Skeletal muscle is the most important part of the animal body, maintaining the body movement and providing human with the necessary meat products. The difference in the regulatory mechanisms during skeletal muscle growth and development can cause differences in muscle yield and meat quality. Long intergenic noncoding RNA (lincRNA) is a class of RNA located in the intergenic region, which is longer than 200 nt and has a lower protein-encoding potential. The expression of lincRNA has space-time specificity and tissue specificity, and exerts biological functions in epigenetic regulation, transcriptional regulation and post-transcriptional regulation.In recent years, the popular high-throughput RNA-seq technology has identified a number of lincRNA associated with skeletal muscle development, model organism studies such as human and mouse have shown that lincRNA is involved in the regulation of skeletal muscle growth and development, muscle cell growth and proliferation, migration, differentiation and apoptosis. In this review,we summarized the evolutionary conservation,the characteristics of lincRNA, and its research progress in the regulation of skeletal muscle development and domestic animal.

With the continuous development and application of high-throughput sequencing technology, transcriptome analysis method is developed for mining genes with important function. However, a lot work needs to be done for efficient and accurate transcriptome analysis based on massive sequencing data. Here, we reviewed methods for reads quality control, reads mapping, genome annotation, transcripts assembling, expression quantification, differential expression analysis for RNA-seq data. We summarized the performance and scope of application of the common softwares, algorithms and databases used. We also reviewed analysis methods such as protein regulatory interaction networks as well as weighted gene co-expression networks. Transcriptome analysis has been evolved from identifying differentially expressed gene within-species to utilizing related species as reference to mine the functional genes in target species. By combining with various methods, such as the homologous gene prediction, select signal detection, extreme data analysis, GO annotation and KEGG enrichment and bulked segregant RNA-Seq (BSR-Seq) methods, the results from RNA-seq analysis are more scientific and reliable. With the development of sequencing technology and data analysis methods as well as continuous improvement of database resources, the underline gene regulation and the law of life implied in the sequencing data will be uncovered accurately and deeply in future.

This study was designed to investigate the population genetics parameters and selection signatures of Enshi Black pig. Using the Porcine 80K SNP chip, a series of population genetics parameters, including the relationship coefficient, the inbreeding coefficient, the linkage disequilibrium and the effective population size, were calculated to evaluate the population structure relationship of Enshi Black pig. In addition, the CLR and iHS methods were used to identify the potential selection signatures and then the corresponding selected genes were revealed by bioinformatics analysis. The results indicated that the mean value of relationship coefficient was 0.12 for all individual-pairs in Xianfeng county, the inbreeding coefficient of 16% of all individuals was higher than 0.125. In addition, this study presented a linkage disequilibrium map for the whole genome of Enshi Black pig. Analysis of the past effective population size based on the information of linkage disequilibrium suggested a decline in effective population size, and up to Ne=25 before 5th generation. Based on CLR method, a total of 126 significant potential selection signature regions were detected, which were approximately 51.6 Mb in length, accounting for approximately 2.1% of the total length of the genome. Similarly, a total of 248 significant potential selection signature regions were detected using iHS method, which spanned approximately 78.78 Mb and accounted for approximately 3.2% of the total genome length. Enrichment analysis showed that genes such as LPAR2, NDUFA13, MEF2B and AHR overlapping with the selection signatures region were associated with carcass length (drip loss), sperm formation, skeletal muscle differentiation and total litter size. This study suggests that the genetic basis of Enshi black pig is narrow and inbreeding is obvious, and the effective population size is limited, and it continues to shrink. The candidate genes revealed by sweep analysis can provide some references for the future genetic improvement of Enshi Black pig.

This study was conducted to evaluate the expression patterns and polymorphisms of LHR gene in the hypothalamic-pituitary-ovarian axis (HPOA) and its association with litter size in Small Tail Han sheep, and further understand its effect on lambing traits of Small Tail Han sheep. The real-time fluorescence quantitative PCR (qPCR) method was used to detect the expression of the LHR gene in reproductive and brain tissues of 6 Small Tail Han sheep (3 polytocous and 3 monotocous ewes from the FecB ++ genotype) in this study. The 380 Small Tail Han sheep and total 380 sheep for Small Tail Han sheep, Tan, Sunite, Cele Black, Hu and Prairie Tibetan sheep were selected, and Sequenom MassARRAY^®SNP assay was applied to detect the polymorphism of 7 single nucleotide polymorphism sites (SNPs) of LHR gene. Then the association between LHR and litter size was analyzed in Small Tail Han sheep. The results showed that LHR gene was expressed in the brain, hypothalamus and ovary tissues, and highly expressed in ovary. The expression of LHR gene in ovary, brain and hypothalamus of polytocous Small Tail Han ewes was extremely significant higher than that of monotocous ewes (P<0.01). The genotype frequencies and allele frequencies of the 4 SNPs were extremely significantly different between monotocous and polytocous sheep breeds (P<0.01). Population genetic analysis indicated that 7 SNPs showed moderate polymorphism in most sheep breeds (0.25 < PIC < 0.5); The result of chi-square test showed that 7 SNPs were in Hardy-Weinberg equilibrium in most sheep breeds (P>0.05). Association analysis showed that the polymorphism of one SNP in the LHR gene were significantly correlated with litter size in Small Tail Han sheep (P<0.05), and the polymorphism of 2 SNPs were extremely significantly correlated with litter size in Small Tail Han sheep (P<0.01). The preliminary results of this study showed that there was a correlation between the polymorphisms of 3 SNPs of LHR gene and litter size in Small Tail Han sheep, and the LHR gene may be involved in the regulation of prolificacy in Small Tail Han sheep.

The aim of this study was to investigate the effect of silencing SCAP gene on formation of lipid droplet in dairy mammary epithelial cells. SCAP short hairpin RNA lentivirus vector was constructed, the bovine mammary epithelial cells were packed and infected. SCAP gene silencing cell lines were selected by applying puromycin in culture medium. Effects of SCAP gene silencing on the expression of SCAP gene and protein were detected by Real-time PCR and Western blot, moreover, the effects of SCAP gene silencing and transient transfection of SCAP plasmid on lipid droplet formation were detected using Nile red and Oil red O staining. The results showed that lentivirus recombinant vector with SCAP gene silencing was constructed successfully. SCAP gene silencing stable cell line was obtained via screening of 5 mg·L^-1 puromycin. The expression of SCAP gene in all silent cell lines were significantly reduced(P<0.05 or P<0.01). Compared with control group, the expression of SCAP protein in RNAi-SCAP3 cell lines were decreased by 0.49 fold (P<0.05), the expression of SCD and FAS genes were significantly decreased (P<0.05) in RNAi-SCAP3 cells. Lipid droplet content in RNAi-SCAP3 cell lines was significantly lower than that in control cells (P<0.01). The number of small lipid droplets with size ≤ 2.0 μm were increased, while the number of large lipid droplets with size ≥ 2.5 μm were decreased. After transient transfection of SCAP eukaryotic expression vector into RNAi-SCAP3 gene silencing cell lines, overexpression of SCAP significantly decreased the percentage of small lipid droplets and increased the percentage of large lipid droplets (P<0.05). In this study, SCAP gene silencing cell lines were successfully constructed, and the size of lipid droplets were affected by the expression of SCAP.

This experiment was conducted to study the effects of different training venues on 1 000 m speed race performance, physiological indicators, plasma antioxidant capacity and whole blood glucose metabolism of Yili horse. 12 2-year-old Yili horses with the approximate 1 000 m speed race performance were divided into 2 groups, the sand training group and the grass training group. All the horses were trained by the same training methods and intensity, with the same feeding management and diet nutrition levels for a 30-day training test. The result showed that horses in grass training group used less time in 1 000 m speed race by 4.39%(P<0.05) than those in sand training group. The concentration of SOD,GSH-Px and T-AOC in plasma of horses in grass group were increased by 9.72%(P<0.05), 5.96%(P<0.05), 6.65%(P<0.05) than those in sand training group. The concentration of lactic acid in blood of horses in grass training group was reduced by 14.86%(P<0.05) than those in sand training group. In conclusion, under the conditions of this experiment, the grass training could significantly improve the performance of the 1 000 m speed race of Yili horse, improve antioxidant capacity of plasma before and after exercise, and reduce the accumulation of lactic acid in whole blood.

The research aimed to get 5'UTR sequence of mink DCT gene, characterize its structure, predict the transcriptional regulation elements, detect its promoter activity, and provide a theoretical basis for exploring the role of DCT gene in regulating the fur color formation of mink. 5'UTR of DCT gene of black mink, white mink and coffee mink were amplified and compared. Serial fragments deleted in 5'UTR, pGL3-1-pGL3-7 for coffee mink and pGL3-4-pGL3-6 for black mink, were amplified to construct the recombined luciferase reporter gene plasmid and to measure the promoter activity. The three kinds of minks with different coat colors were detected for the methylation level of CpG island in promoter of DCT gene by bisulfite sequencing PCR. 8 203 bp sequence of 5'UTR in DCT gene was cloned and a transposon with 204 bp in length in g.7133-7336 region was discovered. Among the 100 items with high similarity, one was from Soboliphyme baturini in Ecdysozoa and the others were all from Caniformia. P3 and P4 fragments had significant promoter activity (P<0.05). The methylation level in CpG island of coffee mink was significantly higher than that of black mink and white mink (P<0.05), and the promoter activity of individuals with CC haplotype in coffee mink was significantly lower than those with TT haplotype in black mink (P<0.05). The 204 bp transposable element in 5'UTR of mink DCT gene, special Can-SINEs of Caniformia, was inserted into the genome from Soboliphyme baturini into Ecdysozoa. The 32 bp element and the proximal domain in DCT co-activated the promoter, while the GC-box and CpG islands silenced the promoter activity of mink DCT gene. The CC haplotype, formed from the 2 SNPs of T > C mutation at locus of g.-684 and g.-621, resulted in the higher methylation level and the lower promoter activity in coffee mink, and might ultimately decrease the synthesis of eumelanin and form the coffee coat character.

To explore the role of FGF21 and receptors FGFR1, FGFR 2 in hair follicle development, we researched the localization and expression of FGF21 and receptors FGFR1, FGFR2 in the first hair follicle growth cycle of mice. The immunohistochemistry, real-time PCR and Western blot were used to study the expression of FGF21 and receptors FGFR1, FGFR2 mRNA and proteins in mice back skin on 1, 3, 5, 8, 12, 17, 21, 23 days old. The results showed that during the first hair follicle growth cycle of mice, FGF21 was mainly expressed in dermal papilla, matrix cells, inner and outer root sheaths and connective tissue around hair follicles; FGFR1 was located in various structures of hair follicle, and abundantly expressed in the inner and outer root sheaths in the catagen(12-17 days old) and telogen(18-21 days old); FGFR2 was abundantly located in various structures of hair follicle. The expression levels of FGF21 mRNA at 1, 3, 5, 8, 12 and 17 days old were significantly lower than at 23 days old(P<0.01), and the expression at 21 days old was lower than at 23 days old, the difference was not significant(P>0.05); The expression levels of FGF21 protein were higher at 1-5 days and 23 days old. The expression levels of FGFR1 mRNA at 1, 3, 5, 8, 12, 21 and 23 days old were significantly lower than at 17 days old(P<0.01); The expression levels of FGFR1 protein increased from 12 days old, reached the highest at 17 days old, and then decreased. The expression levels of FGFR2 mRNA at 1, 5, 8, 12, 21 and 23 days old were significantly lower than at 17 days old(P<0.01), and the expression at 3 days old was lower than at 17 days old, the difference was not significant(P>0.05); FGFR2 protein was highly expressed at 1-17 days old. The results showed that during the first hair follicle growth cycle of mice, FGF21 plays an important role in the proliferation and differentiation of fibroblasts involved in hair follicle formation, and induces hair follicles to enter the catagen from the anagen. FGFR1 plays an important role in the proliferation and differentiation of inner and outer root sheath cells, and induces hair follicles to enter the telogen from the catagen. FGFR2 plays an important role in cell proliferation and differentiation in hair follicles, and induces hair follicles to enter the telogen from the anagen and catagen.

The main purpose of this study was to investigate the influence of the adding of ubiquitin-conjugating enzyme(E2) inhibitors to oocyte zona pellucida(ZP) ubiquitination and sperm-oocyte binding ability in vitro maturation of porcine oocytes. There was 6 groups in this study:control group,DMSO,5,10,15 and 20 μmol·L^-1 NSC697923 treatment groups. We analyzed the effect of different concentrations of ubiquitin-conjugating enzyme inhibitor NSC697923 on the expression of ubiquitination in porcine oocytes ZP with Western blot in vitro maturation. The difference of sperm-oocyte binding ability among different groups was examined by Hoechst staining. The result showed that:1) In vitro maturation of porcine oocytes, the treatment groups with 10,15 and 20 μmol·L^-1 of NSC697923C significantly reduced oocyte maturation rate (P<0.05), ZP hardening time (P<0.05) and the sperms-oocyte rate (P<0.05). 2) The ZP proteins in the control group and each treatment groups showed different degrees of ubiquitination at 61, 81 and 106 ku, while the treatment group with 15 and 20 μmol·L^-1 of NSC697923 significantly reduced the ZP ubiquitination protein level (P<0.05).In conclusion, ubiquitin-conjugating enzyme changes the level of ubiquitination of mature oocytes ZP and the ability of sperm-oocyte binding in vitro maturation of porcine oocytes.

The objective of this study was to explore the link between lncRNAs and the mechanism of male infertility of cattle-yak by analyzing the gene expression profile about lncRNA in testis tissue of adult healthy yak and cattle-yak (3-4 years old). This study provided the base for further interpreting the mechanism of male infertility of cattle-yak.Testis tissues of 3 adult healthy yaks and 3 adult healthy cattle-yaks were collected to construct the cDNA libraries. And, the structure and function of genes were analyzed by using high-throughput sequencing and differentially expressed lncRNAs (DElncRNAs) were screened between yak and cattle-yak. Then the cis-targeted and tran-targeted genes of DElncRNAs were predicted by exploring the positional relation and binding energy between lncRNA and mRNA, and the GO and KEGG pathway enrichment analysis was also made. The results displayed that 20 363 and 24 133 candidate lncRNA transcripts of yak and cattle-yak were respectively obtained, 6 178 DElncRNAs of which were selected.Compared with yak, there were 2 470 up-regulated and 3 708 down-regulated lncRNAs in cattle-yak. Meanwhile 2 676 candidate target genes of DElncRNAs were obtained. The analytical results indicated that these genes participated in 58 functional classifications and participated in 306 signal pathways. And, they were mainly involved in such signal pathways as axon guidance, endocytosis and hedgehog. In conclusion, these findings inferred that some lncRNA-mediated target genes (PTGDS, IGF2, MEST, GLIS3, NTOCH2, HOXA10, HOXA11) were associated with male infertility of cattle-yak, and lncRNA may played an important role in the reproductive development of cattle-yak. Moreover, the gap analysis of lncRNA was made by using RNA-Seq technology,which provided more complete data for further exploring the mechanisms of male infertility of cattle-yak on transcriptome.

The study aims to explore the co-culture conditions of sheep trophoblast cells (STCs) and the endometrial luminal epithelial cells (ELECs), and provides experimental basis for chorionic trophoblastic coenocyte formation. Harvesting healthy sheep uterus and placental tissue from 45 to 60 days of gestation at the slaughter house, and the enzyme digestion method has been used to isolate and culture the STCs and ELECs in vitro, and they were identified by cellular immunofluorescence. Simultaneously, the optimal co-culture conditions for STCs and ELECs were determined by Giemsa staining. Then, the empty plasmid pEGFP-C1 was transfected into ELECs for 48 h, and then co-cultured with STCs. The morphology of chorionic trophoblastic coenocyte was observed by confocal laser scanning microscopy. The results showed that STCs CK-7 and ELECs CK-18 were positive.When the ratio of STCs to ELECs cells was 2:1, the number of multinucleated cells reaches the maximum when the co-culture time was 48 h, and it was significantly different from the control group (P<0.05). The results of immunofluorescence showed that CK-7 expressed red fluorescence in STCs, and there were pEGFP-C1 labeled green fluorescence in the cells, and the nucleus was blue-stained, and the 3 fluorescent co-expressions showed white light. This study established the conditions for co-culture of STCs and ELECs, and showed that ELECs were involved in the formation of polynuclear cells in sheep trophoblasts.

The objective of this study was to investigate the effects of dietary chlorogenic acid (CHA) and hesperidin (HDN) on growth performance and intestinal function in weaned piglets. Forty Landrace×Yorkshire×Rongchang healthy castrated weaned male piglets ((28±2) d, (8.97±1.48) kg) were randomly divided into 4 groups (10 repeat per group, 1 piglet per repeat):control group (CON), Bacilli peptide group(BT), HDN group, CHA group. The experiment lasted for 33 d including 5 d adaptation period. The growth performance, biochemical indicators of plasma and liver, intestinal mucosa morphology, the expression of genes and proteins related to intestinal mucosa and colon microflora were determined. The results showed that:1) Compared with control group, the addition of CHA and HDN in basal diet of weaned piglets significantly increased the average daily gain, average daily feed intake, thymus index and small intestine length index (P<0.05), and significantly improved the total antioxidant capacity and the activity of total superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase (P<0.05). 2) The height and integrity of jejunum villus of piglets in CHA, HDN and BT groups were better than those in CON group (P<0.05). 3) The β-defensins 2 concentration of piglets in CHA group were higher than those in CON group (P<0.05). The TNF-α mRNA level in jejunum villus of piglets in CHA group was higher than those in HDN and CON groups (P<0.05). The IL-8 mRNA level in jejunum villus of piglets in HDN group was higher than those in BT group (P<0.05). There was no significant difference in HSP₉₀ and TLR4 protein levels and TGF-β1 mRNA abundance among 4 groups (P>0.05). 4) CHA and HDN could maintain the diversity of the colonic microbiological flora of weaned piglets and protect the intestinal micro-ecological balance. The result indicate that dietary supplementation of chlorogenic acid and hesperidin increased average daily gain, average daily feed intake, jejunal mucosal villus height, and significantly reduced the ratio of feed to meat of weaned piglets. Chlorogenic acid plays an anti-bacterial role by promoting the expression of BD2 in intestinal epithelium and plays an anti-inflammatory role by enhancing TNF-α. Chlorogenic acid and hesperidin can maintain the diversity of intestinal microflora in weaned piglets.

The aim of this study was to explore the effects of probiotic Saccharomyces cerevisiae (S.c) and inactivated S.c on the expression of sheep-defensin-1 (SBD-1) in rumen explants of sheep. After different concentrations (10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ CFU·mL^-1) of S.c and inactivated S.c were respectively co-cultured with explants for 24 h, the expression of SBD-1 mRNA and protein in rumen explants were detected by qPCR and ELISA to determine the optimal concentration for the induction of SBD-1 expression by S.c and inactivated S.c. Then, the sheep rumen explants were stimulated with the optimal concentration of S.c and inactivated S.c for 2, 4, 8, 12, 16, 20, 24 h, respectively, and the SBD-1 mRNA and protein were also detected by qPCR and ELISA, so as to screen out the optimal time for the induction of SBD-1 expression by S.c and inactivated S.c. The results showed that S.c and inactivated S.c could significantly promote the expression of SBD-1 in rumen explants (P<0.05). When the rumen explants of sheep were stimulated by the concentration of 10⁷ CFU·mL^-1 for S.c and 10⁸ CFU·mL^-1 for inactivated S.c for 16 h, respectively, the expression level of SBD-1 were the highest, and which were significantly different from the control (P<0.01). Comparing the induction effects between S.c and inactivated S.c under optimal induction conditions, the S.c were better. Therefore, S.c and inactivated S.c could promote the SBD-1 expression in rumen explants of sheep, and the expression of SBD-1 reached the maximum when the rumen explants of sheep were stimulated by the concentration of 10⁷ CFU·mL^-1 for S.c and 10⁸ CFU·mL^-1 for inactivated S.c for 16 h. The induction effect of S.c is better than that of inactivated S.c.

The aim of this study was to isolate and identify bovine rotavirus (BRV) from the diarrhea stool samples of Yak. BRV-positive samples detected by RT-PCR was used to inoculate into MA-104 cells for virus isolation and identification, and the complete genes of VP4, VP6 and VP7 were sequenced to determine their molecular characteristics. The cytopathic effect occurred after three continuous blind passages, and the stable time that cytopathic effect occurred was found after seven passages. The virus titer was 10^8.39TCID₅₀·mL^-1 after plaque purification. The isolate, named HY-1 strain, was confirmed to be BRV by RT-PCR, indirect immunofluorescence and electron microscopy. The complete VP4, VP6 and VP7 genes of HY-1 strain were sequenced successfully, showing that HY-1 strain belong to G6P[11] I2 genotype. The phylogenetic analysis showed that the complete VP4, VP6 and VP7 segments of HY-1 strain were closely genetic related to Chinese bovine DQ-75 strain and Indian bovine M-1 and RUBV319 strains, respectively, which indicated that HY-1 strain may be a reassortant strain. Compared with the domestic G6 and P[11] BRV strains, VP4 and VP7 of HY-1 isolate had significant variation in the important amino acid regions. In this study, a BRV strain was successfully isolated from yak with G6P[11] genotype. To the best of our knowledge, this is the first report on the identification of G6P[11] BRV in China.

Epizootic haemorrhagic disease (EHD) is an infectious, non-contagious insect transmitted disease of ruminants. To investigate the infection of epizootic hemorrhagic disease virus (EHDV) among ruminants in Yunnan Province of China and study the virus genetic characteristics, monitoring points were set up in Shizong. Virus isolation was carried out through the inoculating BHK-21 cells with EHDV nucleic acid positive blood samples collected from the sentinel herds. Serotype of the isolated virus was identified by serum neutralization test; ORF of Seg-2 and Seg-3 of the virus were amplified with specific primers, then cloned and sequenced; antibody levels and viral nucleic acid in the EHDV infected animals throughout the year were tested by C-ELISA and qRT-PCR. Results were as follows:In August 2013, an EHDV strain (YNSZ/V269/2013) was isolated from Sentinel Cattle in Shizong, Yunnan. The serum neutralization test showed that the YNSZ/V269/2013 was an EHDV-7 virus. Sequence analysis of Seg-2 and Seg-3 showed that the isolated virus belonged to EHDV-7 Eastern type and had the closest genetic relationship with the Serotype 7 strain in Japan or Australia. The animal serum antibody increased rapidly after being infected by EHDV-7 and the antibody reached the highest point after 3 weeks and lasted for a longer time; meanwhile the nucleic acid content of the virus declined rapidly, 7 weeks later could not be detected in blood samples. This study reported the first isolation of EHDV serotype 7 in China and the virus sequence characterization of Seg-2 and Seg-3. The research results provide the basis for further development of the whole genome sequencing of Chinese EHDV-7 virus, epidemiological investigation, establishment of diagnostic methods and pathogenicity research.

To clarify the epidemiological characteristics of diarrhea in pigs around Shanghai, especially the prevalence of coronavirus, we used viral metagenomic method to detect viruses in stools from 90 diarrhea piglets from 6 swine farms in parts of Shanghai. And then a multiple sequence alignment was carried out using Clustal W and Geneious, and the phylogenetic trees were generated by the distance-based maximum likelihood method using MEGA 7.0. The data showed that the intestinal virus community of diarrhea piglets was mainly composed of Picornaviridae (63.67%), Astroviridae (12.68%), Caliciviridae (4.07%), and Parvoviridae (2.51%). We found that the farm C was the only farm where PEDV is the main cause of piglet diarrhea, and the positive rate of PEDV infection in farm C was as high as 43.33%(13/30). The genetic evolution analysis showed that one of the three PEDV ORF3 gene sequences obtained in this study belonged to the G2 genotype, and the homology with other reference PEDV strains was higher (96.44%-99.70%), which was relatively conservative; the other two ORF3 gene sequences belong to the G1 genotype, and the homology was relatively lower (95.11%-99.41%). In the comparison analysis of protein sequence with the traditional strain CV777, we found that in addition to the piglet91 strain showed a mutation of F2S, three ORF3 sequences both showed consistent mutations, namely:V21A, I70M, V79I, F80V, L85I, L92F. This characteristic may be regarded as the basis for identification of PEDV traditional strains and the popular strain genotype. The enterovirus of diarrhea piglets from surrounding areas of Shanghai were rich in composition. The infection status in the different pigs were significantly different. Apart from PEDV, astrovirus and calicivirus are also the main pathogens of diarrhea piglets. As one of the main pathogens of piglet diarrhea in this particular region, the ORF3 gene of PEDV has unique molecular characteristics compared with traditional isolates. The relationship between mutation site and viral virulence of the newly detected virus needs further experimental research.

This study was designed to investigate the effects of TLR4 signaling pathway in RAW264.7 cells treated with lipopolysaccharide (LPS) in two different virulent Pasteurella multocida serotype A isolates (high-virulent PmCQ2 and low-virulent PmCQ6) respectively. The activation of TLR4 pathway, expression of TNF-α and IL-12p40, phosphorylation of IκBα, and nuclear translocation of NF-κB, were identified in LPS-stimulated RAW264.7 cells. Results showed that the expression of TLR4, TNF-α and IL-12p40 was significantly increased in LPS-stimulated RAW264.7 cells. Furthermore, both LPS_PmCQ2 and LPS_PmCQ6 up-regulated IκBα phosphorylation, and activated TLR4-triggered nuclear translocation of NF-κBp65, but there was no significant difference between the two treatments. The LPS of bovine Pasteurella multocida Serotype A high-and low-virulent strains can induce the TLR4-mediated immune response, and there is no difference in TLR4-mediated IκBα-NF-κB signaling pathway, indicating that there may be no significant difference in virulence between LPS_PmCQ2 and LPS_PmCQ6 on RAW264.7 cells. These results suggested that for RAW264.7 cells, the virulence of Pasteuralla multocida is dependent on others virulence factors, not LPS.

The protein components of Haemaphysalis flava eggs were investigated, and the key nutrients were analyzed in the development of embryo, in order to lay a foundation for screening antigen molecules that can interfere with embryonic development. Firstly, the egg protein components of fresh Haemaphysalis flava were analyzed by LC-MS/MS, and based on the salivary gland transcriptome translation library, the midgut transcriptome translation library and the Uniprot database, each protein components were identified. A total of 221 unique peptides were detected from the yolk protein extract of Haemaphysalis flava, and 53 kinds of proteins were identified, among which 12 were highly confident proteins. The functional proteins that can be identified were actin and vitellogenin 2 (Vg-2). Vg-2 is completely same with the primary structure of vitellins (Vn). The protoplasts of the eggs contain less protein, and there is only one type of vitellogenin (Vg-2).

Aiming to find the transcription difference of albendazole resistant strain of Haemonchus contortus,before and after administration. In this study, Samples of bBZ (before albendazole administration) and aBZ (after albendazole administration) were analyzed using Illumina Hiseq4000 RNA sequencing platform and their transcriptome libraries were constructed. Screening for differentially expressed genes(DEGs) and functional annotation and enrichment analysis by GO and KEGG databases,at the same time,they were verified by real-time PCR. The results showed that:a total of 851 DEGs between aBZ and bBZ, of which 584 and 267 genes were up-and down-regulated, respectively. The DEGs were then searched against the GO and KEGG database for enrichment analysis. GO analysis showed that 458,418 and 367 DEGs were annotated into biological process, cellular component and molecular function. KEGG analysis showed that 173 differentially expressed genes were assigned to 75 KEGG pathways, and clustered significantly in ribosome synthesis, apoptosis-multiple species, PPAR signaling pathway, and so on. This experiment initially screened DEGs between bBZ and aBZ of Haemonchus contortus. The study provides a foundation for exploring the molecular mechanism of resistance to Haemonchus contortus, screening for molecular markers of drug resistance detection and the establishment of early differential diagnosis methods for drug resistance.

The purpose of this study was to explore the effect of RIP1 on apoptosis of RAW264.7 cell by assessment the apoptosis related indicators of RAW264.7 cell treated with RIP1 RNAi vector or/and BCG infection. The RIP1 adenovirus RNAi vector was constructed and transfected into BCG-infected RAW264.7 cell line. Flow cytometry was used to detect the apoptotic rate, mitochondrial membrane potential, cell reactive oxygen species and cell cycle of RAW264.7 cell. The expression of RIP1 and apoptosis associated proteins were examined by Western blot. The results showed that BCG-infection can significantly induce RAW264.7 cells apoptosis, which was accompanied with up-regulation of PIP1 protein. In addition, a RIP1 adenovirus RNAi vector showed an ability to reduce the BCG-induced macrophage apoptosis along with an increased mitochondrial membrane potential and reduced reactive oxygen species (ROS). It also down-regulated Bax expression and up-regulated Bcl-2 expression of RAW264.7 cell infected with BCG. Intriguingly, cell cycle of RAW264.7 cell was arrested in G₁ phase. In conclusion, RIP1 involves in BCG-induced apoptosis by down-regulating mitochondrial membrane potential, increasing the production of ROS and the ratio of apoptosis-related proteins (Bax/Bcl-2) and arresting the cell cycle in G₁ phase in BCG-infectied RAW264.7.

This study was aimed to investigate the morphology, histological structure and development of the thymus of the Chinese yellow quail at different ages, and to provide morphological basis for the establishment of immune program during the growth of yellow quail. The thymus index, histological structure and ultrastructure of thymus of yellow quail from birth to 38 weeks old were studied by anatomical, histological and electron microscopy techniques. The results were follows:the weight, index, length and diameter of thymus gradually increased with age, reaching a maximum for each of these measurements at the age of 4 weeks, decreased between 5 and 9 weeks of age and then remained unchanged between 10 and 38 weeks of age. Cortex area/medullar area was gradually decreased with the age. The number of Hassall's corpuscle was gradually increased with age, reaching a maximum at the age of 6 weeks and then decreased with age. Electron microscope observation showed that:the natural apoptosis was found in thymocyte. The main changes of apoptotic cells appeared in nucleus. The chromatin were condenses. The proliferation of mitochondria happened to apoptotic cells. It was seen that a sustained developmental period occurred at 0-4 weeks of age, a maturation period occurred at 5-9 weeks, a involution period occurred at 10-38 weeks during the development of quail thymus.

In order to study the effect of low level cadmium (Cd) exposure on osteoclast (OC) differentiation, RAW264.7 cell (mononuclear macrophage lineage) was used as materials. In the presence of macrophage colony stimulating factor (M-CSF) and receptor-activated nuclear factor κB ligand (RANKL), treatment of different concentrations of Cd were conducted for 4 days. CCK-8 assay was used to detect changes in the viability of osteoclasts and their precursor cells. Tartrate-resistant acid phosphatase (TRAP) staining assay was used to observe the osteoclastogenesis. Laser scanning confocal microscopy was used to observe morphological changes of osteoclasts. The expression of osteoclast marker proteins and mRNA expression were detected by Western blot and qRT-PCR. The results showed that with the increase of Cd concentration, cell viability was significantly inhibited (P<0.01),which showed a concentration-effect relationship. Compared with the control group, the number and area of osteoclast production were significantly or extremely significantly decreased (P<0.05 or P<0.01). In the 2 and 5 μmol·L^-1 Cd treatment groups, the formation of osteoclasts' sealing zone was inhibited. The osteoclast-specific protein and mRNA expression levels of 2 and 5 μmol·L^-1 Cd groups were significantly or extremely significantly decreased (P<0.05 or P<0.01), which showed the dose effect. The above results indicate that low micromolar cadmium exposure can inhibit the differentiation of osteoclasts.

Sarcocystis gigantea is a globally ubiquitous pathogen that infects sheep. The formation of cyst in striated muscle induces a largely waste of commercial mutton which causes livestock husbandry losses. In this study, we tried to explore the special antigens for serological diagnosis of sarcocystosis. We screened a series of candidate antigens in S. gigantea through Western blot and mass spectrum identification. Whereafter, Genome Walking technology was applied to acquire two flanking sequences of these antigens. Ultimately, purify recombinant proteins was used to evaluate diagnostic value by bioinformatics analysis and Western blot. Results were as follows:Enolase of S. gigantea was finally supposed to a candidate diagnostic antigen, named SgENO. Subsequently, 1 181 bp expressive sequence of SgENO was cloned, and recombinant protein rSgENO was obtained. Nevertheless, it was found to have high homology with enolase amino acids of other Apicomplexa (71%-92.1%), rSgENO could cross react with Toxoplasma gondii and Neospora caninum positive serum. We cloned and expressed the S. gigantea enolase, and the recombinant protein had a cross reaction with other parasitic protozoa positive serum, which made it an irrelevant antigen for Sarcocystis specific diagnose. However, this protein may be a candidate antigen for vaccines.