The rich fossil record of Equus over the past 55 million years were punctuated by many episodes of origin, migration, extinction and evolution, which has made them as a textbook example of evolution. The ancestors of Equus originated in Northern America continent in the Eocene period, called Hyracotherium. As the climate changes, the Bering Strait sea level rise and fall. The Equus groups colonized the Old World several times crossing Beringia but they left no descendants there. Equus genus continued to evolve in North America. Until the Pliocene, the most recent common ancestor of present-day Equus emerged, called Pliohippus. Within the following 3 million years, their ancestors rapidly expanded and occupied the ecological niches in different geographical areas, radiating into 3 main subgenus (horse, donkey and zebra). The main reason for facilitating the speciation was the acute chromosomal rearrangements. With the publication of the horse whole-genome sequences and the donkey draft genome sequences, and the progress of biomolecule level, the identification of Equus species karyotype diversity is more profound. More and more scientists believe that the mechanism of which Equus interspecies hybridization can produce offspring is based on the high similarity of genome sequence. And their hybrid offspring infertility is caused by the karyotype diversity which makes the meiotic recombination breakdown. This assumption still needs to be confirmed by experiments.

Mycoplasma bovis is considered to be one of the important pathogenic agents of cattle. It can persist in the host and causes a series of chronic diseases including bronchopneumonia, mastitis, otitis, genital disorders, arthritis, meningitis or keratoconjunctivitis. Due to the resistance to antibotic therapy and the lake of commercially available diagnostic tools and effective vaccines, M. bovis-induced diseases are difficult to prevent and control. The main reason of persisitent infection caused by M. bovis is to evade the host innate and adaptive immunity through high frequent variation of membrane surface antigens, avoiding phagocytosis, cell invasion, biofilm formation and modulating host immune responses. Deeply understanding immune evasion mechanism of M. bovis is helpful to develop the new dignostic tools and novel vaccines, and it is also beneficial to prevent and control the diseases.

The study aimed to investigate the effect of MC4R gene silencing on gene expression in bovine fibroblast，further reveal the relationship between MC4R gene and carcass quality, energy balance and energy metabolism in beef cattle. Bovine fibroblasts with MC4R-knockdown were used for this study. Differentially expressed genes between experimental group (MC4R knockdown) and control group (MC4R non-knockdown) were identified by deep sequencing. GO annotation cluster analysis and KEGG pathway enrichment analysis were performed to enrich the differentially expressed genes involving in energy metabolism, fat transport processes and their regulation pathway. For validating the reliability of RNA-sequencing, mRNA expression of 10 differentially expressed genes randomly selected were validated by quantitative real-time PCR (qRT-PCR). Moreover, single nucleotide polymorphisms (SNPs) of the targeted genes were tested by the bovine SNP microarray, and SNP-based genotypes were tested for the 136 Simmental cattle. On the basis of genotyped data, further association analysis between SNP and carcass traits were carried out using PLINK software. 7 799 differentially expressed genes in MC4R knockdown cells were detected using deep sequencing, including 1 703 up-regulated genes and 6 096 down-regulated genes. Significant GO annotation analysis identified 265 differentially expressed genes involving in energy metabolism and fat transport processes from 2 different MC4R knockdown level groups. Function cluster analysis further screened out 29 genes associated with the energy metabolism. KEGG pathway enrichment analysis indicated that the 265 differentially expressed genes were involved in 40 different signaling pathways. Of which, MC4R gene and other 4 genes (Leptin, AGRP, NPY and POMC) closely associated with the energy metabolism regulation were all involved in the adiponectin signaling pathway. And, MC4R knockdown significantly influenced the expression of 22 genes in this pathway. As the qRT-PCR data, mRNA expression of the 10 genes (up-regulated genes: CFB, RSAD2, IFIT3, CHI3L1 and BOLA-N; down-regulated genes: UPK1B, IER2, LASS3, TGFβ3 and AXUD1) showed non-significant difference with the results of RNA-sequencing, which was proved that the results of deep sequencing were accurate and reliable. 16 SNPs were detected from 11 genes by the bovine 50 K SNP microarray. The association analysis between SNPs and carcass traits showed that TGFβ3 gene on chromosome 10 significant influenced the eye muscle area, carcass weight, thickness of the waist and thigh meat and striploin weight of Simmental cattle (P<0.05), while extremely significantly influenced the eye meat weight and tendorloin weight (P<0.01). The RHOJ gene on chromosome 10 also had a significant influence on the eye muscle area of Simmental cattle (P<0.05). The KRT17 gene on chromosome 19 and VCAN gene on chromosome 7 could significantly affect the meat shearing force of Simmental cattle (P<0.05 and P<0.01, respectively). Additionally, MASP1 gene on chromosome 1 significantly influenced the meat color (P<0.05).The results indicate that MC4R knockdown significantly influence the expression of genes involving in the processes of energy metabolism, protein metabolism, fat metabolism and energy homeostasis regulation, further MC4R gene regulate the energy balance, fat metabolism and carcass characteristics in beef cattle.

The aims of the study were to clone the CDS of krüppel-like factors (KLF5, KLF6, KLF7), determine their expression in various tissues, and analyze their correlation with intramuscular fat (IMF), which might provide basis for the functional research on KLFs in regulating lipid metabolism in yak. Seven healthy, 4-6 years old male Maiwa yaks were selected as experiment animals. After fasting slaughter, the tissue samples of spleen, lung, kidney, subcutaneous fat and longissimus dorsi muscle were collected for the total RNA extraction. The sequences of KLF5, KLF6 and KLF7 genes were cloned by RT-PCR, and analyzed using bioinformatics, respectively. The mRNA expression of KLFs were determined using real-time fluorescent quantitative PCR (qPCR). Pearson correlation coefficient was used for analyzing the correlation between the mRNA expression of KLFs and IMF content in yak. The CDS of KLF5, KLF6 and KLF7 were 1 086, 855 and 909 bp, encoding 361, 284 and 302 amino acids, respectively. The yak KLFs sequences were closest associated with bovine, followed by goat, sheep, pig, and more distant with chicken. The tissue expression of the KLF5, KLF6 and KLF7 were higher in lung, and lower in longissimus dorsi muscle. The expression of KLF5 was significantly correlated with the expression of KLF6 and KLF7, respectively. However, all of mRNA expression of KLFs were not correlated with IMF content in longissimus dorsi muscle of yak. These data provided basic information for the functional research of KLF5, KLF6, KLF7, and would be beneficial for understanding the regulatory mechanism of lipid metabolism in yak.

This research aimed to clone and express the porcine transcription factor E74-like factor 4 (pELF4) gene in vitro and to prepare its polyclonal antibody. The pELF4 gene was cloned by RT-PCR (Reverse Transcription-Polymerase Chain Reaction), and the characteristics of its nucleotides and coding amino acids were analyzed by the bioinformatics softwares. The recombinant expression vector pET28a-pELF4 was constructed by inserting the pELF4 gene into the prokaryotic expression vector pET28a. Then pET28a-pELF4 was transformed into E. coli Rosetta(DE3) and to investigate its expression under different temperatures, IPTG concentrations and induction time. The expressed proteins were analyzed by SDS-PAGE and Western blot. The anti-pELF4 immuno-globulin egg yolk antibody(IgY) was prepared by immunizing hens after purification of the recombination pELF4 protein (rpELF4). The results showed that the ORF (Open reading frame) of pELF4 gene (GenBank Accession number: KU097322) was 1 989 bp, coding 662 amino acids. The similarity between pELF4 gene and that of human, cattle and mouse were 89.4%, 90.7% and 81.1%, respectively, therefore it was closer to the animals such as cattle while it was further to human and mouse. The relative molecular weight of rpELF4 was 95 ku, and it existed in the form of protein and inclusion body. The amount of inclusion body reached highest when the expression combination was 37℃, 0.8 mmol•L^-1 of IPTG and 8 h.The chicken anti-rpELF4 antibody titer was above 1︰256 000 fold at the 13rd days post the 4th vaccination. The recombinant protein pELF4 was successfully expressed by prokaryotic expression system, the chicken anti-pELF4 polyclonal antibodies with the wonderful activity and specifity was prepared, which provided basic material for the research of the biofunction of this protein and the related diseases.

The objective of this study was to obtain the complete mitochondrial DNA (mtDNA) sequence of Sichuan Sheldrake duck, and to provide reference data for protection and utilization of this genetic resources of duck. Sixteen pairs of primers were designed by Primer 6.0 according to the mitochondrial genome sequences of duck homologous species. The genomic sequence of Sichuan Sheldrake duck’s mtDNA were then amplified by PCR and sequencing technology. The results showed that the length of Sichuan Sheldrake duck’s mtDNA genome was 16 604 bp, which composed of A (29.19%), C (32.82%), G (15.79%) and T (22.20%). The whole genome contained 22 tRNA genes, 2 rRNA genes, 13 protein-encoding genes and a non-encoding control area (D-loop area). The analysis by online software of tRNAscan-SE 1.21 and RNA structure 5.6 found that 22 tRNA genes were the standard secondary structures with 4  constant arms which like a clover. The D-loop region of Sichuan Sheldrake duck’s mtDNA genome contained Poly (c), TAS, E-box, F-box, D-box, Bird-similarity box and CXB1 conserved box. The neighbor joint (NJ) phylogenetic tree was established by MEGA6.0 basing on the D-loop region and the complete mtDNA sequence, respectively, and the results showed that the Longsheng duck was more closer to Sichuan Sheldrake duck.

The objective of this study was to explore the expression and localization of chromatin remodeling factor BPTF and BRG1 in black and white sheep skin tissues. Skins were collected surgically from the hindquarters of 3 black sheep and 3 white sheep. The expression level and distribution of BPTF and BRG1 were detected by quantitative real-time-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry to investigate the underlying mechanisms of coat color in white and black skin tissues of sheep. qRT-PCR and Western blotting results suggested that BPTF was expressed at significantly higher levels in black sheep compared with that of white sheep（P<0.05）; BRG1 was expressed at very significantly higher levels in black sheep compared with that in white sheep（P<0.01）. Immunohistochemistry demonstrated BPTF and BRG1 staining in the root sheath and dermal papilla in hair follicle of sheep skins. Therefore, it come to the conclusion that BPTF and BRG1 may involve in the coat color formation of sheep.

To study the expression pattern of goat beta-defensin 124 (gDB 124) in different organs and the location of gDB 124 in testis and epididymis of adult bucks. Testis, caput epididymidis, corpus epididymidis and cauda epididymidis were collected from 1-week-old male kids, 2-6-month-old male kids and 18-month-old bucks. Part of the tissues were gathered from heart, liver, spleen, lung, kindey and longissimus dorsi of bucks at the age of 18 months. Analysis of gDB 124 mRNA expression pattern was detected by qRT-PCR. Location of gDB 124 in the reproductive organs was determined by immunofluorescence. The results of qRT-PCR showed that gDB 124 mRNA was detected in testis, caput, corpus, cauda, heart, liver, spleen, lung and kidney of bucks at the age of 18 months except the longissimus dorsi，and the expression level in caput of epididymis was extremely higher than that in other tissues (P<0.01). Moreover, gDB 124 mRNA expression level in testis of buck at the age of 6 months was significantly higher than those at other ages. And the expressions in caput, corpus, cauda of epididymis were increased with the increase of the age. There was no obvious difference at gDB 124 mRNA expression levels between testis and epididymis of 1-week-old bucks. gDB 124 mRNA expressions in caput of epididymis were extremely higher than those in testis, corpus, cauda of epididymis of bucks at 3-6 and 18 months old (P<0.05).The result of immunofluorescence suggested that gDB 124 protein was detected in testis and epididymis, and mainly located in false stratified columnar epithelium cells of caput of epididymis. The order of the expression level was caput >corpus>cauda> testis, which was consistent with expression pattern of gDB 124 mRNA in testis and epididymis. In conclusion, the expression of gDB 124 was in the spatio-temporal expression.

This study aimed to clarify quantitative distributions of free amino acids (FAA) and small peptides (PAA) in gastrointestinal tract of sheep and the mRNA expression of CAT1 (cationic amino acid transporter), EAAT3 (acidic amino acid transporter), y⁺LAT2, ASCT2(neutral amino acid transporters), PepT1 (small peptide transporter), APN (aminopeptidase) and DPEP2 (dipeptidase) in gastrointestinal tract of sheep. A total of 6 healthy Small-tail Han sheep at the age of 18 months were slaughtered to collect chyme and tissues of rumen, duodenum, jejunum, ileum and cecum. Then concentrations of the most of FAA and PAA in supernatant of chyme were detected and the mRNA expression of related genes were quantified by RT-PCR. The results showed that: 1) The concentrations of the most of FAA from high to low was jejunum, duodenum, ileum, rumen, cecum. The concentrations of FAA in jejunum and duodenum were significant higher than that in other parts(P<0.05). Proportions of FAA in these parts were different. The concentrations of the most of PAA from high to low was duodenum, jejunum, ileum, cecum, rumen. Except for cecum and rumen, they were significant different from each other(P<0.05). Proportions of PAA in these parts were different. The proportion of PAA in total amino acids was the significantly lowest in jejunum(P<0.05). 2) CAT1 mRNA in jejunum had the highest abundance, but there were no significant difference among 5 parts. EAAT3, y⁺LAT2, PepT1 and APN mRNA had a similar expression pattern that they all had the highest abundance in ileum. ASCT2 mRNA had the highest abundance in duodenum, and DPEP2 mRNA had the highest abundance in cecum. Results indicate that concentrations and proportions of FAA in these parts are different, the same as PAA. The major secretion site of FAA is jejunum. The major absorption site of FAA is ileum. The major production site of PAA is duodenum. The major digestion site of FAA is jejunum. The major absorption site of FAA in mesenteric system is ileum. The absorbility of cationic amino acids in 5 parts are similar. Acidic amino acids and the most of neutral amino acids are absorbed major in ileum, but some of neutral amino acids are absorbed major in jejunum. This study provides the theory basis for amino acid nutrition in small intestine of sheep and for digestion and absorption regular pattern of protein of sheep.

This research aimed to study the effect of dietary nutrient levels on the development of late pregnancy ewes and embryos. Sixty-six primiparous and healthy Hu ewes with double lambs were provided with TMR diets ad libitum for the first 90 days of pregnancy, and the ewes ((44.45±2.2) kg body weight) were randomly divided into 3 groups with 11 replicates for each group and 2 ewes per pen. Each group was offered the dietary with the same concentrate supplement and forage but the different ratio of concentrate/forage for 5∶5, 4∶6 and 3∶7(DM basis), respectively from 90th days of pregnancy. Three ewes from each group were slaughtered on the 140th days of gestation for the slaughter experiment. The results showed as follows: 1) With the nutrient levels decreasing, the body weight of ewes had decreased trend (P<0.10). No significant changes were found in abdominal circumference during late pregnancy of ewes (P> 0.05), but the ewes’ body weight gain were significantly affected (P<0.05). 2) The dietary nutrient levels had no significant effect on the weight of pregnancy appendages, amniotic fluid and fetal in late pregnancy of ewes (P>0.05), but the fetal weight had the decreased trend (P<0.10) with the nutrient levels decreased. 3) Body size and organs weight of fetal in late pregnancy were not affected by the different nutrient levels (P>0.05), except the heart weight (P<0.05),and the body height, body length and the weight of liver, lung, stomach, small intestine had the decreased trend (P<0.10). The late pregnancy dietary nutrient levels resulting from concentrate supplement/forage could affect the ewes’ body weight gain, and the ewes would reduce their body weight gain to maintain the development of ewe pregnancy appendages, embryo body size, tissues and organs when it did not exceed the tolerance range of maternal, but which still impeded the normal development of embryo.

This experiment was conducted to study the substitution effect of green sweet sorghum stalks for diet on the growth performance, carcass yields and meat quality in geese, and explore the feeding technology using green sweet sorghum stalks substituting diet. Three hundred and sixty 28-day-old Sichuan White geese (male and female in half) with an initial average body weight of (1.11±0.008) kg were randomly allocated into 6 treatments (control group, experimental group I-V) containing 6 replicates with 10 birds per pen. The birds in control group was provided the basal diet ad libitum without the green sweet sorghum stalks; the birds in group I-V were fed 96%, 92%, 88%, 84% and 80% of basal diet fed in control group, respectively, and green sweet sorghum stalks were provided ad libitum. The experiment lasted until 70 days of age. The results showed that: 1) The final body weight and average daily gain in control group was not significantly different from those in group I and II (P>0.05), the average daily gain in control group and group I were higher than those in group III, IV and V (P<0.05). 2) No significant difference was observed in dressing percentage, breast muscle percentage and leg muscle percentage among the 6 treatment groups (P>0.05); compared with control group, abdominal fat percentage in group I-V and skin subcutaneous fat percentage in group I, IV and V were significantly decreased (P<0.05), the gizzard index in group I-V was significantly higher than that in control group (P<0.05). 3) No significant difference was observed in the content of crude protein, intramuscular fat and inosine monophosphate in breast muscle among the 6 treatment groups (P>0.05); the content of total amino acids, essential amino acids, delicious amino acids, saturated fatty acids and unsaturated fatty acids in the breast muscle in group I-III were not different from those in control group (P>0.05). Collectively, it seems to be feasible to replace partial diet with green sweet sorghum stalks for geese, and 4％-8% of diet can be replaced with green sweet sorghum stalks without negative effects on growth performance, carcass yields and meat quality in Sichuan White geese from 28 to 70 days of age.

The aim of this study was to clone and express the SpaA gene of Erysipelothrix rhusiopathiae, and analyze its immunogenicity, thus provide the basis for the development of a new swine erysipelas vaccine. The SpaA gene was amplified using PCR and then connected to the pGEX-6P-1 vector. The positive recombinant plasmid was transformed into Rosetta (DE3) of E. coli and SpaA protein was expressed by induction of IPTG. The expression product was analyzed using SDS-PAGE. The fusion protein was purified by GST affinity chromatography and identified by western blot. The mice were immunized with purified SpaA protein, and then the IgG antibody and cytokine levels of Th1, Th2 were measured with an indirect ELISA. All immunized mice were challenged by Erysipelothrix rhusiopathiae with different pathogenicity, and the pathological changes and the protective rate were observed. The results indicated that the SpaA gene of Erysipelothrix rhusiopathiae was successfully expressed in E. coli, and the molecular weight of purified target protein was 75 kD, which can specific recognize antiserum of Erysipelothrix rhusiopathiae. SpaA can induce high levels of specific IgG antibody in mice, and the levels of TNF-β, IFN-γ, IL-5, IL-10 were significantly increased. The immune challenge-protection rate was 100%. There were obvious differences between pathological changes in control group and experimental group. The experiment suggests that SpaA recombinant protein has good antigenicity, which can stimulate the humoral and celluar immunity, and produce powerful immune protection. Therefore, it can be used as a candidate antigen for developing subunit vaccine of swine erysipelas.

To investigate subcellular localization and effect on type Ⅰ interferon (IFN) response of nonstructural protein 7 (Nsp7) of porcine epidemic diarrhea virus (PEDV), the Nsp7 gene sequence was predicted by bioinformatics, the Nsp7 gene of Chinese PEDV epidemic strain was cloned and inserted into the eukaryotic expression vector pCAGGS. The expression and subcellular localization of Nsp7 in transfected cells were determined by Western blot and indirect immunofluorescence assay respectively. The effect of Nsp7 on type Ⅰ IFN response was evaluated by dual luciferase reporter gene assay, ELISA and virus replication-inhibition bioassay, respectively. Gene cloning and sequence analysis results showed that PEDV Nsp7 gene was 249 bp in length, and it was highly conserved among different PEDV strains. Western blot and indirect immunofluorescence assay showed that Nsp7 was highly expressed and localized mainly in cytoplasm. Dual luciferase reporter gene assay indicated that Nsp7 strongly inhibited the IFN-β promoter activity. ELISA results showed that Nsp7 could significantly inhibit the expression of IFN-β in the protein level. VSV replication assay revealed that Nsp7 significantly inhibited type Ⅰ IFN antiviral activity induced by poly(I:C). Our results implied that Nsp7 was a highly conserved protein of PEDV and exhibited antagonistic function on type Ⅰ IFN response. The results have laid a foundation for exploring the innate immunity evasion mechanism of PEDV and guiding the clinical application of vaccines.

In order to decipher the mechanism of PPV-VLP presentation by dendritic cells (DC_s), CD172a⁺ CD11R⁺ DC and CD4^- CD8⁺ T cell were isolated from spleen of unvaccinated and CSFV vaccinated pigs, respectively, using magnetic beads. Purified splenic DCs were preincubated for 1 h with primaquin, cycloheximide, chloroquine, lactacystin, brefeldin A, leupeptin, or pepstatin. Then PPV-VLPs-E290 was added, and the DCs were incubated for 4 h in the continued presence of these different drugs. The presentation assay of PPV-VLPs-E290 was monitored by cytotoxic activity of CD8⁺ T cells, which was measured using the Lactate dehydrogenase (LDH) release assay. The results showed that incubation of DCs with PPV-VLPs-E290 in the presence of primaquin and leupetin did not block PPV-VLPs-E290 presentation. However, the present efficiency of DCs pretreated with chloroquin, cycloheximide, brefeldin A and lactacystin was severely reduced. These results showed that DCs can cross-present PPV-VLPs-E290. Both acidification of late endosomes and protease hydrolysis are necessary for PPV-VLPs-E290 processing.

To investigate the mechanism of chicken stress granule (SG) formation and the role of Ras GTPase- activating protein-binding protein 1 (G3BP1) in SG formation and virus infection, immunofluorescence assay was utilized to evaluate G3BP1 accumulation in Newcastle disease virus (NDV) infected HeLa cells. GFP-tagged chicken G3BP1 and its 4 domains were transfected into HeLa cells, respectively, before exposed to various stressors. Finally, G3BP1 was transfected into cells before NDV infection. Western blot and TCID₅₀ test were utilized to evaluate virus replication. The results showed that NDV infection induced endogenous G3BP1 accumulating as SGs. Transfection of exogenous chicken G3BP1 into either HeLa, DF-1 or CEF cells induced SGs in the presence or absence of environmental stress. Then the role of 4 domains of chicken G3BP1 in SG formation was evaluated. The results showed that RNA-binding domain was responsible for SG accumulation. In contrast, NTF2-like domain inhibit SG formation. Finally, it confirmed that G3BP1 overexpression enhanced NDV replication, indicating the pro-viral role of SG in NDV infection. These results demonstrated that chicken G3BP1 plays an important role in SG formation. Chicken SG promotes NDV replication.This study lay the foundation for the interactions between NDV and chicken cells.

To screen siRNAs that can suppress the replication of duck hepatitis A virus 1 (DHAV-1) and explore the application of RNAi technology for the control of DHAV-1. The RNA-dependent RNA polymerase (RdRp) gene was amplified by PCR. pEGFP-RdRp was constructed for fusion expression of EGFP-RdRp protein. According to the sequence of RdRp, three gene-specific siRNAs were designed and corresponding shRNA was inserted into pmiRZipΔ to construct pRdRp-shRNA. pRdRp-shRNA and pEGFP-RdRp were co-transfected into HEK293T cells for effective siRNA screening with fluorescent microscopy, flow cytometry, and quantitative fluorescence PCR. More effective siRNAs were selected for preparation of recombinant lentivirus using lentivirus vector pmiRZip. The suppressing effect of entivirus-Mediated siRNA was determined by calculating the TCID₅₀ and RdRp gene expression of DHAV-1 in the duck embryo fibroblast (DEF) cells infected with recombinant lentivirus followed by DHAV-1. The results indicated that the three siRNAs screened all could suppress the RdRp gene expression, and the shRNA2 containing GDD motif had the highest efficiency. The recombinant lentivirus corresponding to shRNA2 reduced the TCID₅₀ of DHAV-1 by 6.2 lg and RdRp gene transcription by 89.6%, with the suppressing effect continued for at least 120 h. This work provides a new idea for the clinical control of duck virus hepatitis.

The aim of this study was to investigate the difference of bacterial flora and diversity of the saliva and midgut contents of Rhipicephalus microplus with the extension of feeding time, and the proliferation and migration characteristics of dominant genera in saliva and midgut. The saliva (S) and midgut contents (M) collected from partially (P) or fully (F) engorged adult female ticks under sterile environment, the total DNA of bacteria were extracted and PCR amplification. Using Illumina MiSeq sequencing platform, two-terminal sequencing of V3-V4 areas of 16S rDNA was carried. Then OTUs (operational taxonomic units) picking, taxonomy assignment, and diversity analysis were carried on the effective Tags. The results showed that 134 317 sequences were obtained, the total of OTUs were 1 304, OTUs obtained from four samples (SP, SF, MP and MF) were 336, 332, 269, 289, respectively. 152 OTUs had high similarity in 4 samples. Proteobacteria and Firmicutes were the most predominant phyla in all samples. Proteobacteria had higher abundance in midgut than saliva. On the contrary, Firmicutes was highly abundant in saliva. Coxiella, Acinetobacter, Methylobacterium, Brevundimonas, Rickettsia and Escherichia were the major genera. Coxiella and Acinetobacter were highly abundant in all samples. The abundance of Coxiella was higher in partially engorged than fully engorged ticks. Acinetobacter had higher abundance in midgut than saliva. These findings suggested that the saliva and midgut of R. microplus had complicated microbial community structure. The distribution of bacteria were different in different tissues, and the abundance of bacteria changed with the feeding time.

Using ESE-Quant tube scanner testing platform, we established a real-time fluorescence loop-mediated isothermal amplification (RTF-LAMP) method for rapid detection of hCD39. The method employed a set of six specially designed primers for amplification of nucleic acid under isothermal conditions at 63 ℃ for 45 min. The amplification process of LAMP was monitored by the addition of SYBR Green Ⅰ dye prior to amplification. The fluorescence intensity of SYBR Green Ⅰ dye was used as the standard of the results. Results of the study showed that the sensitivity of the method was 3 fg•μL^－1, and there was no cross-reactivity with the genes of related and unrelated detected, such as hCD46, LEA29Y. More importantly, this method could be completed testing of buccal swabs specimens of transgenic pigs. The purpose of our study was to develop a simple, economical, rapid method with good sensitivity and specificity. The method could be no damage to the living body samples for testing, suitable for quick and easy on-site operation.

This experiment was conducted to study the residue dynamics of oil adjuvant in eggs and chicken. One hundred and twenty 1-day-old Jingfen layer chickens were raised. Every group was allocated into 12 replicates and 10 layer chickens per replicate. All the right wing shoulder muscle was injected in accordance with the recommended injection dose of oil adjuvant vaccine immune program. 3 eggs were collected randomly from every replicate of group at 159, 166, 180, 201, 235, and 271 d to measure the residual of n-alkanes and PAHs. Six chickens were chosen randomly from group to slaughter at 280, 330, 380, 430 d, collecting right wing shoulder muscle, left thorax muscle, right thorax muscle, left leg muscle, right leg muscle, liver and kidney to measure the residual of n-alkanes and PAHs. The results showed as follows: The alkanes with smaller molecular weight of mineral oil, such as n-hexadecane, n-heptadecane, n-octadecane, were easier to metabolize and transfer to other part of chicken. The amount of mineral oil in injection site was most, the residue amount of liver and kidney also more by general circulation, and residue elimination rate was slower. n-alkanes and PAHs in all samples were declined as time goes on. Then it had not detected PHAs in egg. The mineral oil transferred to eggs is too little, which has no effect on the egg quality. The mineral oil residue time in the chicken body is long, the long chain alkanes eliminated and migrated need more time to metabolized completely in the chicken.

The aim of this study is to gain a clear understanding of the expression distribution of ACE 2 through both in vivo and in vitro experiments and also to study ACE 2’s anti-injury effect in dairy cow mastitis induced by long-term high concentrate feeding. In vitro, mammary tissues samples from the lactating Holstein cows and Mac-T cells were used. The mRNA and protein expression of ACE 2 in bovine mammary gland was determined using RT-PCR and Western blotting. The location of ACE 2 protein in Mac-T cell was determined using immune fluorescence method. In vivo, 6 healthy lactating Holstein cows were divided into 2 groups: normal feeding group (40% concentrate, dry matter basis) and high concentrate feeding group (60% concentrate). After 20 weeks’ feeding, mammary biopsy samples and milk samples were collected. Milk LPS content, somatic cell number, as well as some inflammatory indicators including NAGase, AKP and MPO were tested. Histopathological test was also performed. The mRNA expression level of ACE 2, ACE and their receptors AT 1 and MasR in mammary tissues were analysed using RT-qPCR. The contents of AngII and Ang1-7 were also determined using ELISA.The results showed that both mRNA and protein expression of ACE 2 were found in dairy cow mammary gland, ACE 2 was located in the cytoplasm and on the membrane of the Mac-T cell. With a long-term high concentrate feeding, somatic cell number and LPS content in milk significantly increased (P<0.01); the activities of NAGase and AKP were significantly higher than those in the control group (P<0.05), MPO activity had no significant differences between the 2 groups; mammary gland tissues in the high concentrate group cows found inflammatory injury changes. The mRNA expression level of ACE, ACE 2, AT 1, MasR increased, and the mRNA ratio of ACE 2/ACE was higher than the control group. Ang1-7 content was significantly higher in breast tissue (P<0.05), AngII content was significantly lower ( P<0.05).Conclusion: It is the first time that the presence of ACE 2 in bovine mammary gland has been confirmed. When mammary inflammatory injuries are induced by long-tern high concentrate feeding, the local RAS in mammary is activated, ratio of ACE/ACE 2 varies and the ACE 2-Ang1-7-MasR pathway takes the dominat position, that is to say that the anti-injury effect of ACE 2 becomes significant. ACE 2 helps to protect the mammary from inflammatory injuries by degrading Ang II to Ang1-7.

In order to explore the regulation mechanism of probiotic Saccharomyces cerevisiae inducing Sheep Beta-Defensin-1 (SBD-1) gene expression in sheep rumen epithelial cells (RECs), we studied the NF-κB and MAPKs signal pathways. Firstly, the RECs of sheep were cultured successfully as in vitro experimental model, and then selected the concentration and incubation time of S. cerevisiae, inducing SBD-1 expression highest, to preliminarily study the subsequent signaling pathways. Real-time fluorescence quantitative PCR (RT-qPCR) was conducted to determine mRNA expressive variation of the toll like receptor 2 (TLR2), myeloid differentiation factor88 (MyD88) and related factors of Nuclear factor-kappa B (NF-κB) and Mitogen-activated protein kinases (MAPKs) pathways in the established model to induce SBD-1. And then four specific inhibitors of NF-κB and MAPKs pathways were chosen, which were PDTC (NF-κB signaling pathway inhibitor), SB202190 (P38 signaling pathway inhibitor), PD98059 (ERK1/2 signaling pathway inhibitor) and SP600125 (JNK signaling pathway inhibitor). Four kinds of inhibitors were used to treat cells by single or combined with each other and then further induced culture, while using RT-qPCR methods for detecting expression level of RECs SBD-1 mRNA after inhibitor treatment. The results showed that the cells stimulated by S. cerevisiae resulted in the transcription of NF-κB, P38, JNK, ERK1/2, TLR2 and MyD88 were significantly increased (P<0.05). And PDTC, SB202190, SP600125 and PD98059 significantly inhibited (P<0.01) the SBD-1 transcription of stimulated cells that previously treated with the inhibitor or inhibitors, and the SB202190 had the best inhibitory effect. The results suggest that the pathways of S. cerevisiae inducing SBD-1 transcription may be related to TLR2-MyD88-NF-κB/MAPKs, but the TLR2-MyD88-P38 of TLR2-MyD88-MAPKs may be as main signaling pathway.

This study aimed to analyze the projects of porcine reproductive and respiratory syndrome virus (PRRSV) funded by National Natural Science Foundation of China (NSFC) since 1995. The results demonstrated that the number of PRRSV funded by NSFC showed a general upward trend from 1995 to 2016, which increased most significantly from 2008 to 2015. The main bodies of the supporting institutions were universities, among them, Huazhong Agricultural University had the largest number of projects, while China Agricultural University had the largest total funds. In addition, the PRRSV research funded by NSFC mainly focused on veterinary infectious diseases. The number of projects directed by leaders who had two or more grants accounted for 43.8% of the total 128 projects. Papers published in Science Citation Index (SCI) journals covered heredity and variation, replication, pathogenesis, immune mechanism, as well as comprehensive prevention and control of PRRSV, indicating PRRSV research funded by NSFC has reached the international leading level.

The aim of this study was to explore the effect of octamer-binding transcription factor 4(Oct 4) during yak oocytes maturation and embryos development, which was achieved by examined the expression of transcription factors Oct 4 at different stages. In this study, yak cumulus-oocyte complexes (COCs) were collected and matured in vitro, the embryos were produced by the method of in vitro fertilization (IVF) and the development competence of oocytes and embryos at different stages were evaluated. The expression and distribution of Oct 4 in the level of mRNA and protein in COCs and embryos at different stages were detected by quantitative real-time PCR (qRT-PCR) and indirect immunofluorescence. The results showed as follows: 1) The rates of embryos developed to stages of 2-4-cells, 5-8-cells, 9-16-cells and blastocyst were (64.66±1.76)%, (43.33±1.76)%, (24.66±2.40)%, (9.33±0.67)%, respectively. 2) The expression of Oct 4 gene could be detected at all stages during yak oocytes maturation and embryos development, which was highest in 9-16-cells embryos, followed by blastocyst, 2-4-cells embryos and 5-8-cells embryos, while it were lowest in immature COCs and mature COCs.3) The result of indirect immunofluorescence method showed that the protein of Oct 4 was mainly distributed in the cytoplasm of COCs and embryos,which was weaker in the nucleus.Analyzing by immunofluorescence IOD(integrated optical density) showed that the IOD of 9-16-cells embryos was highest, followed by blastocyst, 2-4-cells embryos, which was lowest in 5-8-cells embryos. The results showed that Oct 4 could express in various periods of yak oocytes and embryo development, while the expression levels of Oct 4 were different, which indicated that Oct 4 might participate in the embryonic genetic transcription activation at stages of 9-16-cells, which might take part in maintaining tipotency of inner cell mass in blastocyst.