miR-2284z sponge. In addition, the RNaseR enzyme resistance test and qRT-PCR were performed on the randomly selected 6 circRNAs. The expression trend was consistent with the sequence results, which indicated that these obtained circRNAs were the circular transcript.The expression profile and signaling pathways of circRNA in brain tissues of Zang cattle and Sanjiang cattle were obtained by high-throughput sequencing technology, and the interaction model of circRNA-miRNA-mRNA network was initially constructed, which provided reliable data for further researching the biological process and molecular function of circRNA in Zang cattle hypoxia adaptability, as well as the regulation mechanism of circRNA in hypoxia adaptability in large mammals.

The aim of this study was to screen CART receptor and clarify its expression characteristics in dominant follicles (DF) and subordinate follicles (SF). CART and proteins associated with CART were identified by using immunomagnetic Protein A/G Co-IP; The membrane proteins were predicted, transmembrane times were analyzed, GPCRs were obtained by HMMTOP V2.0; CART and screened GPCRs were modeled homologously by using SWISS-MODEL and PDB database, PDBQT file of model molecules was obtained, respectively. The quality of constructed model and each amino acid residue were evaluated by scoring function; Inputting CART and PDBQT file of receptors to be analyzed in ZDOCK interface for molecular docking, respectively, complex three-dimensional space model and score function value were obtained; Expression and localization of CMKLR1 in bovine DF and SF were analyzed by qRT-PCR and immunohistochemistry. One hundred and eleven proteins were obtained by Co-IP, which contained 10 membrane proteins (A2M, C5, CMKLR1, COX2, DDOST, HEATR5A, B3AT, ADT2, RPN2, SLC4A1); CMKLR1 had 7 transmembrane helix structures, which belonged to GPCRs; Molecular models of CART and CMKLR1 were constructed using SWISS-MODEL technology, complex three-dimensional space model was obtained by ZDOCK molecular docking, and the highest score function value was 1 977.34. qRT-PCR analysis showed that expression level of CMKLR1 mRNA in SF was significantly higher than that in DF (P<0.05); Immunohistochemical analysis showed that CMKLR1 was expressed in GCs and membranelayer cells layer of DF and SF, and specific color intensity showed that expression levels of CMKLR1 in SF GCs and membrane cells were higher than those in DF, which were consistent with qRT-PCR results. The results show that protein homology modeling and molecular docking techniques are feasible for receptor screening. CMKLR1, as a candidate receptor for neuropeptide CART, has a significantly higher expression level in SF than DF. This study is of great significance for the identification of CART receptors and for elucidating the mechanism of CART regulating bovine follicular development.

The objectives of this study were to evaluate the effects of platelet-activating factor (PAF) supplementation in in vitro maturation (IVM) medium on yak (Bos grunniens) oocyte development competence and gene expression. A total of 1 025 yak cumulus-oocyte complexes (COCs) were assigned to 4 groups. COCs in different groups were matured in IVM medium with different concentrations of PAF (0, 10^-8, 10^-7, and 10^-6mol·L^-1), followed by in vitro fertilization (IVF) with cattle sperm and in vitro culture (IVC). The maturation rate, cleavage rate and blastocyst rate of oocytes, and the relative expression level of pro-apoptosis (BAX), anti-apoptosis (BCL-2), epidermal growth factor (EGF) and its receptor (EGFR), transcription factors (OCT-4 and NANOG) and c-fos genes in COCs and embryos were compared among different groups. The results showed that oocytes in IVM medium with 10^-7mol·L^-1 PAF had higher maturation rate((92.16±0.19)%), cleavage rate((77.20±0.85)%) and blastocyst rate((46.71±0.68)%) than those in the other groups (P<0.05). The results of qPCR showed that the expression level of BCL-2, EGF, EGFR, c-fos were significantly up-regulated (P<0.05), but the expression level of pro-apoptotic gene (BAX) was significantly down-regulated in matured COCs (P<0.05) in 10^-7 mol·L^-1 PAF group. The expression level of EGF, EGFR and OCT-4 in blastocysts were significantly up-regulated in 10^-7 mol·L^-1 PAF group (P<0.05). The expression level of BCL-2, BAX and NANOG genes in 2-, 4-, 8-cell, morula and blastocyst stages were not different among different groups, but the expression of BCL-2 was down-regulated and BAX was up-regulated in morula and blastocyst. In conclusion, the optimal PAF concentration in IVM medium (10^-7mol·L^-1) could improve oocyte maturation and embryo development, and regulate the expression of apoptosis and proliferation related genes of the yak.

The aim of this study was to assess the nutritional value of solid-state fermentation rapeseed meal(FRSM) by metabolic test in growing-finishing pigs. Sixteen healthy Duroc×Landrace×Yorkshire crossbred barrows with initial body weight of (71.25±1.23) kg were randomly assigned to 4 dietary treatments,with 4 replicates per treatment and 1 pig per repeat. The 4 diets were corn-soybean meal basal diet, rapeseed meal (RSM) diet, fermented rapeseed meal (FRSM) diet (35% nitrogen(N) of basal diet were substituted by RSM or FRSM) and N-free diet. In order to evaluate the nutritional value of fermented rapeseed meal, the metabolic trial lasted for 7 days, the adaptation period was 3 days and the formal period was 4 days.All feces and urine were collected during formal period in order to determine the digestibility of nutrients. Results showed that:1)The pH of fermented rapeseed meal mixture decreased by 1.68 after fermentation, and the contents of crude protein(CP), water-soluble protein(WSP), acid-soluble protein(ASP) and crude fat(EE) increased by 1.91%, 52.88%, 44.40% and 24.27%, respectively; The contents of 17 amino acids were increased. The neutral detergent fiber(NDF) of FRSM decreased by 5.21%; Isothiocyanate(ITC) degradation rate was 92.20%, oxazolidinone(OZT) degradation rate was 100.00%; Tannin and phytic acid decreased by 35.81% and 24.22%, respectively. 2) The apparent digestibility of dry matter of FRSM was 75.28%, which was significantly higher than that of RSM (P<0.05);Digestible energy, nitrogen apparent (true) digestibility and nitrogen apparent (true) availability of FRSM were 13.84 MJ·kg^-1, 70.89% (72.84%) and 68.12% (71.12%), respectively, which were all extremely significantly higher than those of RSM(P<0.01). The apparent digestibility of calcium, phosphorus and crude ash were not significant different between FRSM and RSM (P>0.05). 3) All amino acids (except cysteine) apparent (true) digestibility of FRSM were significantly or extremely significantly higher than those of RSM (P<0.05 or P<0.01). In summary, by solid state fermentation of RSM with two bacteria, the content of anti-nutritional factors were reduced, the nutrient digestibility and utilization rate of rapeseed meal were increased, and the feeding value of rapeseed meal was effectively improved in growing-finishing pigs.

This study was conducted to investigate the effects of coated methionine (CM) and coated folic acid (CFA) supplementation and their interactions on growth performance, nutrients apparent digestibility, energy and nitrogen metabolism, and synthesis of rumen microbial protein in finishing Doper×Thin-tailed Han F₂ crossbred male lambs. Forty Doper×Thin-tailed Han F₂ crossbred male lambs with similar body weight ((30.08 ±1.34) kg) and good condition were arranged into 4 groups in a randomized design with a 2×2 factorial arrangement. One factor was supplemented CFA (0 mg,CFA-or 4 mg·(kg·d)^-1,CFA+) in the diet according to the body weight of the experimental animals,and the other factor was supplemented CM (0 g,CM-or 0.1 g·(kg·d)^-1,CM+). The feeding experiment lasted for 85 days with 15 d of adaptation and followed by 70 d of trial period. A digestibility trial was performed 10 days before the end of the trial period. The results showed that:1) Average daily weight gain and feed conversion ratio were significantly improved during 1-30 d with CFA supplementation in the ration of finishing lambs(P<0.05); There was a significant effect of CM supplementation on increasing feed conversion ratio (P=0.036), but no significant effect on average daily weight gain (P>0.05) during 1-30 d;Average daily weight gain and feed conversion ratio were significantly increased during 31-60 d and 1-60 d with CFA or CM supplementation in the ration for finishing Doper×Thin-tailed Han F₂ crossbred male lambs (P<0.05). The significant interaction between CFA and CM supplementation was found in feed conversion ratio during 1-60 d (P=0.007). 2) The apparent digestibilities of dry matter, organic matter, neutral detergent fiber and acid detergent fiber were significantly increased by adding CFA in the ration (P<0.05), the apparent digestibility of organic matter was tended to increase with CM added in the ration (P=0.066), and the apparent digestibility of dry matter was tended to increased (P=0.095). There was no significant interaction effect on the apparent digestibility of the nutrients (P>0.05). 3) The addition of CFA or CM significantly decreased urinary energy loss, enhanced digestibility and metabolizability of energy (P<0.05). The apparent digestibility of nitrogen and the deposited nitrogen were significantly increased (P<0.05) by significantly reducing fecal nitrogen, urinary nitrogen and total excretion nitrogen (P<0.05). A significant interaction effect between CFA and CM addition was found in reduction of nitrogen excretion and elevation of nitrogen digestibility (P<0.05). 4) The addition of CFA or CM significantly increased the urinary excretion of purine derivatives and the synthesis of rumen microbial protein (P<0.05). The results showed that the addition of CM or CFA improved the digestibility of dry matter and organic matter, enhanced digestibility and metabolizable ratio of total energy, increased apparent digestibility of nitrogen and nitrogen deposition, thus promoted the growth of Doper×Thin-tailed Han F₂ crossbred male lambs.

This experiment was conducted to study the effect of feeding level on growth performance, serum biochemical parameters and body energy deposition of Arctic foxes during the growth period. Forty-six 85-day-old healthy male Arctic foxes with average body weight of(3 198±281)g were selected, including 6 Arctic foxes as slaughter trial control at the beginning of the trial, another 40 Arctic foxes randomly divided into 4 groups with 10 replicates per group and 1 fox per replicate. The foxes were offered diet for ad libitum (AL) (Group I), 80% (IR 80) (Group II), 60% (IR60) (Group III) and 40% (IR 40) (Group IV) of ad libitum intake, respectively. The experiment was 7 days for adaption and 55 days for trial period. The parameters of growth performance, serum biochemical parameters and body energy deposition were evaluated by means of feeding, serological and slaughter trials and chemical analysis methods. The results showed as follows:1) 100-day-old body weight in group IV was extremely significantly lower than those in group I and II (P<0.01), and no significant difference was found between group IV and III (P>0.05). Body weight at the age of 115,130,145 days and average daily gain(ADG) in group IV were extremely significantly lower than those in the other groups(P<0.01),whereas no significant difference was detected among the three groups (P>0.05). With the decrease of feeding level, average dry matter intake (ADFI) showed a extremely significant decrease trend (P<0.01). Feed to gain ratio (F/G) in group III was extremely significantly lower than those in group I and II(P<0.01), whereas no significant difference was found in F/G between group III and IV(P>0.05). 2) Serum glucose(GLU) in group IV was obviously higher than that in group III(P<0.05), whereas no significant difference was found among group I, II and IV(P>0.05). Serum CHO and LDL-C in group IV were extremely significantly higher than those in the other groups(P<0.01), whereas no significant difference was found among group I, II and III(P>0.05). Serum TP and ALB in group IV were significantly higher than those in group I and III(P<0.05), whereas no significant difference was found between group II and IV(P>0.05). There were no significant effects on serum TG, HDL-C, GLOB, IgA, IgM,IgG,C3, C4 and INS contents within different feed intake levels(P>0.05).3) With the decrease of feeding level, pelt fat deposition and its energy deposition had significantly decreased trend, which was significantly higher in group I and II than those in group III and IV(P<0.05). Pelt weight gain and total energy deposition in group II were significantly higher than those in group IV(P<0.05), whereas no significant difference was found among group I, II and III(P>0.05). Carcass weight gain in group IV was extremely significantly lower than that in the other groups (P<0.01), whereas no significant difference was found among group I, II and III(P>0.05). There were no significant effects on the pelt protein deposition and its energy deposition, carcass fat deposition and its energy deposition, carcass protein deposition and its energy deposition as well as total energy deposition of carcass fed different feed intake levels(P>0.05). Feeding 60% of ad libitum intake (IR60) reduces the contents of serum GLU and lipid parameters, ensures the normal growth and health state of body, improves the feed efficiency, thus increases the benefit of breeding production of Arctic fox during the growth period.

The guanine nucleotide binding protein Gα12 is involved in the regulation of various cellular responses, but there are few studies on foot-and-mouth disease virus (FMDV). In this study, we had used FMDV to infect PK-15 cells, and found that the mRNA and protein levels of Gα12 were up-regulated, indicating that Gα12 may have some relationships with FMDV replication. To investigate the effect of Gα12 on the replication of FMDV, quantitative real-time PCR, Western blot and TCID₅₀ were used to detect the effect of overexpression of Gα12 and knockdown of Gα12 on FMDV replication. The results showed that the mRNA level, protein level and virus titer of FMDV were decreased after overexpression of Gα12. However, when knockdown Gα12, the replication of FMDV showed the opposite effect, indicating that Gα12 inhibited the replication of FMDV. This study found for the first time that Gα12 has the effect of inhibiting the replication of FMDV, and provides a new idea and theoretical basis for the study of anti-FMDV, which is of great significance for FMVD-related research.

It is difficult to distinguish the clinical symptoms caused by Seneca virus A (SVA) and foot-and-mouth disease virus (FMDV), and the study of inserting foreign genes of FMDV epitope with SVA as avector has not been reported at present. This paper aims to construct a recombinant SVA strain with chimeric FMDV epitopes and identify it. The genes encoding B cell epitopes of type O FMDV and albumin signal peptide (Human albumin signal peptide, HAS) were inserted into the SVA genome by gene cloning technique. The recombinant plasmid was successfully constructed and the recombinant virus was rescued after transfection. The results of RT-PCR and gene sequencing showed that the target gene was inserted correctly, and the effective expression of chimeric antigen protein was identified by Western blot and cellular indirect immunofluorescence. Genetic stability analysis showed that the recombinant virus still had stable B cell epitopes during the passage of 25 generations. The results of plaque phenotype and one-step growth curve of the virus showed that the proliferation characteristics of the recombinant virus were similar to those of the parent strain. In this study, the recombinant SVA virus which can express FMDV epitopes was successfully constructed and rescued, which provides a theoretical and experimental basis for the research of chimeric virus and vaccine based on SVA.

A competitive and quantitative chemiluminescent enzyme immunoassay (CLEIA) for rapidly detecting antibody against gB protein of Pseudorabies Virus(PRV) was established by using the purified recombinant gB protein expressed in E. coli as coating antigen and horseradish peroxidase labelled monoclonal antibody (MAb) against gB protein as enzyme labelled antibody, and calibrator diluted from national reference to draw standard curves for quantitative detection. The successfully established PRV-gB-CLEIA which could complete the test within 45 min had no cross-reaction with the standard positive serum of other five viral antigens such as swine fever and could detect the national reference with the maximum dilution ratio of 1:2 048. The coefficient variation of intra-assay was between 1.13% and 9.47%, and inter-assay between 2.43% and 14.07%. By comparing the detection results of 180 clinical serum samples collected, the positive coincidence rate between the method and neutralization test was 94.00%, and the negative coincidence rate was 96.92%, and the total coincidence rate was 96.11%, which was obviously superior to commercial ELISA kit. The PRV-gB-CLEIA established in this study could be used for the rapid and quantitative detection of PRV gB antibody.

This experiment was conducted to study the epidemiological and phylogenetic characteristics of swine pseudorabies virus (PRV) currently circulating in China. A total of 1 328 samples from pigs with suspected PRV infection were collected from 28 provinces of China in 2018 for PRV-gE detection by PCR and virus isolation; DNA sequencing was also performed on the gB, gC and gE genes of the PRVs isolated herein and the sequences were used for DNA alignments as well as phylogenetic analysis. The results showed that:1) A total of 92 samples collected herein were positive for PRV-gE by PCR, with an average positive detection rate of 6.93%. Thirteen PRV strains were finally isolated from these 92 PRV-gE positive samples; 2) Phylogenetic analysis on either gB, gC and/or gE showed that the 13 PRVs isolated herein were close to the epidemic PRV strains in China after 2012 but were not close to those circulating in China before 2012; most of the PRV strains isolated from China were located on a different evolutionary branch with those from the other countries such as NIA-3, Bartha, and Kaplan; 3) There were so many amino acid changes with the gB, gC and/or gE proteins in the 13 PRVs isolated herein compared to those in the viruses isolated from the other countries as well as those isolated from China before 2012. These results indicated that the PRVs currently circulating in China were mainly the variant strains that showed different phylogenetic and genetic characteristics from those prevalent in China before 2012 as well as those circulating in the other countries, representing a serious problem.

To explore the effects of macrophage migration inhibitory factor of Psoroptes ovis (PoMIF) on Th1/Th2 and Th17/Treg balance of rabbit peripheral blood mononuclear cells (PBMC) in vitro, the PoMIF gene was amplified from the extracted total RNA from P. ovis mites by the reverse transcription-polymerase chain reaction (RT-PCR), and the recombinant protein rPoMIF was expressed with prokaryotic expression system, then the oxidoreductase and tauromerase activities of rPoMIF was evaluated. PBMCs isolated from healthy New Zealand rabbits were cultured with different concentrations of rPoMIF, and the optimum concentration of rPoMIF was screened. Finally, the expression levels of Th1, Th2, Th17 and Treg-specific transcription factors of T-bet, GATA-3, RORc, Foxp3 and specific cytokines of IFN-γ, IL-4, IL-17A and IL-10 in PBMC incubated with the optimal concentration of rPoMIF for 0, 1, 6, 12, 24 and 36 h were detected by real time fluorescence quantitative PCR (qPCR). The results showed that the full-length of PoMIF was 363 bp, containing 357 bp open reading frame; rPoMIF was mainly in the supernatant of Escherichia coli (E. coli) lysate, with a molecular size of 32 kD (including an extra 19 kD for the attached His-tag fusion peptide); and it has tautomerism activity; the mRNA levels of Th1-specific T-bet and IFN-γ decreased firstly and then increased after treatment, while Th2-specific GATA-3 and IL-4 decreased at all tested time points, and the ratio of T-bet/GATA-3 and IFN-γ/IL-4 increased at 12, 24 and 36 h; the mRNA levels of Th17-specific RORc and IL-17A decreased at all tested time points, while Foxp3 and IL-10 from Treg cells increased at all tested time points, and the ratio of RORc/Foxp3 and IL-17A/IL-10 decreased at all tested time points. In conclusion, rPoMIF can cause the shifts of Th1/Th2 and Th17/Treg towards Th1 and Treg in PBMC of rabbits.

This study was conducted to reveal the resistance mechanism of Staphylococcus aureus to β-lactams, which may associate with thickening of the cell wall. From 2016 to 2018, we collected milk samples from clinical and subclinical mastitis in some dairy farms in Ningxia region. Separation and identification of S. aureus were conducted by using chromogenic medium, microscopic examination and PCR. The resistance in S. aureus isolates to 14 antibacterials was detected by micro-dilution method, to understand the resistance rate and multi-drug resistance of S. aureus isolates in the region. Transcription levels of pbpB, murG, glmU and atlR genes that are related to cell wall thickening were detected by qRT-PCR. Furthermore, phenotypes were examined by the transmission electron microscopy. Our goal was to discover the mechanism of cell wall thickening. The results indicated that we isolated and identified 261 strains of S. aureus, including 9 strains of Methicillin-resistant Staphylococcus aureus (MRSA). Antimicrobial susceptibility determination showed that the isolates have high resistance rate to β-lactams, the resistance rate to ampicillin was 79.69% and that to penicillin was 78.54%. Multi-drug resistance was distributed by isolates with 3, 7 and 8 resistance, in particular, one of the isolates could tolerate 14 antibacterials. The results of qRT-PCR showed that 4 related genes were significantly up-regulated (P<0.01 or P<0.001). The cell wall of JY21 (Methicillin sensitive Staphylococcus aureus, MSSA) was significantly thicker than the control group at the concentration of 64 and 128 μg·mL^-1 penicillin (P<0.001), and the cell wall was rough with nodular processes. Whereas, with the penicillin concentration increased from 64 μg·mL^-1 to 128 μg·mL^-1, and the cell wall was no longer significantly thickened (P>0.05). MRSA WLD10 showed no significant cell wall thickening (P>0.05). In conclusion, these data illustrated that cell wall thickening is a resistance mechanism for S.aureus to β-lactams in Ningxia. Simultaneously, the reason for cell wall thickening is mainly the excessive synthesis of peptidoglycan and the decrease of cell autolysis. Cell wall thickening is a critical resistance mechanism for the MSSA JY21. However, for MRSA WLD10, it's not the case.

The aim of this study was to investigate the morphological changes of hypothalamic cells in pre-puberty mice exposed to electromagnetic radiation at 1.8 GHz. The trial was randomly divided into three groups and each group consisted of 30 repetitions. Microwave irradiation frequency we used was 1.8 GHz and three groups were treated respectively on 0 (Control group), 1 (Low dose test group) and 2 mw·cm^-2 (High dose test group). Radiation was made on maternal mice during the entire period of gestation and lactation, and on the offspring until the day of postnatal 30. The mental state of mice were observed. The body mass, the cerebral mass of mice was recorded. Brain samples were collected at 30, 45 and 60 days of age. The histological changes of the hypothalamus were observed. The contents of neurotransmitter glutamate (Glu), aspartic acid (Asp), γ-aminobutyric acid (GABA) and glycine (Gly) in the hypothalamus were detected by HPLC. Results were as follows:(1) At the age of 30 days, the results showed that the body weight and brain weight in two experimental groups were lower than that of the control group (P<0.05), and the content of Asp, Glu, Gly and GABA in the brain of mice increased. Changes of ultrastructure including vacuolization in the hypothalamic cells, intracellular mitochondria edema, and dilated vascular space. (2) At the age of 45 days, the morphology of the cells in the low-dose test group recovered, but mitochondrial swelling still existed. Besides, the level of Asp and Gly in the brain decreased (P>0.05), and the level of Glu and GABA increased (P>0.05). In the high-dose test group, massive atrophic cells and swollen intracellular mitochondria still could be observed, and the content of Asp, Glu, Gly and GABA in the brain increased (P<0.05). (3) At the age of 60 days, the morphology of the cells in the low-dose group recovered. The mitochondria returned to normal, and there was no significant difference of the content of Asp, Glu, Gly and GABA in the brain between two groups (P>0.05). In the high-dose group, the hypothalamic cells basically recovered. Swollen mitochondria was nearly repaired and the nuclear was the normal appearance. The concentration of Asp in the brain decreased, and the concentration of Glu, GABA, and Gly increased. Overall, radiation damages the hypothalamic cells in mice, and these damages could be restored after radiation stops.

The aim of this study was to discuss the effect of hypoxia-inducible factor 1α(HIF-1α) on renal tubular epithelial-to-mesenchymal transition (EMT) in yak. First of all, the primary culture and identification of yak renal tubular epithelial cells were conducted. Then the third generation cells were chosen to be treated with drugs (DMOG group) and untreated (control group). The expression changes of EMT related genes were detected at both mRNA and protein levels, and the morphological changes of cells were observed. The results showed that renal epithelial cells could be better obtained by using the collagenase Ⅰ+Ⅱ; Immunofluorescence results showed that CK18 expression was positive, but Vimentin and CD31 were negative; The results of qRT-PCR and Western blot showed that mRNA and protein expression levels of HIF-1α, TGF-β1 and α-SMA in DMOG group were extremely significantly higher than those in control group (P<0.01), while the expression of E-cadherin was significantly lower than that in control group (P<0.05). After drug intervention, the cell morphology changed from paving-stone shape to long shuttle, which was identified as mesenchymal cells. Therefore, this study had successfully established a method for isolation and culture of yak renal tubular epithelial cells, and proved that the up-regulation of HIF-1α expression could promote the renal tubular epithelial-to-mesenchymal transition of yak.

To study the effects of Zhukuqin compound extractive fluid on intestinal intraepithelial lymphocyte (iIELs) and the transcription of INF-γ and IL-4 mRNA in the duodenum of diarrhea mice. A total of 72 mice were randomly divided into six groups equally:control group, infection group, positive drug group (Astragalus polysaccharide oral liquid, the concentration was 1.2 mg·mL^-1), and drug group Ⅰ, Ⅱ, Ⅲ (Zhukuqin extractive fluid, the concentrations were 0.1, 1 and 10 mg·mL^-1, respectively). After continuously pre-administration for 7 days (0.5 mL, once daily dosing), except the control group, the mice in each group were intraperitoneally injected with E. coli (5×10⁷CFU). The time point was selected as 0 h when mice appeared diarrhea in the infection group. Six mice were killed for preparing pathological sections, the number of iIELs in the small intestine was counted, and the mRNA level of INF-γ and IL-4 in the duodenum were analyzed by quantitative real-time PCR at 6 h and 4 d, respectively. The diarrhea protective rate of positive drug group and drug group Ⅰ, Ⅱ, Ⅲ were 66.7%, 50.0%, 66.7%, and 75.0%, respectively. At 6 h, compared with control group, the number of iIELs in infection group was significantly increased, and the mRNA level of INF-γ and IL-4 were observably reduced and strengthened, respectively (P<0.01); Compared with infection group, the number of iIELs in positive drug group and drug group Ⅰ, Ⅱ, Ⅲ were decreased, and the mRNA level of INF-γ and IL-4 in positive drug group and drug group Ⅲ were augmented and lessened, respectively. At 4 d, compared with control group, the quantity of iIELs in the duodenum and ileum in infection group were markedly heightened, while that in the jejunum was dropped, and the mRNA level of INF-γ and IL-4 were dramatically enhanced (P<0.01); Compared with infection group, the number of iIELs in the duodenum and ileum in positive drug group and drug group Ⅰ, Ⅱ, Ⅲ were significantly diminished (except that in duodenum of group Ⅰ), and that in the jejunum was aggrandized, and the mRNA level of INF-γ and IL-4 in positive drug group and drug Ⅲ group were decreased. The results of the current study show that Zhukuqin compound extractive fluid can protect the diarrhea mice, via regulating the number of iIELs in the small intestine and balancing the transcription of INF-γ and IL-4 mRNA in the duodenum of mice.

We used RNA-seq to measure the transcription differences in renal tissues between gout and healthy goslings. Five goslings with typical visceral gout characteristics were selected as "gout group", while six healthy goslings were selected as the "control group". As a result, the transcription expressions of gout goslings in renal tissues were significantly different from that of healthy goslings. A total of 1 749 differential expression genes were found. Among these, 501 genes were up-regulated and 1 248 genes were down-regulated in the gout group. The GO and KEGG search revealed the differential expression genes mainly enriched in chemokine activity, T cell receptor binding, antigen binding and immune response, and participate in processes of cytokine-cytokine receptor interaction, intestinal immune network for IgA production, cell adhesion and so on.

Cholangiocellular carcinoma is a malignant tumor derived from hepatic bile duct epithelial cells, which occurs in many species. In this study, two cases of canine liver tumors were diagnosed by histopathology and immunohistochemistry. Macroscopic of liver tumor is a spherical white mass, histopathological examination the tumor tissue consists of tumor cells arranged in a glandular tube, in the center of the gland have some necrotic and exfoliated cells,and some have mucus-like substances, the ducts are separated by thin fibrous connective tissue, and the tumor tissue has large area necrosis. The tumor cells are mostly oval, the nucleus is large, oval, and mitotic image is more common, CK19 was positively expressed in tumor cells, while CK18 and AFP were negatively expressed. According to the results of histopathology and immunohistochemistry, 2 cases of canine liver tumors were Cholangiocellular carcinoma, this study accumulated pathological data for the diagnosis of canine liver tumors.