As a new genetic evaluation method in livestock and poultry, genomic selection(GS)has been applied in dairy cattle breeding and gained remarkable success. With the improvement of reference genomes of sheep and goats and the availability of SNP chips with different densities, the application of GS in sheep and goats has also being undertaken in New Zealand,Australia,France,etc.. In this review, we summarized the current status of application of GS, factors affecting the accuracy of GEBVs and the advantages of GS in sheep and goat breeding. Moreover, we also discussed potential problems, aiming to provide a reference for carrying out GS in sheep and goat breeding in China.

Muscle and adipose are important for the production and quality of the carcass in pig. The close relationship between the two tissues affects not only their metabolism, but also the energy metabolism and homeostasis of the whole animal body. But at present, little is known about the interactions between muscle and adipose tissues, especially in livestock. This review will discuss about the interaction between muscle and adipose tissues from movement, cytokines, metabolites, especially the new cell messenger, such as exosome and miRNA, in order to present a basis for the formation of pig meat quality traits and the regulation of energy homeostasis in a new perspective.

Adiponectin (AdipoQ) is an adipocytokine mainly secreted by adipose tissue. Recent studies have shown that adiponectin played an important role in regulating lipid metabolism in animals. Adiponectin firstly binds to C terminal of adiponectin receptors, namely AdipoR1 and AdipoR2, N terminal of adiponectin receptor is combined with signal adapter protein (APPL1 and CK2) subsequently, then affect the activity or expression of related kinases (LKB1 and SIRT1), finally activate AMPK and PPARα signaling factors to promote fatty acid oxidation and inhibit lipid synthesis, thereby exerting lipid-lowering effects. At present, the research on lipid metabolism regulated by adiponectin have focused on human and rodents, and some progress has been made in the study of related molecular regulation mechanisms. This article reviews the discovery, structure, tissue distribution, signal adapter proteins of adiponectin, as well as the mechanism of adiponectin regulating animal lipid metabolism in order to provide a partial reference for further exploring the role of this gene in the regulation of lipid metabolism.

The mammalian ovary is a dynamic parenchyma organ. The ovarian follicle occurs continually physiological process of recruitment, selection and dominance during mammalian estrous cycle. Ovarian follicle is mainly composed of the oocyte, granulosa layers and theca layer. The follicular development is co-regulated by endocrine factors, autocrine and paracrine communication among the oocyte, granulosa and theca cells. microRNAs (miRNAs) post-transcriptionally regulate gene expression through base pairing with the 3'-untranslated region of target mRNA, leading to either mRNA cleavage or translational repression. miRNAs have been acknowledged to play an important role in ovarian reproduction and endocrinology, including oogenesis and follicular growth, as well as proliferation, apoptosis, differentiation and steroidogenesis of granulosa cells. This review summarized the recent advances in the fields of identification of miRNAs related to the regulation of ovarian function, the role of miRNAs in folliculogenesis, follicle development, as well as proliferation, differentiation, apoptosis, steroidogenesis of granulosa cell and theca cell. This review will lay the foundation for understanding the regulatory mechanism of mammalian reproduction based on summing up the role of miRNAs on follicle development.

The objective of this study was to investigate the plausibility of joint genetic evaluation in Duroc, Landrace and Yorkshire pigs in China. According to the genetic connectedness, a total of 9 genetic connectedness groups consisting of 39 national nucleus breeding farms from 3 pig breeds were established, respectively. The phenotypic data related to the growth and reproduction traits from 2011 to 2016 were collected. In total, 126 070, 187 975 and 491 350 records of the age to 100 kg (AGE) and backfat to 100 kg (BF), 36 542, 78 409 and 195 031 records of total number born (TNB) were analyzed for Duroc, Landrace and Yorkshire pigs, respectively. The genetic parameters and breeding values for single herd and each genetic connectedness group were estimated by REML and BLUP, respectively. The results showed that the heritabilities estimated by joint genetic evaluation were more accurate than single-herd evaluation. Similarly, the accuracy of the estimated breeding value (EBV) was improved as well. Compared to single-herd evaluation, the average accuracies of EBV obtained by joint genetic evaluation for Duroc, Landrace and Yorkshire pigs were increased by 20.9%, 11.9% and 17.3% for AGE, 21.3%, 16.3%, 21.0% for BF, and 31.2%, 14.4%, 6.6% for TNB, respectively. With the joint genetic evaluation, the population size of breeding herd was increased, and the genetic variation was enlarged, resulting in the improvement of accuracy of EBV. Therefore, the joint genetic evaluation for genetic connectedness groups in Duroc, Landrace and Yorkshire pigs in China is feasible.

The present study aimed to compare the growth, carcass and meat quality traits of progeny from Duroc (♂)×Luchuan pigs (♀) (referred as Dulu pigs, DLC) and progeny from Duroc (♂)×(Longlin (♂)×Luchuan (♀)) (referred as Dulonglu pigs, DLLC), and to confirm the feasibility of three-way crosses. DLC (n=295) and DLLC (n=91) pigs with an average age of 7 months old and a body weight of about 75 kg were slaughtered, and the carcass traits including carcass straight length, carcass slanting length, backfat thickness, loin eye muscle area, and meat quality traits including muscle color score, pH, drip loss, marbling and intramuscular fat content were measured. The results showed that:1) The nursey weight of Dulu pigs ((17.00±3.73) kg, n=460) was significantly higher than that of Dulonglu pigs ((14.62±2.60) kg, n=262) (P=3.74×10^-7); The average daily gain of Dulonglu pigs from weaning to slaughter ((402.23±48.94) g·d^-1, n=74) was significantly higher than that of Dulu pigs ((366.03±57.86)g·d^-1, n=242) (P=8.96×10^-7). 2) The carcass straight length and carcass slanting length of the Dulu pigs ((78.68±3.78) cm, (67.14±2.81) cm, n=189) were significantly lower than those of the Dulonglu pigs ((80.55±3.13) cm, (68.26±2.59) cm, n=88) (P=5.79×10^-5, P=0.003); The average adjusted loin eye muscle area of Dulonglu pigs ((31.65±4.29) cm², n=91) was slightly greater than that of Dulu pigs ((30.84±6.70) cm², n=295), but the difference was not significant. 3) There were no significant differences in meat quality traits between DLLC and DLC pigs. The study shows that the Dulonglu pigs display a faster growth rate and better carcass performance than Dulu pigs, and maintained the excellent meat quality, which ensuring a better economic benefits. Therefore, the present study provides a new way for the utilization of local pig breeds, especially for high-quality pork production.

The aim of this study was to investigate the effects of 3 small molecule compounds (RS-1, Chir99021 and PD173074) on the DNA precise repair efficiency in porcine fetal fibroblasts. In this study,the activator RS-1 for Rad51, the inhibitor Chir99021 for glycogen synthase kinase 3 (GSK-3) and the inhibitor PD173074 for fibroblast growth factor receptor (FGFR1) were used to culture porcine fetal fibroblasts, and then the percentage of green fluorescent cells was detected by flow cytometry to verify the effect of 3 small molecule compounds on DNA repair efficiency. The results showed that:1) After treatment with different doses of Chir99021 and PD173074, the expression levels of PFFs DNA damage repair factors LIG4, NHEJ1, XRCC5, XRCC6 and Rad51 were significantly down-regulated (P<0.05), and after treatment with different doses of of PD173074, the expression level of PNKP factor was significantly up-regulated (P<0.05).2) The low dose of Chir99021 significantly increased the efficiency of homologous recombination (P<0.05) mediated by homologous recombination (HR) repair(P<0.05). High dose of Chir99021 could significantly improve the efficiency of homologous recombination mediated by single strand annealing (SSA) repair(P<0.05).The low doses of RS-1 and PD173074 significantly increased HR efficiency (P<0.05), but RS-1 significantly down-regulated SSA repair efficiency at high concentration (P<0.05), while PD173074 had no significant effect on SSA efficiency (P>0.05). Furthermore, the single-stranded oligonucleotide-mediated DNA repair (ssODN) was not affected (P>0.05) by small molecule compounds. This study indicates that the application of small molecule compounds with appropriate concentrations will facilitate the generation of gene knock-in cell lines, which laying the foundation for obtaining genetic modification animal models.

The study aimed to compare and analyze the sebum protein components of high and low residual feed intake (RFI) Pekin duck strains, and to reveal the molecular mechanism of fat deposition differences among different RFI strains. In this paper, the sebum of 12 Pekin ducks in high RFI line and 12 Pekin ducks in low RFI line of 6 weeks old were selected as experimental material, then the protein components in sebum of different RFI strains were analyzed by using the iTRAQ proteomics and bioinformatics mathods. The results showed that a total of 859 proteins were identified, 25 proteins were highly expressed in high RFI strain, and 34 proteins were highly expressed in low RFI strain; GO analysis showed that differentially expressed proteins were mainly involved in organic acid metabolism, lipid response, lipid synthesis, and so on; Protein network interaction analysis found that the number of differentially expressed proteins involved in the lipid metabolism process was the highest; The expression of 3 genes coding differentially expressed proteins were analyzed by qRT-PCR, and the results were consistent with the corresponding proteins expression. The results showed that fatty acid synthase, lipid droplet protein, apolipoprotein and fatty acid binding protein were highly expressed in the sebum of ducks in high RFI strain, which improved the fat deposition ability of Pekin ducks in high RFI strain. The results of this experiment lay the foundation for further study of the molecular mechanism of differences in sebum of ducks of different RFI strains.

The study aimed to use metabolomics techniques to screen the differential metabolites in whey and serum of ovarian quiescence cows and to determine the relationship between differential metabolites and the ovary quiescence of cows. In the study, 14 estrus cows and 14 ovarian quiescence cows from 45-60 d postpartum were selected for testing. The whey and serum of cows in the two groups were tested using ¹H-NMR technique. Differential metabolites in whey and serum of cows between the two groups were screened by multivariate statistical analysis and univariate analysis. Bioinformatics analysis was performed to elucidate the relationship between metabolic pathways involved in differential metabolites and ovarian quiescence of dairy cows. The results showed that 13 differential metabolites in cows between the two groups were screened in whey and serum, respectively. Compared with estrus cows, the contents of 6 differential metabolites increased in the whey of ovarian quiescence cows, which were succinate, creatine phosphate, glycine, myo-inositol, glycolate,orotate; and the contents of 7 differential metabolites decreased, which were alanine, creatinine, O-phosphorylcholine, lactose, taurine, galactose and glucose-1-phosphate. The contents of 4 differential metabolites increased in the serum of ovarian quiescence cows, which were β-hydroxybutyrate, acetate, glutamine, glycine; and the contents of 9 differential metabolites reduced, which were alanine, succinate, citrate, creatinine, O-phosphocholine, glucose, myo-inositol, tyrosine and histidine. The differential metabolites in whey of cows between the two groups were mainly involved in the metabolism of taurine and hypotaurine, the metabolism of galactose, and the biosynthesis of primary bile acids. The differential metabolites in serum were mainly involved in alanine, aspartate and glutamate metabolism, glyoxylic acid and dicarboxylic acid metabolism, citric acid cycle, phenylalanine, tyrosine and tryptophan biosynthesis.In this study, the metabolic profiles and differential metabolites in whey and serum of ovarian quiescence cows were obtained. The results showed that the main abnormal pathways of glucose, lipid and amino acid metabolism played a role in postpartum ovarian quiescence in dairy cows, which provided a new direction for further studying the mechanism and prevention strategy of postpartum ovarian quiescence in dairy cows.

The aim of this study was to establish the culture system of porcine myogenic adipocytes cell in vitro, and to understand the mRNA expression regulation of adipogenesis related genes during adipogenic differentiation process and to provide experimental materials for further studying on the mechanism of intramuscular fat deposition in pig. Samples of longissimus dorsi muscle of the 15 days old piglets were collected and the precursor adipocytes cell was obtained by digested with type Ⅱ collagenase, and the cell were separated, primary and passage cultured and adipogenic induction. The morphology of cell were observed and the growth curve was determined. The adipogenesis was measured by Oil red O staining and the mRNA expression of adipogenesis related genes, such as PPARγ, FABP4, C/EBPα, C/EBPβ, ZFP423, SREBP1, PRMD16 and HSL were detected by qRT-PCR during induced differentiation. The results showed that a small number of myogenic preadipocytes cells began to adhere to the cell wall at about 6-7 h, and the shape of the cells were round, small shuttle and irregular, and became uniform in fibroblast-like appearance after cell passage. The growth curve was typical "S" shaped. All genes detected in this study were expressed at early stage of adipogenic differentiation after adding differentiation medium, in which the mRNA expression of FABP4, C/EBPα, SREBP1 and PPARγ at early stage was very significantly higher (P<0.01) and then was decreased significantly (P<0.01). There was no significant change for the mRNA expression of C/EBPβ,ZFP423 and PRMD16 genes during different differentiation stages. The mRNA expression of HSL was significantly reduced with the differentiation process (P<0.01). Oil red O staining was red after induction for 12 days. In conclusion,The system of the isolation, culture and adipogenic differentiation of porcine myogenic adipocytes cell was successfully established, and the expression and regulation of key genes involved in adipogenesis was investigated during cell differentiation process, which provide the foundation for further studying on the mechanism of intramuscular fat deposition and improvement of meat quality in pig production.

This study aimed to present a method to clarify the effects of exogenous glutathione (GSH) on bovine embryo development. Bovine embryos fertilized in vitro were cultured in media with 3 mmol·L^-1 GSH for 7 days in treatment group and without GSH for 7 days in control group,respectively. The rates of cleavage, morula and blastocyst formation of embryos, and the total cell number of blastocysts were calculated in both groups. The contents of reactive oxygen species (ROS) and GSH in zygotes at different stages(8,18,24,32 hours post-insemination)were detected by fluorescence staining and liquid chromatography mass spectrometry (LC/MS). Additionally, the changes of isotope labelled GSH (GSX) contents of bovine zygotes and culture media were tracked by LC/MS after GSX was added in culture medium. Results showed that:1)Exogenous GSH significantly reduced ROS contents of embryos(18,24,32 hours post-insemination)(P<0.05,P<0.01). 2)The morula rate (58.67% vs. 48.46%), the blastocyst rate (50.83% vs. 34.88%) and the total cell number of blastocysts (102.26 vs. 76.27) in treatment group were significantly higher than that in control group (P<0.05).3) GSH contents of zygotes at 3 stages in treatment group were significantly higher than that in control group (P<0.05). Meanwhile, GSH contents of zygotes in both groups decreased along with the developmental process(18,24,32 hours post-insemination). 4)After addition of GSX, the contents of GSH of zygotes in treatment group increased significantly (P<0.05). In addition, GSX concentrations in culture media decreased steadily with the extension of culture time(18,24,32 hours post-insemination). Conclusively, exogenous GSH improves the development rate of bovine embryo and blastocyst quality with scavenging of ROS. Moreover, LC/MS is a reliable method for quantitative analysis of GSH and isotope labeled GSH. Therefore LC/MS can be used to explore the mechanism of exogenous GSH improving the development of embryo cultured in vitro.

In order to reveal the mechanism of sperm mitochondrial sheath formation and improve the fertility of bulls, testis and cauda epididymidis of 3 health Holstein bulls aged 15-17 months were collected. The round and elongated sperm cells were collected throung frozen section and (laser capture microdissection,LCM) technique. Epididymal spermatozoa were obtained by floating method. TRizol was used to extract sample RNA. The differences between transcriptomes of germ cells during the spermiogenesis of dairy cows has been analysed and verified by qPCR. To understand the function of the Pld6 gene differentially expressed during spermiogenesis, the structure and function of the Pld6 protein were predicted by bioinformatics analysis. The results showed that, during spermiogenesis, the transcription of Pld6 gene increased during the development from round to elongated spermatids, and then decreased or even shut off during sperm maturation. The gene was enriched in the mitochondrial fusion term and associated with distribution and morphological structure of mitochondria on the midpiece of sperm tail. The 10-32th amino acids of Pld6 was a transmembrane region in cattle. The tertiary structure of Pld6 was horn-like with a conserved β-sheet active site H(X)K(X4)D (HKD) inside, which was similar to the mouse Pld6 structure. And there was a closest evolutionary trend of Pld6 between mouse and cattle. Therefore, according to the studies about mouse, it is concluded that, during the process of bovine spermaiogenesis, increasing expression of Pld6 gene induces the cluster of mitochondria in the midpiece of sperm tail, which promotes the formation of normal mitochondrial sheaths and plays an important role in ensuring bovine sperm viability and fertilizing ability.

The objective of the present study was to investigate the effects of contaminated corn by Fusarium graminearum on the antioxidant levels and NOS of ovary of weaned piglets and the mitigation of lactulose and hydrogen-rich water. Twenty-four weaned piglets of three-hybrid (Duroc×Landrace×Large White) of 21 days old with average body weight (7.25±1.02) kg were randomly divided into 4 groups (control group, contaminated corn group, lactulose relieving group, hydrogen-rich water relieving group), and 6 weaned piglets in each group. The piglets in control group were fed the basal diet, while piglets in contaminated corn, lactulose relieving and hydrogen-rich water relieving groups were fed the moldy feed with the deoxynivalenol (DON) and zearalenone (ZEA) contents of 0.4 mg·kg^-1 and 0.55 mg·kg^-1, respectively. In addition, the lactulose and hydrogen-rich water relieving groups were additionally intragastrically administered twice daily with lactulose (500 mg·kg^-1) and hydrogen-rich water (10 mL·kg^-1), respectively. The experiment lasted for 25 days. After 25 d, piglets were slaughtered, ovaries were collected and weighed. The ovary of piglets was observed histologically, the activities of CAT, GSH-PX, T-SOD, SDH, Caspase-3, NOS and content of NO in ovary were detected. The expressions of nNOS, iNOS and eNOS were also detected by immunohistochemistry. The results showed that:1) The relative ovary weight of the piglets in the contaminated corn group was significantly higher than that in the control group (P<0.05). Compared with the contaminated corn group, the relative ovary weight of piglets in the lactulose relieving group and the hydrogen-rich water relieving group had the downward trend. 2) There were no obvious pathological changes in the ovary of piglets in the 4 groups, but the number of growing follicles in the contaminated corn group were significantly higher than those in the control group (P<0.05). The number of growing follicles in the lactulose and hydrogen-rich water relieving groups decreased(P>0.05) compared with the contaminated corn group. 3) Compared with the control group, the activity of CAT in the contaminated corn group increased significantly, the activities of GSH-PX and SDH decreased significantly (P<0.05) and the T-SOD activity had a decreasing trend, the Caspase-3 activity, NO content, total NOS activity significantly increased (P<0.05). 4) Compared with the contaminated corn group, GSH-PX and SDH activities in the lactulose relieving group significantly increased (P<0.05). GSH-PX and SDH activities in the hydrogen-rich water relieving group had a tendency to increase; CAT activity in lactulose and hydrogen-rich water relieving groups decreased(P>0.05), T-SOD activity increased(P>0.05); Caspase-3 activity and NO content in lactulose and hydrogen-rich water relieving groups decreased significantly (P<0.05). Total NOS activity in lactulose relieving group had a tendency to decrease, while it in hydrogen-rich water relieving group decreased significantly (P<0.05). 5) The results of immunohistochemistry showed that three kinds of NOS (nNOS, iNOS, eNOS) were localized in oocytes, granulosa cells and theca cells of follicles at all stages, and showed cell-specific expression. DON and ZEA in contaminated corn may mediate the decrease of anti-oxidation levels in piglet ovaries, which leads to deeper apoptosis, while lactulose and hydrogen-rich water effectively relieve the harmful effects of DON and ZEA by increasing antioxidant levels in piglet ovaries.

The objective of this study was to compare the structure and composition of rumen microorganism of goats with different true digestibility of feed phosphorus using Illumina MiSeq sequencing technology. Twenty-eight Nubian Black goats, aged 10 months on average and weighed (24.25±2.47) kg, were selected as the experimental animals. After two digestion tests, the content of phosphorus in feed and feces was determined, and the true digestibility of phosphorus in all goat feed was calculated by differential method. Among them, 6 goats with high phosphorus true digestibility were selected as HP group and 6 goats with low phosphorus true digestibility as LP group. Rumen fluid from each goat in HP and LP groups was collected and then total genomic DNA was extracted. High variable region of bacterial 16S rRNA gene was amplified by PCR using bacterial universal primers. The amplicon sequence data was analyzed by the Illumina MiSeq sequencing platform, and bioinformatics analysis was performed using QⅡME software. The results showed that:1) The content of phosphorus in blood of goats in HP group was significantly higher than that in LP group (P<0.05). 2) At the phylum level, Firmicutes and Bacteroidetes were the dominant bacteria in the rumen microorganisms of goats in the two groups; 3)At the genus level, the relative abundances of 10 genus were significantly different between the two groups (P<0.05). Only the relative abundances of Prevotella, Desulfovibrio and Selenomonas in HP group were significantly higher than that in LP group(P<0.05), but the relative abundances of Ruminococcaceae_UCG-010, Ruminococcus_2, Ruminococcaceae_NK4A214_group, Saccharofermentans, Ruminococcaceae_UCG-014, Butyrivibrio and Clostridium were significantly lower in HP group than that in LP group (P<0.05). The relative abundance of rumen microorganisms was significantly correlated with the phosphorus true digestibility in goats. There are significant differences in structure and composition of rumens microorganisms among goats with different phosphorus true digestibility.

To explore the function of miR-222 on effecting the replication of caprine parainfluenza virus type 3 (CPIV3). The miR-222 mimics were synthesized and transfected into the MDBK cells, then infected with CPIV3. Twenty-four hours after infection, the cell cultures were harvested to determine the expression of CPIV3 genome and IFN mRNA. The molecular mechanism of effecting CPIV3 replication was explored using bioinformatics software, reporter gene system, RT-qPCR, Western blot and RNAi analysis. The results showed that the overexpression of miR-222 promote interferon (IFN) production to inhibit CPIV3 replication. Prediction the targets of miR-222 discovered that interferon regulatory factor 2 (IRF2) has a putative miR-222 target site and miR-222 mimics markedly decreased the luciferase level. Furthermore, transfection with miR-222 mimics decreased IRF2 expression in MDBK cells at both the mRNA and protein levels. Knockdown of miR-222 target inhibited CPIV3 replication. These findings indicate that overexpression of miR-222 promote the host antiviral innate immune response by targeting IRF2.

This experiment was conducted to study the effects of arctigenin (ACT) on viral replication and histopathology after porcine circovirus type 2 (PCV2) infection of piglets. Sixteen 30-day-old healthy piglets were randomly assigned to 4 groups. Drug treated groups were intramuscular injected of ACT on dose of 20 and 50 μg·kg^-1 respectively. Control group and virus challenge group were intramuscular injected with normal saline. After continuous injection for 3 days, the two drug treated groups and virus challenge group were inoculated with PCV2 and control group were inoculated with normal saline. The peripheral blood samples were collected on day -2, 0, 7, 12, 21 and the viral load in those serums were detected by qPCR. The piglets were sacrificed on day 21 after PCV2 infection, and the lung, spleen, tonsilla, mesenterium lymph nodes, inguinal lymph nodes were collected for PCV2 quantitative detection and histopathological observation. The results showed that both ACT treated groups could significantly inhibit the copy number of PCV2 in piglets peripheral blood after 7, 14 and 21 days of infection (P<0.05 or P<0.01). The results of viral load in tissues showed that, PCV2 proliferation in the tissues of inguinal lymph nodes (P<0.05) and mesenteric lymph nodes (P<0.05) were significantly inhibited in the drug group 1 (20 μg·kg^-1), and the viral proliferation in inguinal lymph nodes (P<0.001), mesenteric lymph nodes (P<0.01), tonsils (P<0.05) and lungs (P<0.05) were significantly inhibited in the dose group 2 (50 μg·kg^-1). Further histopathological sections and histopathological scoring results showed that two doses of ACT could reduce the pathological damage of the above-mentioned organ tissue caused by PCV2 infection. These results showed that ACT had a significant inhibitory effect on the proliferation of PCV2 in artificially infected piglets and could reduce the pathological changes caused by PCV2 infection.

To study the activation of ITIM motif of PRRSV M protein to IFN-β production, the full-length ORF6 gene fragment and the full-length ORF6 gene fragment without ITIM motif were amplified using the PRRSV Hn-1/06 cDNA as template by PCR method, respectively. The two gene fragments were respectively inserted into the eukaryotic expression vector pcDNA3.0 to yield recombinant plasmids, designated by pcDNA3.0-GP6 and pcDNA3.0-GP6ΔITIM (lacking of ITIM motif). Then, the two recombinant plasmids were used to transfected the Marc-145 cells, respectively. The TRIF, MyD88, TRAF6, IRF3, IRF7, NF-κB and IFN-β mRNA levels of the transfected Marc-145 cells were detected by qRT-PCR. The results showed that the TRIF, IRF3 and IRF7 mRNA levels of the Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were up-regulated significantly (This is the general trend,which may not be completely consistent with individual time points.The same as below), while the MyD88 and TRAF6 mRNA levels of the Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were down-regulated significantly, when compared with the Marc-145 cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid. The two recombinant plasmids were used to transfected the Marc-145 cells transfected with Poly (I:C), respectively. The TRIF, IRF3, IRF7, NF-κB and IFN-β mRNA levels of the transfected Marc-145 cells were detected by qRT-PCR. The results showed that the TRIF, IRF3 and IRF7 mRNA levels of the Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were up-regulated significantly, while the NF-κB and IFN-β mRNA levels of the Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were up-regulated significantly at 36 h, when compared with the Marc-145 cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid. The recombinant plasmids pcDNA3.0-GP6 and pcDNA3.0-GP6△ITIM were respectively transfected into the Marc-145 cells silenced with SHP-1-siRNA-781 or SHP-2-siRNA-1335, then the TRIF, IRF3, IRF7, NF-κB and IFN-β mRNA levels of the transfected Marc-145 cells were detected by qRT-PCR. The TRIF, IRF3, IRF7,NF-κB and IFN-β mRNA levels of the SHP-1 silenced Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were down-regulated significantly at any timepoints, compared with the SHP-1 silenced Marc-145 cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid, the TRIF, IRF3, IRF7, NF-κB and IFN-β mRNA levels of the SHP-2 silenced Marc-145 cells transfected with pcDNA3.0-GP6 recombinant plasmid were down-regulated significantly at early phase, then recovery to normal levels at later phase, when compared with the SHP-2 silenced Marc-145 cells transfected with pcDNA3.0-GP6ΔITIM recombinant plasmid. In summary, the ITIM motif of PRRSV M protein may induce the IFN-β production by interacting with SHP-1 in TLR3-TRIF-dependent signaling pathway, however the relationship between ITIM motif and SHP-2 needs to further study. This study provides an important reference for understandin the role of ITIM motif.

The aim of this study was to investigate the effect of nuclear localization signal (NLS) mutation in the M protein on the pathogenicity of Newcastle disease virus (NDV). The parental virus and the M/NLS mutant virus were used to infect 4-week-old SPF chickens. The clinical symptom and pathological change, the virus shedding in larynx and cloaca, the virus titers in the tissues, the microscopic lesions, the expression change of M protein and the cytokine genes in immune organs of virus-infected chickens were examined. The results showed that chickens infected with the parental virus exhibited typical ND clinical symptom and pathological change, and had continuous and high virus shedding in the larynx and cloaca. By contrast, chicken infected with the mutant virus showed slight clinical symptom and pathological change, and had delayed and low virus shedding. The survival rate of parental and mutant virus-infected group was 0% and 70%, respectively. Compared to the replication ability and virus titers of the parental virus in different tissues, the mutant virus only replicated in the immune organs and trachea and simultaneously had very low virus titers. In addition, the mutant virus caused unconspicuous pathological change and low expression of M protein in the immune organs in comparison to the parental virus, and also reduced expression level of IL-1β, IL-6, IL-10, IFN-β and IFN-λ in immune organs, indicating that M/NLS mutation significantly reduced the cellular immunologic response induced by NDV. This study revealed for the first time the attenuated pathogenicity of M/NLS mutant NDV, and provided useful information for further investigating the nuclear localization function of M protein in NDV pathogenicity.

The aim of the present study was to develop a visual chip for simultaneously detecting avian leukosis virus (ALV), chicken Marek's disease virus (MDV), infectious bursal disease virus (IBDV), Newcastle disease virus (NDV), avian influenza virus (AIV), and infectious laryngotracheitis virus (ILTV) using gold label silver stain method. The gene sequences of ALV-env、MDV-meq and IBDV-vp₂ were cloned using T-A method. These sequences, also the sequences of AIV-np, NDV-f, ILTV-tk stored by our laboratory, were used for probes design, following which the simultaneously detecting ALV-MDV-IBDV-AIV-NDV-ILTV chip was developed. Following the PCR amplification using a pair of biotin-labeled primers, the chip was used for naked-eyes observation after hybridization and silver stain. The chip was then validated by specificity and sensitivity identification, stability evaluation, and clinical verification. The sequences of ALV-env, MDV-meq, IBDV-vp₂ were cloned successfully. The hybridization, at 40℃ for 2 h, of 50 μmol·L^-1 oligonucleotide probes with the biotin-labeled PCR products resulted in visible silver stain signal. No cross reaction was observed among ALV, MDV, IBDV, NDV, AIV and ILTV by specificity test. The specificity of the chip was confirmed by its negative hybridization with the PCR products from infectious bronchitis virus. The minimum-does of the plasmid as PCR template was 1 pg·μL^-1 by sensitivity test. The chip can be stored at room temperature for 60 d, or at 4℃ for 75 d according to stability test. The results of chip test consistent with that of PCR/RT-PCR method. The present study successfully developed a visual chip for simultaneously detecting ALV,MDV,IBDV,NDV,AIV and ILTV with an optimization reaction condition, which will facilitate the clinical application of the chip in the future.

To explore the immune protective effect of recombinant Tm16 and GST proteins against Coenurus cerebralis in goats, forty-four goats were divided into four groups, including rTm16 group, rTm-GST group, rTm16+rTm-GST group and control group, which were immunized 3 times by subcutaneous injection, respectively. Second vaccination was performed 4 weeks after the first vaccination, and 6 months later the last vaccination was performed. The serum was regularly collected from each goat, and the specific IgG response to each group was measured by indirect ELISA. Two weeks after the final vaccination, each goat was orally challenged with 5 500 eggs of Taenia multiceps. One hundred and five days post infection all goats were slaughtered, the cysts in brain and muscle were collected and counted. Result were as follows:Compared with the control group, the cysts of goat in group rTm16, rTm-GST and rTm16+rTm-GST reduced by 50%, 62.5% and 87.5%, respectively. Combined immunization of rTm16 with rTm-GST induced significant protection (P<0.05)against challenge of T. multiceps in goats. Result of iELISA showed that the specific IgG response against rTm-GST was significantly weaker than that against rTm16 (P < 0.05). However, the specific IgG response against rTm-GST was not significant compared with that against rTm16 in combined group (P>0.05). This revealed that the combined use of rTm16 and rTm-GST could induce higher antibody levels in goats, thus obtaining significant protective immunity.

This study was conducted to explore the characteristics of progesterone on duodenal smooth muscle activity and to establish a mathematical model to reveal its mechanism. In this experiment, rabbits were grouped according to the dosage of progesterone containing 0, 0.5, 1.0, 1.5 and 2.0 mg·kg^-1. The corresponding dose of progesterone was injected intramuscularly into the ovariectomized and electrode implanted rabbit models. The changes of myoelectric activity in the duodenum of the experimental group were collected by BL-420F biological function test system. Using myoelectric activity index as dependent variable and progesterone dose as independent variable, a mathematical model was constructed to reveal the mechanism of progesterone on duodenal smooth muscle activity. The results showed that:1) In the dose range of 1.0-2.0 mg·kg^-1, the amplitude, frequency and myoelectric activity index of duodenal myoelectric activity increased firstly and then decreased with the increase of progesterone dose, that is, concentration-dependent regulation mode. 2) A functional model of progesterone dosage and myoelectric activity index was constructed, which accord with the basic change characteristics of Gauss function. The accuracy of the function fitting analysis showed that the R²=0.998 0 and P=0.001 4, suggesting the fitting accuracy of the model was very high. 3) According to this function, the myoelectric activity index changed significantly in the range of 0.72-1.98 mg kg^-1 of progesterone, ranging from 1.64 to 4.92 mV min^-1. At the same time, the dosage of progesterone of 1.14, 1.35 and 1.56 mg·kg^-1 were the boundary point. The change trend of myoelectric activity index was continuous exponential type increase, logarithmic type increase, exponential type decrease and logarithmic type decrease. The above results suggest that the regulation of progesterone on the electrical activity of duodenal smooth muscle is based on a certain level of myoelectric activity in the duodenum. The results of this study provide a very important theoretical basis and reference for clarifying the mode of progesterone regulating duodenal movement and the development and use of progesterone drugs.

This study was conducted to investigate the effects of different levels of calcium on the endoplasmic reticulum apoptosis pathway in fluoride-induced kidney tissue injury in rats. One hundred and twenty male SD rats were randomly divided into 5 groups:Control group (C) (conventional feed), High fluoride group (F) (conventional feed, containing 150 mg·kg^-1 F^-), High fluoride + Low calcium (L) (conventional feed containing 150 mg·kg^-1 F^-+0.5% CaCO₃), High fluoride + Medium calcium group (M) (conventional feed containing 150 mg·kg^-1 F^-+1% CaCO₃) and High fluoride + High calcium group (H) (conventional feed containing 150 mg·kg^-1 F^-+2% CaCO₃). After eating and drinking freely for 120 days, renal tissue cells were collected for pathological examination. The expression of endoplasmic reticulum pathway related genes and proteins were detected by RT-PCR, immunohistochemistry and western blot, respectively. Results showed that:(1) Fluorosis caused glomerular swelling, widening of the renal balloon space, swelling of proximal tubule epithelial cells and vacuole degeneration in rats. Low and medium doses of calcium reduced fluoride-induced kidney toxicity, while the high-dose calcium group enhanced the injury. (2) Fluoride significantly increased the mRNA expression of Caspase-12, Caspase-3 and JNK, while the expression of IRE1, ASK1 and TRAF2 was significantly decreased, which are endoplasmic reticulum apoptosis pathway related genes. However, low and medium doses of calcium relieved the fluoride-induced adverse effects. (3) In addition, fluoride significantly decreased the protein expression of Bcl-2 and increased the protein levels of Caspase-12, JNK, Bax, Bax/Bcl-2. Supplementation of low and medium doses of calcium alleviated the over-expression of endoplasmic reticulum pathway related proteins, thereby inhibiting the toxic effects of fluoride. In summary, fluoride activated the Caspase-12 signaling pathway and the JNK signaling pathway of endoplasmic reticulum pathway, which accelerated the apoptosis of renal tissue cells. However, low and medium doses of calcium relieved the toxic effects of fluoride by inhibiting the expressions of endoplasmic reticulum apoptosis pathway related genes and proteins.

The aim of our study was to explore the curative effect and compatibility mechanism of Gui Qi Yimu decoction. The chemical constituents and action targets of three kinds of traditional Chinese medicines were studied by pharmacological database and analysis platform of TCMSP. The compound target network, protein interaction PPI network and target pathway network were constructed. Through screening, a total of 24 constituents, 249 targets related to three kinds of traditional Chinese medicines were selected. PPI network contains 182 targets, including TNF, IL8, HSP90AA1, MAPK1, F3, ENSBTAG00000014921, PSMD3, TP53 and other key targets. There are 73 gene Noumenon (GO) entries, including 61 biological process items, 9 molecular functional items and 3 cellular component entries. In addition, there are 11 KEGG pathways, including tumor pathway, T cell receptor signal pathway, Toll like receptor signal pathway, apoptosis, calcium signal pathway, and so on. The results of the study preliminarily verified the basic pharmacological effects and related mechanisms of Guiqi Yimu decoction, which laid a foundation for further study of its action mechanism.

Osteoblasts play bone-forming roles in the process of bone metabolism. Calcium-chelated bone marrow peptides (MP-Ca) was prepared and its effects on the proliferation and osteogenic activity of osteobalst (OB) were investigated and compared with calcium-free peptides of sheep bone marrow (MP). Prepared MP-Ca was identified via Fourier transform infrared spectrometer. The osteoblasts isolated from the skull of newborn SD rats were identified through alkaline phosphatase (ALP) and alizarin red staining. Different concentrations of test substance, including the positive control 17 β-estradiol, were added to the medium and the CCK-8 method was used to determine the optimal concentration for the subsequent study. The proliferation and the osteogenic activity of OB were expressed as OD_{450 nm} and the content of ALP and osteocalcin (BGP) in cell lysate prepared from MP-Ca, MP, estrogen treated OB cells at optimal concentrations. MP and MP-Ca showed no completely overlapped infrared spectra and apparent shifts of absorption peaks were observed, which indicated that MP-Ca was different form MP and it was prepared successfully. After ALP staining the isolated and purified OB cells appeared positive reaction. The results showed that OD_{450 nm}, ALP and BGP levels of the MP-Ca treated cells were significantly higher than those of the MP treatment (P<0.05 or P<0.01), but they were significantly lower than those of 17β-estradiol treated group (P<0.05 or P<0.01). It can be concluded that although inferior to 17β-estradiol, MP and MP-Ca were effective in promoting the proliferation of osteoblasts in vitro and enhance their bone formation activity, and MP-Ca was superior to MP.

The aim of this study was to investigate the effects of 6-bromo-indirubin-3'-oxime(BIO) on apoptosis of mouse mammary epithelial cell induced by lipopolysaccharide (LPS) and its regulatory mechanism. The cells from the blank control group, DMSO group, BIO alone treatment group and different concentrations of BIO (5, 25, 50 nmol·L^-1 treatment) + LPS co-treatment group were collected, respectively. Apoptosis rate was analyzed by flow cytometry. Bak, Bax, Bcl-2, Bcl-xl, Caspase-3 and Caspase-8 mRNA expression were detected by qRT-PCR. Bcl-2, Caspase-3 and Caspase-8 protein expression were tested by Western blot. Results were as follows:BIO reduced the apoptosis rate of MMECs induced by LPS. BIO decreased LPS-induced activities of apoptosis genes Bak and Bax, apoptotic pathway proteins Caspase-3 and Caspase-8 (P<0.001) were also down-regulated. BIO increased the activities of anti-apoptosis genes Bcl-2 and Bcl-xl. BIO inhibited apoptosis of MMECs induced by LPS through regulating Bax, Bak, Bcl-2, Bcl-xl, Caspase-3 and Caspase-8 activity.

To investigate the effect of polysaccharides from ZhuQin Formula (ZQp) on the proliferation and autophagy of spleen lymphocyte in immunocompromised mice, the optimum concentration and time of ZQp on the proliferation of spleen lymphocyte in mice was screened by CCK-8 method. The test was divided into blank group, 3-MA (autophagy inhibitor) group, 3-MA + ZQp group, Western blot was used to determine the effect of ZQp on the expression of autophagy related protein Beclin1 and LC3B in mice under the inhibition of 3-MA; The test was divided into blank group, cyclophosphamide induced immunocompromised model group and model +ZQp group, Western blot was used to determine the effect of ZQp on the expression of autophagy related protein Beclin1 and LC3B in immunocompromised mice. Test results were as follows:1)The ZQp with the concentration of 10^-1μg·mL^-1 acted on mouse spleen lymphocytes for 12 h, and the cell proliferation ability was the strongest; 2)The proliferation ability of spleen lymphocyte in mice with cyclophosphamide immunodeficiency was reduced, and the proliferation of lymphocyte was enhanced by ZQp; 3)When 3-MA inhibited autophagy of spleen lymphocyte, the expression of autophagy related protein Beclin1 decreased, and ZQp could increase the expression of Beclin1; When the expression of autophagy related protein LC3BⅡ/LC3BⅠincreased, ZQp could reduce the expression of LC3BⅡ/LC3BⅠ; 4)The expression of autophagy related protein Beclin1 and LC3BⅡ/LC3BⅠ increased in spleen lymphocytes of immunocompromised mice. ZQp could decrease the expression of Beclin1 and LC3BⅡ/LC3BⅠ protein. The results suggest that:Cyclophosphamide-induced immune dysfunction in mice may be related to the splenic lymphocyte proliferation and development impair and excessive activation of autophagy lead to type Ⅱ programmed cell death. ZQp can inhibit excessive autophagy, enhance the proliferation of lymphocytes, and regulation the immune function by regulating the expression of Beclin1 and LC3BⅡ/LC3BⅠ protein.

This study was conducted to analyze the effects of Nühuang granule on histopathology and cell cycle in spleen of immunosuppressed mice. Fifty healthy Kunming mice were randomly divided into blank group, model group, and high-, middle-, and low-dose Nühuang granule groups, with 10 rats in each group. All groups were treated with intraperitoneal injection of cyclophosphamide (80 mg·kg^-1, once a day and for 5 days) to replicate the immunosuppressive model except for the blank group. After model replicated successfully, each group was gavaged with normal saline or drugs for 7 days consecutively. After injection of cyclophosphamide, the spleen index, the number of blood leukocytes, the percentage of lymphocytes and monocytes of mice were decreased. Spleen tissues were damaged and the percentage of G0/G1 phase cells was reduced, and the cells in S phase was increased significantly. While the spleen index, number of blood leukocyte, percentage of lymphocyte and mononuclear of mice were increased, spleen tissue structure was improved, the percentage of spleen cells in G0/G1 phase was increased significantly, and the cells in S phase decreased after Nühuang granule was gavaged to the test mice. Among them, the effect of intermediate dose group was best. Nühuang granule can improve the spleen tissue structure in the immunosuppressed mice. It also can increase the percentage of G0/G1 phase cells, decrease the percentage of S phase cells, and increase the spleen index of test mice.

The aim of this study was to clone the lysine-specific histone demethylase 1A (KDM1A) gene, identify its expression in various tissues of yak, and to analyze the expression pattern in different growth periods of yak testis. The total RNA was extracted by collecting the heart, spleen, liver, ovary, lung, cerebrum, kidney, uterus, large intestine, testis and stomach from healthy yaks aged 4-5 years old.In addition,the testes at different developmental stages were collected including fetus (5-6-month-old), infancy (1-2-year-old), sexual maturity (4-5-year-old) and old age (9-10-year-old).RT-PCR was adopted for amplification of the complete CDS of KDM1A gene in yak.Meanwhile the structure and function of KDM1A gene in yak were analyzed by a series of bioinformatics softwares.Then the expression of KDM1A in different tissues and testes at different developmental stages were detected by RT-qPCR.The results showed that the KDM1A was obtained by cDNA cloning,the length of which was 2 401 bp.And the length of the KDM1A gene was 2 331 bp in CDS, encoding 776 amino acids.It was high homology identity to that of corresponding cDNA from bovine,which showed that the KDM1A gene was conservative in the process of evolution.The expression profile of KDM1A was wide in yak tissues,but there are some differences in various tissues.For example,the expression level was the highest in liver and testis.The expression level of KDM1A mRNA during testis development presented the tendency of going up firstly and going down secondly. The complete CDS of KDM1A gene was successfully cloned and the KDM1A had a significant different expression among the 4 periods of yak testis,which indicted that the KDM1A gene might play an important role in testis development of yak.