Mitochondria are an organelle that widely exists in eukaryotic cells and provide energy for the body. Mitophagy is of great significance on clearing dysfunctional mitochondria, maintaining mitochondrial dynamic equilibrium and cellular homeostasis. In recent years, the effect of mitophagy on female reproduction in animals received increasing attention. Based on the physiological function of mitophagy, the effect of mitophagy on female reproductive function in the aspects of main reproductive organs and tissues, oocytes, granulosa cells and embryos of female animals were reviewed in this article.

Herpesvirus is a type of double-stranded DNA virus with a capsule structure, which is mainly composed of a double-stranded DNA genome, capsid, tegument, and envelope. At present, more than 100 types of herpes virus have been found, which can be divided into α-, β-, γ-herpesvirus subfamilies. It infects a wide range of hosts, such as amphibians, birds, mammals, primates and humans, and so on. It mainly damages the skin, mucous membrane, and nerve tissue, and seriously affects the health of human and other animal. Infected cell protein 22 (ICP22) is a multifunctional protein encoded by US1 or its homologous genes and can interact with cells and/or viral components to exert a wide range of functions. It can be involved in the establishment of latent infection period of viruses, the interaction with RNA polymerase Ⅱ (RNA Pol Ⅱ) to affect the transcription of viral and host genes, the formation of viral-induced chaperone-enriched (VICE) regions, and the nuclear budding of progeny viral particles, involving apoptosis, autophagy, and antiviral response, etc. This article reviews the research progress of herpes virus US1 and its homologous genes, and the encoding protein ICP22. We hope it will provide new ideas and directions for further research on the interaction mechanism of ICP22 with other virus and host proteins involved in physiological and pathological processes, and we also hope it will provide a certain reference value for the research of animal herpes virus, especially the new drugs targeting ICP22.

Melanogenesis is regulated by many genes and miRNAs. This study aimed to explore the effect of interaction between miR-186-5p and α-MSH on melanogenesis in sheep melanocytes. The miR-186-5p expression vector was transfected into sheep melanocytes and α-MSH was also added to the cells at the same time. Expression patterns of associated genes(MITF, TYR, TYRP1, TYRP2) were detected by qRT-PCR and Western blotting. The expression and localization of MITF protein in sheep melanocytes were further detected by immunohistochemistry. The melanin contents and the migration capability of melanocytes were measured by spectrophotometry and scratch test, respectively. Three experiment groups were set up, containing miR-186-5p transfected group, miR-186-5p+α-MSH interaction group and negative control group, and there were 3 replicates in each group. The results showed that the expression of MITF, TYR, TYRP1 and TYRP2 were down-regulated, the production of melanin was inhibited in miR-186-5p transfected group, while the above effects of miR-186-5p were alleviated by adding α-MSH to the cells. In addition, miR-186-5p also could inhibit cell division and block cell migration, but α-MSH could not affect the negative effects of miR-186-5p. In summary, the results indicated that in sheep melanocytes, both miR-186-5p and α-MSH were involved in regulating melanogenesis.Between them, miR-186-5p mainly played a negative regulatory role, while α-MSH was involved in the positive regulation of related genes and relieved the inhibitory effect of miR-186-5p. It was worth noting that miR-186-5p played an important role in regulating cell proliferation and migration, while α-MSH did not participate in the related regulation.

The objective of this study was to investigate the change regularity of production performance, milk composition, blood physiological and biochemical indexes and nutrient intake and relationship among them in Xinong Saanen dairy goat during lactation period. Fifteen healthy Xinong Saanen dairy goats with similar body weight, parity, milk yield and delivery date were selected. After 54 weeks of single fold feeding, the production performance, milk composition, blood physiological and biochemical indexes, nutrient intake for each individual were accurately measured during the experiment period, and the change regularity model of each index along with lactation period and the correlation model between indexes during lactation period were established. The results showed that:1) The production performance (DMI, milk yield, feed to milk ratio), milk composition (milk protein percentage, milk fat percentage, TS) and nutrient intake (EE, CP, OM, ADF, NDF) were significantly affected by stage of lactation (P<0.05). 2) The change regularity of milk yield, milk fat percentage and milk protein percentage along with lactation period were successfully fitted through Wood model; CP was fitted by the quadratic curve; The DMI, feed to milk ratio, SNF, TS and NDF were fitted by the cubic curves. The best fitting results were obtained for milk yield, milk fat percentage and feed to milk ratio, their degree of fitting was above 0.94. 3) For production performance, DMI, milk yield and feed to milk ratio were extremely significantly correlated (P<0.01); For milk composition, milk protein percentage, milk fat percentage, SNF and TS were extremely significantly correlated (P<0.01); For nutrient intake, DMI, OM, CP, NDF and ADF were extremely significantly correlated (P<0.01). Significant correlation indexes could be fitted by quadratic curve, cubic curve, linear model, exponential model and S curve. The average annual milk yield of Xinong Saanen dairy goat was 504.91 kg. The production performance, milk composition, blood physiological and biochemical indexes and nutrient intake of dairy goats were significantly correlated, and were also significantly affected by lactation period. The lactation curve fitted by Wood model was Y=1.233t^0.414×e^-0.041t. Feed to milk ratio could be predicted by milk yield according to the fitting curve, and milk fat percentage, milk protein percentage and TS content could be predicted by each other. Our findings are of great significance to understand the basic physiological status and production rules of dairy goats, and provide an important guidance for the rational production, accurate feeding and efficiency improvement of dairy goats at different lactation stages.

This study aimed to estimate the genetic parameters of milk production and type traits for Chinese Holstein in Hebei province and to provide reference for breeding programs. Based on 8 891 primiparous Chinese Holstein in 133 farms in Hebei province from 2012 to 2018, the genetic parameters for 3 milk production traits (milk yield, milk fat percentage, milk protein percentage) and 26 type traits were analyzed by using AIREML and EM algorithm of the DMU software combined with animal model considering herd-year-season, first calving age, identification year-season and appraisers as fixed effects, and individual additive genetic effect as random effect. The results showed that the heritability estimates ranged from 0.15 (milk yield) to 0.32 (milk fat percentage) for milk traits and from 0.01 (final score) to 0.28 (rear attachment width) for type traits. The estimated genetic correlation coefficients between milk production and type traits ranged from -0.43 (milk yield and bone quality) to 0.31 (milk yield and rump), -0.59 (milk fat percentage and rump) to 0.20 (milk fat percentage and fore teat placement) and -0.34 (milk protein percentage and fore teat length) to 0.23 (milk protein percentage and rear teat placement). The genetic correlations of most type traits with milk yield were positive, but negative with milk fat percentage and milk protein percentage. Strengthening the selection of milk production traits and type traits with medium and high heritability, especially for final score and rear udder traits,is beneficial to the improvement of production performance.

To screen and verify the miRNA regulating the expression of cattle CEBPA (CCAAT/enhancer binding protein alpha, CEBPA) and their effects on the proliferation and differentiation of intramuscular precursor adipocytes. In this study, multiple online softwares were used to screen miRNA with potential regulation relationships with CEBPA genes. qRT-PCR technology was used to detect CEBPA gene expression levels in various tissues of Guanling cattles. By constructing CEBPA-3'UTR-WT and CEBPA-3'UTR-MUT dual luciferase reporter vectors, and then it was used to verify the regulatory relationship between candidate miRNAs and CEBPA genes. After Guanling cattle intramuscular preadipocytes were isolated and cultured, qRT-PCR, Western blot, Oil Red O staining and CCK-8 were used to explore the effect of miRNA regulating CEBPA on the differentiation and proliferation of bovine intramuscular preadipocytes. Bioinformatics analysis revealed that bta-miR-23a, bta-miR-23b-3p, bta-miR-181a, bta-miR-181b, bta-miR-181c, bta-miR-181d and CEBPA genes had potential regulating relationship. CEBPA gene was expressed in the visceral tissues, muscle tissues, fat, hypothalamus and part of the reproductive organs of Guanling cattles, the expression level in the longissimus dorsi of 2-year-old cattle was significantly higher than that in 1 day old calf (P<0.01). The results of the dual luciferase reporter carrier system indicated that bta-miR-23a, bta-miR-23b-3p, and bta-miR-181a could regu-lation the cattle CEBPA gene 3'UTR; Cell test results showed that the expression level of CEBPA gene in the differentiation of cattle intramuscular precursor adipocytes gradually increased with time, and bta-miR-23a, bta-miR-23b-3p and bta-miR-181a showed extremely significantly higher expression levels at 0 d differentiation than other periods (P<0.01), the expression trends of bta-miR-23b-3p and bta-miR-181a were opposite with CEBPA gene during the whole differentiation period; Overexpression of miR-23a, miR-23b-3p, miR-181a could extremely significantly reduce CEBPA gene mRNA and protein expression (P<0.01), and reduce the content of lipid droplets in cattles intramuscular precursor adipocytes. bta-miR-23a, bta-miR-23b-3p, bta-miR-181a could promote cell proliferation in stages. The results indicate that bta-miR-23a, bta-miR-23b-3p, bta-miR-181a can promote the proliferation of intramuscular precursor adipocytes of cattles in stages, and indirectly affect the differentiation of this cell by directly negatively regulating the CEBPA expression.

The aim of this study was to clone and analyze the complete coding region sequence of TYR gene, further reveal the relationship between SNPs and its mRNA differential expression level in skin and coat color phenotype in mink (Neovison vison). A total of 301 blood samples of 7-month-old male minks (Jinzhou black, Mingwei silverblue and Red eye white) were collected, and the polymerase chain reaction (PCR) method was used to clone 5 exons of the mink TYR gene in sections and the complete coding sequence (CDS) was spliced. The PCR products were directly sequenced to screen SNPs in exon 1, 4 and 5 of TYR gene in minks with three kinds of coat color phenotypes. The back skin tissues of 9 7-month-old male minks (3 black, gray and white coats each) were collected, and quantitative real-time PCR (qRT-PCR) technology was used to detect TYR mRNA expression levels in skin of minks with 3 coat colors. The results showed that the mink TYR gene sequence was 2 391 bp in length, containing 5 complete exons, the full-length coding region was 1 596 bp, which encoded 531 amino acids containing signal peptide(18 amino acids) and mature peptide(513 amino acids). The sequence had been submitted GenBank database and its accession number was KJ716783. SNPs analysis showed that there was no mutation site in exon 4 and 5 of TYR gene, and two mutations were found in exon 1, c.441G>A and c.138T>A. However, c.441G>A was synonymous mutation and existed only in Jinzhou black mink population. The qRT-PCR results showed that TYR mRNA was expressed in the skin of all the three kinds of coat color types of minks, and its expression level in the Jinzhou black mink was extremely significantly higher than that in Mingwei silverblue and Red eye white mink (P<0.01). The TYR mRNA expression level in Mingwei silverblue mink was significantly higher than that in Red eye white mink (P<0.05). The results of present study indicated that the c.138T>A locus and mRNA expression levels of TYR gene might be associated with the coat color phenotype of mink.

This study aimed to compare the changes in tissue morphology and the expression of coding genes in the rumen tissues of the Bactrian camel between embryonic and adult stages, to discover the key genes and pathways affecting the development of rumen of the Bactrian camel, and to explore the desert adaptive mechanism of the Bactrian camel from the digestive system. Three adult Alxa Bactrian camels aged 10-12 years old with good health and three embryonic Alxa Bactrian camels aged 9-10 months old with good health were selected to make paraffin tissue sections, observe the rumen of adult and embryonic Bactrian camels, and compare the differences in tissue structure. The total RNA was extracted from rumen tissue of adult and embryonic Bactrian camels, RNA-Seq analysis was performed by Illumination Hiseq 2000 platform, the RNA-Seq data were performed quality-controlled, alignment, differential genes screening, GO and KEGG analysis. Six differentially expressed genes were randomly selected for RT-qPCR, the results of RNA-Seq and RT-qPCR was compared. The results showed that epithelial cells and muscle fiber cells were clearly visible and densely distributed in the rumen of Bactrian camel at embryonic stage. In the rumen of Bactrian camel at adult stage, obvious muscle fibers could be observed, the diameter of the muscle fibers was wider and the space between the muscle fibers was larger. The RNA-Seq sequencing results showed that at least 10G data was obtained from each sample, the clean ratio were more than 90%, and Q30 data were more than 88%. The embryonic rumen was set as the control group and the adult rumen as the experimental group, 1 207 differentially expressed genes were screened, contained 456 up-regulated genes and 751 down-regulated genes. The hierarchical clustering analysis of differentially expressed genes showed that the similar expression pattern was detected among adult individuals(M1, M2, M3) and among embryonic individuals (T1, T2, T3). The GO enrichment analysis results showed that the up-regulated differentially expressed genes were significantly enriched in 62 GO terms, down-regulated differentially expressed genes were significantly enriched in 366 GO terms. GO terms were mainly enriched in negative regulation of metabolic process, negative regulation of RNA biosynthetic process, negative regulation of gene expression, etc. The KEGG pathway analysis of differentially expressed genes showed that 73 significant KEGG pathways were enriched in MAPK signaling pathway, PI3K-Akt signaling pathway, AGE-RAGE signaling pathway in diabetic complications, insulin signaling pathway, aldosterone synthesis and secretion, etc. At the same time, MAPK12, MAPK13, FABP5, PPARγ, CaMK1 and other genes related to desert adaptation of Bactrian camel were screened. The expression pattern of 6 differentially expressed genes detected by RT-qRCR was consistent with the results of RNA-Seq analysis. The above results indicate that the rumen of Bactrian camel may be related to adipocyte differentiation. And in a desert environment, the Bactrian camel might reduce the metabolic efficiency of rumen, improve blood glucose tolerance through insulin resistance, and promote the synthesis and secretion of aldosterone to regulate blood pressure balance to better survive in the dry and water-deficient environment of the desert.

The study aimed to reveal the variation of transcription products at different RNA expression levels among yak tissues, and provide candidate genes for yak tissue differentiation, systematic genetic regulation and other studies. The cerebrum, cerebellum, buttock fat and buttock muscle tissues of 3 Leiwuqi Yaks with 4.5 years old were collected as experimental material. The expression products of each tissue were scaned through the Illumina 4000 sequencing platform, the SPRINT and JACUSA softwares were used to screen differential RNA editing sites, the classification, annotations and mutation risk assessment of RNA editing sites were analyzed. A total of 24 784 RNA editing sites were found, of which 4 015 sites with editing events were observed in the 4 tissues. The RNA editing sites in the 4 tissues were mainly A-G and T-C editing types. It was found that one of the high-risk editing sites was located in the SON gene, which stopped the translation of the gene prematurely, and caused problems in the binding of RNA to DNA at certain sites in the tissue. The types and distribution of RNA editing sites in cerebrum, cerebellum, buttock fat and buttock muscle tissues of Leiwuqi yaks were predicted and analyzed in this study, which provided a new reference for further study on regulatory genes in yak tissue differentiation and growth.

The aim of this study was to investigate the toxic damage effect of microcystin-LR (MC-LR) on porcine oocytes in vitro and its potential mechanism. The GV oocytes were collected from porcine ovaries obtained from a local slaughterhouse and randomly assigned into 4 groups, 0, 20, 40 and 60 μg·mL^-1 MC-LR were added into maturation medium in vitro, respectively. Then, the effects of MC-LR on the first polar body (PbI) extrusion, spindle structure, reactive oxygen species (ROS), membrane mucin V(Annexin V) and glutathione peroxidase (GSH-Px) activity were examined, respectively. The mRNA expression of oxidative stress and apoptosis related genes (SOD1, SOD2, CAT, GSH-Px, Bax and Bcl2) were also analyzed by RT-qPCR, and three replicates were performed for each experiment. The result showed that the PbI extrusion rate of porcine oocytes decreased in a concentration-dependent manner after MC-LR treatment. When the MC-LR concentration reached more than 40 μg·mL^-1, the oocyte maturation rate extremely significantly decreased(P<0.01), and the spindle structure abnormality rate also extremely significantly increased(P<0.001). Further studies showed that, after MC-LR treatment, ROS levels in oocytes were extremely significantly increased (P<0.01), while GSH-Px activity was extremely significantly decreased (P<0.01), accompanied by extremely significant down-regulation of antioxidant related genes SOD1, CAT and GSH-Px mRNA expressions(P<0.01). Apoptosis detection showed that MC-LR treatment could extremely significantly increase the early apoptosis rate of oocytes (P<0.01), and the ratio of Bax/Bcl2 was extremely significantly increased (P<0.001). The results indicate that MC-LR can induce abnormal spindle structure of porcine oocytes, lead to oxidative stress and apoptosis, and eventually result in a decline of porcine oocyte maturation ability.

The aim of the study was to establish a dual TaqMan Real-time PCR assay that could quantitatively calculate the number of X and Y sperm in dairy goat semen, detect the number and ratio of separated dairy goat X and Y sperm and provide technical support for development and application of gender control technology. The dual real-time PCR assay was established through designing primers targeted to the specific gene F9 and ZFY fragments in the X and Y sperm, a standard curve was established, and TaqMan real-time PCR reaction conditions and system were optimized. The sensitivity and reliability of the method were verified by serial dilution of positive standards and determination of 60 gender controlled semen (3 repetitions) of known purity. The results showed that the dual real-time PCR assay had good specificity and repeatability, and the sensitivities of X and Y sperm detection were 47 and 51 copies·μL^-1, respectively. The assay was used to calculate the numbers and ratios of X and Y sperm of dairy goat gender controlled semen, and the results were not significantly different from the purity of the X and Y sperm provided by the sales company (P>0.05), which indicated that this assay was reliable. The dual real-time PCR assay for calculating the number of X and Y sperm of dairy goats established in this study has good specificity and reproducibility, high sensitivity and reliable results, which provides a fast and reliable assay for calculating the number and ratio of X and Y sperm after semen separation in dairy goat semen.

The study aimed to explore the effects of different concentrations of GnIH on cell cycle, proliferation and related gene expression of duck granulosa cells. Primary duck granulosa cells were cultured in vitro and treated with different concentrations of GnIH (0, 0.1, 1, 10 and 100 ng·mL^-1) for 24 h (n=3). The cell growth status was observed, and the cell cycle and proliferation were detected by flow cytometry and EdU method, and the expression of proliferation-related genes CDK6, CyclinD1, IGF-2, IGFBP-2, p27^kip1 were detected by qRT-PCR. The results showed that the growth status of the cells in the GnIH treatment group at each concentration was good, the morphology was normal, the cell outline was clear, and the number of dead cells were not significantly different between groups (P>0.05). In the 0.1 and 1 ng·mL^-1 GnIH treatment groups, the proportion of cells in the G2 phase increased significantly (P<0.05). With the increase of the concentration of GnIH, the percentage of EdU-positive cells were decreased. In the 0.1 and 1 ng·mL^-1 GnIH treatment groups, the relative expression of CDK6, CyclinD1, IGF-2, IGFBP-2, p27^kip1 genes were downregulated in granulosa cells. The relative expression of these genes were increased in the 10 and 100 ng·mL^-1 GnIH treatment groups. Studies have shown that in the duck granulosa cells cultured in vitro, GnIH could hinder the cell cycle in G2 phase, meanwhile the percentage of EdU-positive cells were decreased and the expre-ssion of proliferation-related genes were downregulated, which could inhibit the proliferation of granulosa cells to affect the reproductive performance of animals.

The aim of the present study is to investigate the effects of dietary N-carbamylglutamate (NCG) supplementation on lambing performance and blood indexes of ewes during early pregnancy. A total of 160 second- and third-parity Hu sheep were randomly assigned into 4 groups which fed with basal diet(control group), basal diet supplemented with 0.05%, 0.08% and 0.11%NCG, respectively. Sheep were fed with experimental diets for 40 days from the day of mating. After that, all sheep were fed with basal diet. The parameters of lambing performance were recorded immediately after farrowing. The plasma amino acids, total nitric oxide synthase (TNOS), inducible nitric oxide synthase (iNOS), nitric oxide (NO), growth hormone, estradiol, progesterone, urea and ammonia were detected on the day 0, 20 and 40 of pregnancy. The results showed that the dietary supplementation of 0.08% and 0.11%NCG significantly increased the number of total lambs and total live lambs per litter compared with the control group(P<0.05), but the birth weight of lamb born alive decreased significantly (P<0.05). On day 40 of pregnancy, the plasma concentration of NO in NCG-supplemented groups were significantly higher than that of control group(P<0.05). In addition,compared to control group, 0.11% NCG-supplemented group had higher plasma concentration of iNOS on day 40 of pregnancy (P<0.05), as well as higher plasma concentration of progesterone on day 20 of pregnancy (P<0.05). Moreover, both 0.08% and 0.11% NCG -supplemented groups had lower level of plasma ammonia than those in control group and 0.05% NCG-supplemented group on day 20 of pregnancy (P<0.05), and on day 40 of pregnancy the plasma ammonia in 0.11% NCG-supplemented group was significantly lower than that of control group (P<0.05). The results suggested that dietary NCG supplementation during the early stage of pregnancy increased plasma concentration of iNOS, NO and progesterone, and thus improved placental nutrition and pregnancy maintenance. The reduced plasma concentration of ammonia by NCG supplementation, may improve the lambing performance by optimizing the uterine environment and enhancing fetal developmental potential.

The study aimed to explore the effects of different dietary nutrition levels on production performance, slaughter indexes and serum biochemical indexes of yaks. Sixty healthy male yaks with the similar age and weight, the average weight was (269.75±35.46) kg, and the yaks were randomly divided into 3 groups, each 20 replicates, and each repeating 1 yak. According to the energy level of the diet, they were divided into high nutrition level (HN) group, medium nutrition level (MN) group, and low nutrition level (LN) group. The test period was 105 days, the pre-test period was 15 days, and the normal test period was 90 days. During the trial period (90 days), the total net energy (NE_mf) of the diet was 5.51, 6.22, 6.94 MJ·kg^-1, respectively. The crude protein level of the diet during the whole trial period was 16.98% for each group. Production performance was detected at the end of the fattening trial. 8 yaks from each gorup was randomly selected for the jugular vein to collect blood, the serum separated quickly, determined the blood biochemical indicators, and the slaughter performance and meat quality of each yak was determined after slaughter. The experiment redults showed that the dietary nutrition level had no significant effect on the average daily gain (ADG) of the yak (P>0.05). However, with the increase of the feed nutrition level, the serum glucose content of the HN group was the highest, which was compared with the LN group and the MN group, increased by 38.86% and 29.69%, respectively (P<0.05). The MN group had the lowest serum urea content, which was 34.31% lower than the LN group (P<0.05), but the difference was not significant compared with the HN group. The content of total protein in serum decreased with the increase of energy level (P<0.05). The LN group had the lowest total cholesterol content, which was 10.98% lower than the MN group. With the increase of energy level, the pre-slaughter live weight, carcass weight and eye muscle area of the yak in the HN group were extremely significantly higher than those in the MN and LN groups (P<0.01); the slaughter rate in the HN group was significantly higher than that in the MN and LN groups (P<0.05). The meat color L^* value was the lowest in the LN group (P<0.05), which was 17.84% and 25.65% lower than the MN and HN groups, respectively. There was no significant difference in water lossing rate and cooking loss among different groups (P>0.05). Under the conditions of this experiment, adding energy is feasible to improve growth performance and meat quality of yak in house. The comprehensive growth performance, slaughter performance, meat quality and serum biochemical indexes are obtained. When the comprehensive net energy level of the diet is 6.94 MJ·kg^-1, the nutritional level of the diet is more suitable for cold-season house feeding and fattening, which can provide the energy level required for high-quality yak meat.

This study aims to establish an iELISA method for the detection of anti-PPRV H protein antibody. Four epitope peptides with good reactivity were selected and synthesized as the coating antigen based on the B cell epitopes of H protein. Optimization of the reaction conditions of each step and screening various buffers were performed and the cut-off value of detection was determined. After optimization, the optimal coating amount of multi-epitopes antigen peptide was 1.5×10^-6 μg·well^-1, the optimal working dilution of serum and the enzyme-labeled secondary antibody was 1:100 and 1:80 000, respectively. The carbonate-hydrogen buffer was chosen as the coating buffer, and PBST buffer was adopted as dilution buffer for the serum and the secondary antibody. The appropriate blocking buffer was determined to be 2% BSA solution and the cut-off value of the assay was 0.25. The test results of clinical sera showed that the coefficients of variation of intra- and inter-assay were less than 10%, which proved its good repeatability. When the assay was tested with the positive sera of sheep pox, FMD, and BT, there was no cross-reaction obtained, indicating that the assay was specific for the detection of the PPRV antibody. The detection results of 306 sera samples showed that the developed ELISA had a relative sensitivity of 92.81% when compared with the commercially available ID Vet cELISA Kit. This study has developed a specific, repeatable, and sensitive iELISA method for detecting antibodies of PPRV H protein, and it is more convenient, timesaving, and labor-saving than cELISA. Therefore, the assay can be used for the detection of antibodies against PPRV.

This study aimed to construct a recombinant plasmid that can simultaneously express the dominant antigen genes of bovine viral diarrhea virus (BVDV) and infectious bovine rhinotracheitis virus (IBRV), and study its immune effect, so as to provide reliable data and theoretical support for the development of BVD-IBR bivalent DNA vaccine. In our experiment, PCR was used to amplify the target genes, E0 and gD, which were sequentially connected to the eukaryotic expression vector pVAX1-IRES to construct the pVAX1-E0-IRES-gD recombinant eukaryotic expression vector. The recombinant plasmid pVAX1-E0-IRES-gD was transfected into 293T cells and the expression of the target genes in foreign cells were detected by indirect immunofluorescence assay. Following, immunization test with the pVAX1-E0-IRES-gD, pVAX1-IRES and BVD-IBR combined inactivated vaccine was inoculated with SPF mice at different doses by intramuscular injection, subsequently, immune effects have been evaluated through antibody level and cytokine detection and splenic lymphocyte proliferation assay. The results showed that the pVAX1-E0-IRES-gD recombinant plasmid was successfully constructed and both antigen genes obtained a high level of expression in 293T cells. In terms of antibody and cytokine levels and the proliferation of spleen lymphocytes, the recombinant plasmid group was significantly higher than that of the empty vector and the negative control group, and the differences were extremely significant, the high-dose immunization group was better than the medium and low dose groups. Also, the high-dose recombinant plasmid immunization group was not significantly different from the BVD-IBR inactivated vaccine group. The results showed that the co-expression recombinant plasmid which contains the BVDV E0 gene and IBRV gD gene was constructed successfully. The expression test in vitro proved that the target protein has good reactionogenicity, and animal immunity tests proved that it can stimulate the body to produce a good immune response.

Tibet orbivirus (TIBOV) is a new member of the genus Orbivirus. The virus was first isolated from Anopheles maculatus collected in Motuo County, Tibet, China in 2009. At present, the recognition of this virus is still very limited. This is the first report on the isolation and identification of a new TIBOV strain YNSZ/V290/2019 from Culicoides collected in Shizong County, Yunnan Province, China. YNSZ/V290/2019 can cause an obvious cytopathic effect on C6/36 and BHK-21 cells. Under the electron microscope, the virus particles are spherical, with a diameter of about 55-75 nm, and have typical morphological characteristics of cycloviruses. The results of agarose gel electrophoresis showed that the genome of the virus was a 10 segment double-stranded RNA, showing the characteristics of "3-3-3-1". The sequence analysis of the highly conserved gene (Seg-3) and proteins (T2) showed that YNSZ/V290/2019 was a member of TIBOV, and YNSZ/V290/2019 shared 80.90%-81.70% nucleic acid sequence identity and 97.20%-97.30% amino acid sequence identity with other TIBOV strains. The sequence analysis of the high variable gene (Seg-2) and proteins (OC1) which determine the serotype of the Orbivirus showed that YNSZ/V290/2019 shared 26.90%-30.60% nucleic acid sequence identity and 28.5%-33.2% amino acid sequence identity with the known TIBOV strains, suggested that the strain may represent a new serotype of TIBOV. Serum neutralization tests were carried out on 40 cattle and 68 goat serum samples collected from Shizong pasture. The results showed that the positive rates of new TIBOV of cattle and sheep were 22.50% and 4.41%, respectively. The discovery of a new type of TIOBV in China enriches the research on arbovirus in China and provides a basis for further study on the distribution, pathogenicity, and host range of infection and development of diagnostic reagents of TIBOV in China.

To detect the African swine fever virus (ASFV) and Classical swine fever virus (CSFV) wild strains rapidly, a specific and sensitive real-time fluorescence quantitative PCR (FQ-PCR) method was established by using the TaqMan MGB probe technique. Specific primers and probes were designed based on the P72 gene of ASFV and 5' non-coding region of the CSFV wild strain to establish the duplex FQ-PCR assay based on TaqMan MGB probe technique. The sensitivity, specificity and stability of FQ-PCR were determined, and 50 clinical samples were simultaneously detected by the FQ-PCR assay, the national standard of CSFV and the OIE recommended detection method for ASFV. The results indicated that the duplex FQ-PCR assay was successfully established and showed a good linear relationship at a template range of 10⁰-10⁶ copies·μL^-1; the specificity of duplex FQ-PCR assay revealed that amplifications were showed synthetic on ASFV and CSFV genes, but other pathogens have no amplifications; variation coefficient of intra and inter assay were 1.18%-2.08%, respectively; the sensitivity of the assay was 10 copies·μL^-1; Fifty clinical samples was detected by the duplex FQ-PCR, the results was consistent with detection of CSFV by national standard and detection of ASFV by OIE recommended method. The duplex TaqMan MGB FQ-PCR assay of ASFV and CSFV wild strain was specific, sensitive, rapid and suitable for identify detection of ASFV and CSFV wild strains.

In this study, we investigated the prevalence, molecular characteristics, and genetic evolution of porcine teschovirus (PTV) in Tibetan pigs in Sichuan. Ninety-six fecal samples of Tibetan pigs were collected from 14 farms in Kangding region of Ganzi Tibetan Autonomous Prefecture, Sichuan Province from 2017 to 2018. The samples were detected by the RT-PCR method, and the viral genome was sequenced and genotyped. The overall positive rate of PTV was 20.8% (20/96, 95% CI=13.2%-30.3%), and the positive rate of PTV was 57.1% (8/14, 95% CI=28.9%-82.3%) in 14 pig farms. Two nearly full-length PTV complete genome sequences were obtained from 2 different clinical samples. The two genomes were of 83.1% nucleotide identity and were named as SWU-E5-2018 and SWU-ZG2-2018. The serotypes of the two sequences were found to be type 3 and type 6 by VP1 sequence analysis. Furthermore, the SWU-E5-2018 was found to be a recombinant strain by recombination analysis, and the recombinant region was located at 552-3 082 nt of capsid protein P1. To have a further understanding of the evolution process of the two PTV sequences, the divergence time estimation was performed using the Bayesian evolutionary analysis software. The result showed that the divergence timepoints of SWU-E5-2018 and SWU-ZG2-2018 was about 2010 and 2011, respectively. It was the first time to investigate the PTV from Tibetan pigs and to confirm the existence and prevalence of PTV in Tibetan pigs, which provided a reference for further study of viral genetic variation and biological characteristics.

To analyze the transcription level of NLRP3 gene in chickens infected with FAdV-4, a pair of NLRP3 specific primers was designed, and the 180 bp NLRP3 gene was amplified by RT-PCR and cloned into the pMD-18T vector to create a recombinant plasmid pMD-18T-NLRP3. Using the constructed pMD-18T-NLRP3 plasmid as standards, a standard curve assay for SYBR Green I real-time PCR was established. After optimization of reaction conditions, a real-time fluorescent quantitative PCR was successfully established to detect NLRP3, and it was applied to measure the transcription level of NLRP3 gene in the organs of chickens infected with pathogenic FAdV-4. The results indicated that the primers designed for this assay were specific to chicken NLRP3 gene; the established assay was able to provide a well-defined quantification standard curve for the chicken NLRP3 standard plasmid, and a linear correlation was observed between the copy numbers of the standard plasmid pMD-18T-NLRP3 and the Cq values. Comparing to the control group, the transcription level of NLRP3 was significantly higher in the liver and spleen (P<0.001) and higher in the cecal tonsils and bursa of Fabricius (P<0.01) of the infected chickens. The chicken NLRP3 SYBR Green I real-time PCR established in this study was able to detect the transcription level of NLRP3 in different organs of chickens infected with FAdV-4, and the inflammation induced by FAdV-4 infection is highly related to NLRP3.

This experiment was conducted to express recombinant Clostridium septicum α toxin without toxin and subsequently evaluate the immunogenicity of the recombinant toxin. Recombinant α toxin gene (GCSA_Δ11) of Clostridium septicum deleted 11 amino acids (212-222) was synthesized based on the sequence reported. Subsequently, GCSA_Δ11 was cloned into the prokaryotic expression vector pET-30a (+) to get the recombinant protein (rCSA_Δ11). The reactivity of rCSA_Δ11 with antiserum of Clostridium septicum crude toxin was detected by Western blot, and mice were used to detect the virulence of rCSA_Δ11. The rabbit antiserum against the recombinant protein was prepared and the neutralizing titer was detected according to the method prescribed in Chinese Veterinary Pharmacopoeia (2015). The results showed that recombinant protein rCSA_Δ11 was expressed at a high level as soluble form with a ratio about 46%, and the protein without virulence to mouse could react with the antiserum of Clostridium septicum toxin; rabbit antiserum after first immunization could neutralize 8-12 mice MLD of Clostridium septicum crude toxin per 0.1 mL, and 32-45 MLD after twice immunizations. After challenge with 1 MLD of Clostridium septicum crude toxin, the rabbits immunized with rCSA_Δ11 were protected with a ration of 100%, whereas all of the rabbits died (4/4) in the control groups. The results suggest that the recombinant of Clostridium septicum α toxin without virulence retains the good immunogenic antigen, which provides important experimental data for the development of Clostridium septicum genetic engineering subunit vaccine.

This experiment was conducted to confirm the sheep pox of the Small-tail Han ewes and explore the characteristics of the pathological changes. Based on clinical diagnosis, the six sheep suspected of sheep pox were diagnosed by the indirect ELISA for detecting antibodies and PCR detection technology. The pathological changes were observed by microscopy, light microscope, and transmission electron microscope. The indirect ELISA showed that all the samples of 6 sick sheep were antibodies positive for Capripox. The PCR results showed that the special 585 bp target gene fragment of sheep pox virus (SPV) was amplified and the SPV was diagnosed as the pathogen of the sick sheep. The pathological changes of clinic and autopsy showed that the skins of sick sheep had hyperemia, papules, erythema, and necrosis in glabrous areas, and the suppuration and ulcer of the tails root were often observed. There were a large number of papules, pox lesions, necrosis, and ulcerations on the mucosa of the digestive tract, especially the mucosa of the tongue and rumen. At the same time, there were pox lesions and necrosis in the lungs, livers and spleen. The histopathological changes indicated that the exudation-proliferation nodules and necrosis-proliferation nodules were mainly changes of pox lesions in the lung, and there were a large number of sheep pox cells around the blood vessels and bronchioles, and the eosinophilic inclusions were often present in the cytoplasm of sheep pox cells. In the diseased mucosa of the digestive tract, the epithelial cells were varying degrees of proliferation and vacuolar degeneration, the lamina propria and submucosa were varying degrees of congestion and edema, and there were also many sheep pox cells and their eosinophilic cytoplasmic inclusions. There were also necrosis-proliferation nodules in livers, spleens and kidneys. The ultrastructural changes of lungs showed that the alveolar epithelial cells were swelling, nuclear concentration, chromatin aggregation, mitochondrial swelling, fractured cristae and cavitation, and there were cytoplasmic inclusions. Those results confirmed that the important pathological changes of the mucosa of the digestive tract and lung are also one of the representative changes in sheep pox. In particular, there were a large number of sheep pox cells in pox lesions, it has important pathological diagnostic significance.

Under cold exposure, O-linked β-N-acetylglucosamine (O-GlcNAc) in skeletal muscle increased significantly, and O-GlcNAc, as a "stress receptor", regulated cell signal transduction and gene transcription. Skeletal muscle, as a secretory organ, produces myogenic IL-6 through contraction, which not only plays an immune role but also can regulate the metabolism of glucose and lipid in skeletal muscle. Therefore, the purpose of this study was to detect the expression of O-GlcNAc related protein and cytokine IL-6 in C2C12 under different durations of cold exposure. To explore the potential relationship between O-GlcNAc and IL-6 under of cold exposure. In this experiment, C2C12 was cultured in vitro and randomly divided into control group (37℃±0.5℃) and cold exposure (32℃±0.5℃) 3, 6, 9, and 12 hours treatment group. Western blot was used to detect the glycosylation level of O-GlcNAc and the expression level of OGT, OGA, and IL-6 under different durations of exposure. The results showed that compared with the normal temperature control group, the expression level of OGT was significantly increased (P<0.05) after 3 hours of cold exposure and the expression level of OGA was significantly increased (P<0.05) after 6 hours of cold exposure; the expression level of O-GlcNAc glycosylation and IL-6 were significantly increased (P<0.01) after 3 hours of cold exposure. The expression levels of O-GlcNAc glycosylation and IL-6 in C2C12 cells were significantly increased under different durations of cold exposure. At the same time, the trend of O-GlcNAc glycosylation and IL-6 expression under different durations of cold exposure was highly similar, suggesting that O-GlcNAc glycosylation participated in the regulation of IL-6 expression under cold exposure.

The purpose of this study was to explore the protective effect of melatonin (MT) on hippocampal inflammatory injury induced by lipopolysaccharide (LPS) in rats. Forty 4-week-old healthy male SD rats were randomly divided into 4 groups:control group (CON group), model group (LPS group), MT intervention group (LPS+MT group) and MT group. Four hours after intraperitoneal injection of 10 mg·kg^-1 MT and/or 10 mg·kg^-1 LPS, the behavior of rats was tested by open field test. The hippocampus was weighed and coefficient of hippocampus was calculated. The pathological changes of the hippocampal region of brain were observed by hematoxylin-eosin (HE) staining. The mRNA expressions of microglial activation markers Iba-1 and CD11b in hippocampus were detected by RT-PCR. The protein expressions of inflammatory cytokines IL-1β, TNF-α, IL-6, IL-10 and TGF-β in hippocampus were detected by Weston blot. The results showed that compared with CON group, the autonomous exploration behavior and motor ability of rats in LPS group were decreased; the arrangement of nerve cells in hippocampus was loose, and the intercellular space of hippocampus was enlarged, cytoplasmic pyknosis was deeply stained, glial cells were infiltrated; the expression of microglia activation markers Iba-1 and CD11b mRNA were significantly increased (P<0.01); the protein expressions of pro-inflammatory factors IL-1β, TNF-α and IL-6 were significantly increased in LPS group (P<0.01); the protein expression of anti-inflammatory factors IL-10 and TGF-β decreased significantly (P<0.01). Compared with LPS group, the autonomous exploration behavior and motor ability of rats in LPS+MT group were increased; the arrangement of nerve cells in hippocampus was compact, and there was no obvious pathological change; the mRNA expressions of microglia activation markers Iba-1 and CD11b were significantly decreased (P<0.01); the protein expressions of pro-inflammatory factors IL-1β, TNF-α and IL-6 were significantly decreased in LPS+MT group (P<0.01); the protein expression of anti-inflammatory factors IL-10 and TGF-β increased significantly (P<0.1). In addition, there was no significant difference in all indexes between MT group and CON group (P>0.05). The above results suggest that MT can inhibit the activation of microglia and reduce the inflammatory response of hippocampus, thus improving the inflammatory injury of hippocampus induced by LPS in rats.

In order to study the molecular mechanism of IPEC-J2 injury induced by Salmonella, we stimulated IPEC-J2 with Salmonella and collected the cells and culture supernatant at pointed times. The expression of tight junction proteins, inflammation-related proteins, cell proliferation-related proteins and the content of lactate dehydrogenase (LDH) were detected by fluorescence quantitative PCR or ELISA to confirm that Salmonella could cause IPEC-J2 damage. We also further explored the regulation of NF-κB/β-catenin signaling pathway in Salmonella induced-IPEC-J2 injury using NF-κB inhibitors and small interference RNA. The results showed that, compared with the control group, the mRNA expression levels of tight junction proteins ZO-1 and Occludin in the Salmonella-infected group were extremely significantly decreased at 6 and 24 h after infection (P<0.01); the production of pro-inflammatory cytokines TNF-α, IL-8, and the release of LDH, were extremely significantly increased at 6, 12 and 24 h after infection (P<0.01), and the mRNA expression of NF-κB p65 was also extremely significantly increased at 1, 3 and 6 h after infection (P<0.01); and the mRNA expression of cell proliferation proteins cyclin D1 and c-Myc significantly decreased at 6, 12 and 24 h after infection (P<0.05), and the mRNA expression of transcription regulatory factor β-catenin was extremely significantly decreased at 24 h after infection (P<0.01). Compared with Salmonella-infected group, the mRNA expression levels of ZO-1 and Occludin in the inhibitor+Salmonella-infected group were extremely significantly increased at 12 and 18 h (P<0.01), while the levels of NF-κB p65, TNF-α, IL-8 and LDH content were extremely significantly decreased at all time points (P<0.01); the levels of β-catenin, cyclin D1 and c-Myc were extremely significantly decreased at each time point in siRNA-β-catenin+Salmonella treatment group (P<0.01), the release of LDH extremely significantly increased at 6 and 12 h (P<0.01), but no significant increase at 18 h after infection (P>0.05). The results showed that Salmonella activated the NF-κB signaling pathway, promoted the production of pro-inflammatory cytokines, and aggravated the IPEC-J2 damage; meanwhile, Salmonella inhibited the β-catenin signaling pathway, decreased the expression of cell proliferation proteins and tight junction proteins, and affected the cell repair, which leads to the imbalance between cell damage and cell repair, and finally presents cell damage. This study revealed the molecular mechanism of IPEC-J2 damage caused by Salmonella from the perspective of NF-κB/β-catenin signaling pathway.

The purpose of the study was to research the effect of Jiawei Gegen Qinlian Decoction (JGQLD) on the regulation of intestine damage and repair in the piglets of damp-heat diarrhea (DHD). Six piglets (8-15 days old) were selected as control group. Twenty-four piglets (8-15 days old) that with the symptom of DHD were divided into 4 groups, including DHD group, low dose, middle dose and high dose of JGQLD group, 6 piglets per group. Piglets in the control group and the DHD group were given normal saline. Piglets in medicine groups were given the JGQLD for 5 consecutive days (the doses were recorded as dried medicinal herb, 2.5 g·d^-1 in the low dose group, 5.0 g·d^-1 in the middle dose, and 10.0 g·d^-1 in the high dose group). The fecal scores of piglets in all groups were recorded at 0, 3 and 5 day. In control group, DHD group and middle dose group of JGQLD, the duodenum, jejunum, ileum and colon of piglets were taken for tissue section and H.E. staining, while the duodenum was selected for proliferating cell nuclear antigen (PCNA) immune-histochemical staining. The mRNA expressions of TLR4, TNF-α, IL-1β and IL-6 in the jejunum of piglets from control group, DHD group and middle dose of JGQLD group were detected by real time PCR. In the DHD group, DHD destroyed the structure of small intestine and colon, especially the duodenum and jejunum. Compared with the control group, the PCNA expression in the duodenum crypt of the DHD group was significantly increased (P<0.05), the mRNA expressions of TLR4 (P<0.05), TNF-α (P<0.01), IL-1β (P<0.01) and IL-6 (P<0.05) in the jejunum of the DHD group were all increased significantly. Compared with the DHD group, the low, middle and high dose of JGQLD significantly decreased the fecal scores of piglets (P<0.05). The structures of duodenum, jejunum, ileum and colon in the middle dose of JGQLD group were improved and there were a few shedding villus in the lumen of duodenum. Compare with the DHD group, the PCNA expression in the duodenum villus of the middle dose group was extremely significantly increased (P<0.01), the mRNA expressions of TLR4 (P<0.05), TNF-α (P<0.01), IL-1β (P<0.01) and IL-6 (P<0.05) in the jejunum of the middle dose group were significantly decreased. JGQLD repaired the intestine damage of piglets with DHD by promoting the proliferation of crypt stem cells and reducing the intestinal inflammatory response.

This experiment aims to study the binding specificity of RGD (Arg-Gly-Asp) motif on the structural protein VP1 of foot-and-mouth disease virus (FMDV) to host cell surface receptor integrin. In this paper, the Biacore 3000 system based on surface plasmon resonance (SPR) was used to study the binding specificity of RGD motif with the α_vβ₆ extracellular domain, α_v extracellular domain and β₆ extracellular domain, respectively. Firstly, the domains of integrin bound to RGD Motif were screened by binding experiments, and then the kinetic analysis of integrin bound to RGD Motif was carried out. The results showed that the synthesized RGD motif was bound to the α_vβ₆ extracellular domain, and the kinetic constants K_a, K_d, and K_D were 42.3 M^-1s^-1, 3.1×10^-4s^-1, and 7.33×10^-6M respectively. It also binds to the α_v extracellular domain, the kinetic constants were 21.8 M^-1s^-1, 2.13×10^-4s^-1,and 9.79×10^-5M respectively. There was almost no binding to the extracellular domain of the β₆ subunit. The results showed that the binding of RGD to the extracellular domain of α_vβ₆ was faster and stronger than that of the extracellular domain of α_v subunit. This study will provide a reference for further study of the interaction between FMDV and host cell surface receptors.

Fourteen strains of canine parvovirus (CPV) were successfully isolated and identified from feces swabs of dogs which were suspected to be infected CPV in Beijing, and their complete NS1 and VP2 genes were sequenced and analyzed. Among the 14 strains identified, the results showed that 7 strains were New CPV-2a and 7 strains were of CPV-2c. Besides, some new amino acid mutation sites were identified (amino acids 19, 33, 293, 588, 624 and 656 of the NS1 and amino acids 13 and 574 of the VP2). Phylogenetic analysis based on the VP2 and NS1 genes indicated that most of the New CPV-2a and CPV-2c strains were closely related to the isolated strains in Nanning, Guangxi, and Changchun, Jilin, indicating that the isolated strains had the same origin as the isolated strains in Guangxi or Jilin. This study provides a useful reference for better epidemiological investigation of canine parvovirus, and also builds a foundation for the in-depth study of the molecular mechanism of canine parvovirus variation and transmission.

This research aimed to diagnose a case of liver melanoma that happened in a broiler chicken. A 25-day-old broiler chicken was observed depressed and extremely thin, and the whole liver was black at autopsy. Hematoxylin and eosin (HE) staining results show a large amount of abnormal cells with different sizes, hyperchromatic nuclei and severe melanin granules deposition in the diseased liver. Immunohistochemistry of the liver incubated with both SOX 10 and HMB-45 antibody show positive results. Based on the results of the pathological and immunohistochemical examination, the broiler chicken was diagnosed with serious liver malignant melanoma. Results of both bile pigment and Prussian blue staining were negative, suggesting that the liver lesion was not caused by bilirubin or hemosiderin deposition. In summary, based on the results of differential diagnosis, the broiler chicken was diagnosed with serious liver malignant melanoma. Results of the present study help to provide reference for the diagnosis of avian melanoma in poultry.