As the only micro organ, the hair follicles grow periodically throughout life, hair follicle is in a relatively active growing and metabolic state. Hair shaft is a sustained developing structure that consists of hair follicle's terminally differentiated cells. The morphological change of hair shaft depends on the structural development of hair follicle, since mitosis, proliferation and differentiation of hair follicle stem cells determine the growth of hair fibre. In recent years, thanks to the improvement and innovation of research methods, great progresses have been made rapidly on studies of hair curvature at the molecular genetics level, and some genetic factors and signal pathways have been uncovered and verified, which act on hair follicle development and hair wave patterns. We introduced the anatomical and morphogenetic properties of hair follicle, and then generalized research advances of hair curvature on the basis of hair follicle's different cell layers and their specific regulatory genes; finally, we also described an interaction network among the canonical pathways and genes related to hair follicle development. The review was presented in order to provide references for the study on molecular mechanisms of wool curling in a dynamic morphogeny.

Guanidinoacetic acid, a white water-soluble amino acid derivative, is a natural precursor of creatine, also known as guanidine acetic acid. At present, guanidinoacetic acid is widely used as a feed additive to increase muscle yield and improve carcass meat quality in animal production. This review summarizes the physiological functions of guanidinoacetic acid, stimulating insulin release, neuromodulation, altering the metabolic utilization of arginine and adjusting the oxidant-antioxidant status, and the function of creatine, a metabolite of guanidinoacetic acid, promoting the differentiation of skeletal muscle and inhibiting differentiation of adipocytes. Which will provide a reference for the follow-up guanidinoacetic acid related research.

The aim of this study was to identify candidate genes affecting the two head types(long-pointed head and short head) in Yushan Black pigs through conducting a genome-wide association study(GWAS) and a genome-wide scan for the selection signatures. Twenty-four pigs with long-pointed head and 12 pigs with short head were selected from the national conservation farm of Yushan Black pigs and were genotyped using Porcine SNP 60K BeadChips. The Fst value of each SNP was calculated to detect signatures of genetic differentiation between the two groups of pigs. Meanwhile, case-control GWAS and linkage disequilibrium(LD) analysis were performed to define confidence intervals and candidate genes based on Fst. The results showed that the most significant signature of genetic differentiation was located on Sus Scrofa chromosome(SSC) 10, and a genome-wide significant locus within the same region was also detected by GWAS. The most likely interval of this locus was defined by the LD block analysis, about 290 kb (from 11.76 to 12.05 Mb) on SSC10. By applying genetic differentiation analysis, genome-wide association study and linkage disequilibrium (LD) analysis, we speculated that candidate genes for head types were RAB3GAP2 and IASR2 in Yushan Black pigs. The findings will contribute to reveal the genetic basis of phenotypic changes in head types in Chinese indigenous pigs.

The objective of this study was to investigate the roles and mechanisms of AMP-activated protein kinase(AMPK) in sheep muscle derived preadipocytes differentiation. The longissimus dorsi(LD) muscle was sampled from lamb of 3 days old, and the perimysium was isolated and digested for preadipocytes isolation by differential adhesion method. The activity of AMPK and Wnt/β-catenin signaling pathways were altered, and then the cells adipogenic differentiation was induced. qRT-PCR and Western blot were used to detect the expression patterns of key genes and proteins in preadipocytes differentiation. Oil-Red-O staining was used to observe the lipid droplet accumulation in mature adipocytes. The results showed that activating AMPK was associated with less Oil-Red-O staining. Meanwhile, the expressions of adipogenic genes and corresponding proteins including PPARγ, C/EBPα and aP2 were down-regulated at significant level of 0.01 when AMPK was activated, while inhibition of AMPK activity showed opposite effects. The activation of Wnt/β-catenin signaling pathway inhibited preadipocytes adipogenesis, decreased the expressions of PPARγ and C/EBPα mRNA at significant level of 0.01, and down-regulated aP2 gene expression at significant level of 0.05, moreover, the corresponding protein contents were also decreased at significant level of 0.01. Furthermore, inhibition of Wnt/β-catenin signaling pathway promoted adipogenesis. β-catenin transcription was not affected by alteration of AMPK activity but β-catenin protein content significantly altered(P<0.01). Finally, AMPK activation increased GSK3β phosphorylation at significant level of 0.01. The result indicated that AMPK regulated sheep muscle derived preadipocytes differentiation through altering GSK3β phosphorylation and affecting the stability of β-catenin.

The study of the association between horse MC1R gene polymorphic loci and coat colors would provide important theoretical reference for coat color breeding of horses. In this study, the CDS of MC1R gene from 132 horses with 10 different coat colors was obtained by cloning and Polymerase Chain Reaction (PCR), Restriction Fragment Length Polymorphsm (RFLP) and sequencing were implemented to analyze the correlation between MC1R gene polymorphism and coat color, the effect of SNPs on its coding protein structure was predicted, and the MC1R gene NJ phylogenetic tree was constructed and analyzed. The results showed that 3 SNPs loci were found in horse MC1R gene, including one synonymous mutation(c.G102A) and two missense mutations(c.C248T, c.G250A).The c.C248T caused change of the 83rd amino acids of MC1R protein from polar hydrophilic serine to nonpolar hydrophobic phenylalanine, predicting that the hydrogen bond between amino acid at this locus and 127th amino acid (serine) at the third transmembrane domain would disappear and the conformation of MC1R protein would be changed. This mutation resulted in 3 genotypes of EE, Ee and ee in MC1R gene.The missense mutation c.G250A caused the change of amino acid from aspartic acid to asparagine. The mutation site had more complex interaction with adjacent amino acids and transmembrane domains. It was speculated that c.G250A and c.C248T had less effect on the MC1R protein function than only the c.C248T mutation, which resulted in the emergence of genotype ee^a. Based on the association analysis of the horse MC1R genotype data (a total of 571 individuals) published by NCBI with their coat color traits, we found that there were two genotypes of EE and Ee in the bay and black horse populations, four genotypes of EE, Ee, ee and ee^ain gray horse population, and two genotypes of ee and ee^a in the chestnut horse population. The distribution of genotypes were extremely significantly different(P<0.01) in black and bay horse populations,showing Hardy-Weinberg disequilibrium, and the allele E was the dominant one. However, allele E was not detected in chestnut horses and e was the dominant allele. The result of phylogenetic tree showed that the individuals with EE genotype had the highest similarity to horse and Przewalskii. These results indicate that the different genotypes produced by the MC1R polymorphic loci are related to the bay,black,chestnut coat colors and the colorful hair color of horses. There was no ee genotype in bay and black horses and no allele E in chestnut horses. This study lay the foundation for the application of genotypes in the directional selection breeding of horse coat color.

The aims of this study were to compare expression quantity of key genes for muscle development before and after long-term high loading exercise in Mongolian horse, and to provide data support for the development of horse endurance training programs.Three female Mongolian horses of 5-years-old were used and continuous high loading endurance exercise were carried out for continue 4 months, and the gluteus medius were collected before and after exercise, respectively. RT-qPCR and Western blot were used to analyze the expression quantity of MYL-2 and TNNC1 genes at transcription and translation levels before and after long-term high loading exercise in Mongolian horses. The results showed that the expression quantity of MYL-2 and TNNC1 genes in Mongolian horses muscle after exercise were significantly higher than that before exercise at mRNA level(P<0.01). The expression quantity of MYL-2 significantly increased after exercise(P<0.01), the expression quantity of TNNC1 increased after exercise at protein level(P>0.05). The long-term high loading exercise promote the expression of MYL-2 and TNNC1 genes related to Mongolian horses muscle development. It is important to form endurance of Mongolian horse.

The purpose of this study was to construct a recombinant inhibin plasmid containing a CpG-ODN fragment with immunogenicity(named as pEGISI-CpG-ODN), and determine its effect on mRNA expression levels of apoptosis-related genes to investigate the roles of CpG-ODN in mouse ovarian granulosa cells (GCs). GCs were obtained from the ovaries of 21-23 days female ICR mice. The experiment was divided into 3 groups:empty vector group (pEGFP), inhibin recombinant plasmid group (pEGISI) and containing CpG-ODN motif recombinant inhibin plasmid group (pEGISI-CpG-ODN). The target fragment CpG-ODN was amplified by PCR. The mRNA expression levels of CpG and the death receptor pathway related genes (Fas, FasL, DR4/5, TRAIL, Caspase8 and Caspase3) in transfected cells were analyzed by quantitative real-time PCR (qRT-PCR). The results showed that a new recombinant inhibin plasmid called pEGISI-CpG-ODN was constructed successfully. Moreover, CpG gene could be highly expressed in GCs after transfection of GCs with recombinant inhibin plasmid (pEGISI-CpG-ODN). Furthermore, the mRNA expression levels of pro-apoptotic genes Fas, FasL, DR4/5,TRAIL, Caspase8 and Caspase3 were very significantly lower in GCs of the pEGISI-CpG-ODN plasmid treated group than those in pEGISI treated group (P<0.01). These results suggested that CpG-ODN might not only resist the function of inhibin but also decrease the expression levels of apoptotic genes in mouse GCs, as a result, it was able to affect follicular development.

This study aimed to select milk biomarkers of pregnancy recognition in dairy cows and provide a new insight and method for early pregnancy diagnosis. Ten healthy Holstein high yield dairy cows with similar body condition and parity were selected. All cows were oestrus synchronized and their milk samples were collected at day 0 and day 17 of artificial insemination. Metabolic profiles of the 2 groups of milk samples were recognized with LC-Q/TOF-MS metabolomics combined with principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA); Differentiated metabolites for the 2 groups of milk samples were selected according to VIP values (VIP>1) from OPLS-DA model and P values (P<0.05) from Kruskal-Wallis test; Their discriminating ability to classification of the 2 groups of milk samples were further observed by ROC curve. The results showed that:1) Milk metabolic profiles at day 0 were obviously different from those at day 17 after artificial insemination. 2) Ten metabolites (mevalonolactone, tryptamine, L-tyrosine, dihydroneopterin phosphate, acetylcysteine, N-Acetyl-L-tyrosine, L-proline, 1BnTIQ, guanosine,heptadecanoyl carnitine) were first confirmed as differentiated metabolites between the 2 groups of milk samples. 3) Only 6 of the 10 differentiated metabolites including 1BnTIQ, N-Acetyl-L-tyrosine, L-tyrosine, guanosine, L-proline and heptadecanoyl carnitine had the obviously separating capacity for the 2 groups of milk samples and were finally viewed as potential milk biomarkers of pregnancy recognition. In conclusion, the 6 metabolites could be better indicators for dairy cows pregnancy diagnosis at pregnancy recognition stage, and provide a new insight into early pregnancy recognition mechanism.

The aims of this study were to observe the expression patterns and the correlation of adiponectin receptors (AdpR) and testosterone (T) synthesis-related genes in male reproductive organs at different developmental stages of Hu sheep. Five Hu sheep at ages of 3, 9, 24 month (M) were randomly selected, respectively. Blood was collected from the jugular vein of each ram to separate the plasma. Tissue samples including hypothalamus, pituitary, testis, caput, corpus and cauda were collected from the different developmental stages of Hu sheep after the slaughter. The concentrations of serum adiponectin (Adp), T, gonadotropin releasing hormone (GnRH) and gonadotropin inhibitory hormone (GnIH) at different ages were analyzed by ELISA, and the expression patterns of AdpR1, AdpR2 and T synthesis-related genes mRNA in male reproductive tract at the 3 developmental stages of Hu sheep were examined by qRT-PCR. The location of AdpR1 in the male reproductive tract was detected by immunohistochemical. The results showed that the levels of Adp, GnRH and GnIH in serum had different change trends. Adp concentration were significantly higher in adult (9 and 24 month) group than pre-pubertal (3 month) group. The mRNA expression of AdpR1 in testis and epididymis cauda at different developmental stages was significantly higher than that of AdpR2 (P<0.05). Moreover, the mRNA expression of AdpR1 in the testis and cauda increased significantly with the increasing age (P<0.05), but no significant difference between epididymis caput and corpus (P>0.05). AdpR1 expressed in the ram testis and epididymis, and was mainly localized in leydig cells, spermatocytes, peritubular myoid cells and principal cells. Furthermore, Adp and T levels in serum had significant positive correlation (P<0.05). Although the content of Adp was negatively correlated with GnIH levels and positively correlated with GnRH, respectively, there was no significant difference among them (P>0.05). Meanwhile, the mRNA expression of AdpR1 were significantly positive correlated with StAR and 3β-HSD, respectively (P<0.05). In conclusion, during the development of the reproductive organs of Hu sheep,Adp may promote production of T by combining AdpR1, and then promote the testis development of Hu sheep.

The experiment was conducted to investigate the effects of dietary Clostridium butyricum on growth performance, antioxidant capacity and immune function in weaned piglets. A total of 180 healthy crossbred weaned piglets (Duroc×(Yorkshire×Landrace), (28±1) days old, (8.07±0.84) kg initial body weight, half male and half female) were randomly allocated to 5 treatment groups, 6 replicates in each group, and 6 weaned piglets in each replicate. Dietary treatments contained:the antibiotic-free basal diet as the negative control group; the basal diet supplemented with 75 mg·kg^-1 aureomycin as positive control group; the concentration of efficacious viable germ in diets were 1.0×10⁸, 1.0×10⁹ and 5.0×10⁹CFU·kg^-1, respectively as Clostridium butyricum treatment groups. The trial lasted for 28 d. Body weight of weaned piglets were measured on 0, 14 and 28 d. During the trial, the diarrhea condition of weaned piglets were observed and recorded. One healthy piglet from each replicate was selected for collecting blood sample at the end of the trial. The levels of immune and antioxidant indexes were determined by commercial kits. The result showed that:1) The average daily gain, average daily feed intake and feed conversion ratio of weaned piglets had no significant differences in all treatment groups during1-14 d, 15-28 d and 1-28 d(P>0.05). 2) With the level of Clostridium butyricum supplementation ascending, the diarrhea rate of weaned piglets significantly linearly increased (P<0.05) during 1-14 d and 1-28 d. The feces score had significant differences among different treatment groups(P<0.05) during 15-28 d and 1-28 d. 3) Compared with negative control group, basal diet supplemented with 5.0×10⁹ CFU·kg^-1 Clostridium butyricum significantly increased the concentration of GSH-Px and T-AOC(P<0.05), basal diet supplemented with 1×10⁹ and 5.0×10⁹ CFU·kg^-1Clostridium butyricum significantly increased the concentration of T-SOD, which linearly significantly increased with the augment of Clostridium butyricum levels(P<0.05), however, there was no significant difference in MDA content among treatment groups(P>0.05). 4) The level of IgG showed a increase tendency with the level of Clostridium butyricum supplementation ascending (P<0.10). Compared with negative control group, basal diet supplemented with 5.0×10⁹CFU·kg^-1Clostridium butyricum significantly reduced the levels of TNF-α (P<0.05) and IL-6 (P<0.01) in serum of weaned piglets, while the level of IL-4 in serum significantly increased (P<0.01). Moreover, the level of IL-4 linearly significantly increased with the augment of Clostridium butyricum levels(P<0.05). There was no significant differences in IL-1β level among treatment groups(P>0.05). The results show that supplementation of 1×10⁹ -5.0×10⁹ CFU·kg^-1 Clostridium butyricum enhance antioxidant capacity and immune function of weaned piglets, which is helpful to the health level and anti-stress ability of weaned piglets.

The aim of this study was to examine the effect of selenium yeast in containing polyunsaturated fatty acids dietary(supplemented perilla frutescens seed) on fatty acid composition and antioxidant capacity in muscle and liver of Hu lambs. Forty-five lambs of 3 months old with the body weight of (23.02±1.36) kg were selected and randomly divided into 3 groups:basic ration group(C), C+10% perilla frutescens seed group (PFS) and C+PFS+yeast selenium group(PFS+Se). The duration of preliminary experiment was 10 days and the duration of the formal experiment was 60 days. Growth performance was measured at the beginning and end of the trial and blood samples were collected for biochemical analysis. Slaughter test was performed at the end of the trail to collect longissimus dorsi muscle and liver samples for measuring the fatty acid composition, antioxidant enzyme activity and antioxidant related genes expression levels. The results showed that:1) There was no significant difference in growth performance and slaughter performance among different treatments (P>0.05). 2) The crude fat content in longissimus dorsi muscle of Hu sheep in the PFS and PFS+Se groups significantly increased (P<0.05), but there was no significantly difference in the content of mineral elements such as Fe, Cu, Na, K and Mg among different treatments (P>0.05). 3) The content of n-3 polyunsaturated fatty acid in liver and longissimus dorsi muscle in PFS group were significantly increased (P<0.05), and the values of n-6/n-3 in muscle and liver was significantly lower compared with the C group (P<0.05). 4) The CAT activity and T-AOC in the serum in PFS+Se group were significantly increased (P<0.05), the content of MDA in liver was significantly decreased (P<0.05), and the expressions of CAT and GPX in liver were significantly increased compared with C group (P<0.05). The above results show that adding selenium yeast in PUFAs dietary can improve the antioxidant capacity of Hu sheep, but it can not promote the PUFA deposition. Therefore, selenium yeast may not be the most effective way to promote the PUFA deposition in Hu sheep fed with perilla frutescens seed diet, and further work is needed to explore the effective antioxidants.

The objective of this study was to reveal the basic chemical profile and lipid molecular structural features, as well the relationship between molecular structural spectral parameters and nutritional values in different batches of corn and its by-products (corn gluten feed, corn germ, corn germ cake and corn gluten meal). This study was conducted to characterize the lipid nutritive values and lipid structure spectral features in different batches of samples using the conventional chemical analysis and Fourier transform infrared spectroscopy (FTIR) techniques, respectively. The spectroscopy parameters included spectral regions and peak heights of lipid CH₃ asymmetric (central peak ca. 2 942-2 934 cm^-1), CH₂ asymmetric (central peak ca. 2 905-2 903 cm^-1), CH₃ symmetric (central peak ca. 2 866-2 850 cm^-1) and CH₂ symmetric (central peak ca. 2 834-2 832 cm^-1) functional groups, unsaturation lipid bands group (CH attached to C-C) (central peak ca. 2 987-2 986 cm^-1), lipid ester C=O carbonyl group (central peak ca. 1 734-1 730 cm^-1) as well as the ratios among them. Multivariate spectral analyses were conducted by the hierarchical cluster analysis (CLA) and the principal component analysis (PCA) for all batches of corn and its co-product samples to investigate the molecular spectral differences. The results showed that:1) The significant differences were found in the lipid nutritive values among different batches and between corn and its by-products (P<0.05), and there was significant interaction was found between the two factors (P<0.05); 2) The result from FTIR spectroscopy analysis showed a significant interaction among the spectrum parameters except for CH functional groups (P<0.05). And a significant difference was found in lipid spectral molecular structures between corn and its by-products (P<0.05). However, no significant difference was found in the height of LECC and the height ratio of CH₂ asymmetric to CH₂ symmetric among different batches (P>0.05); 3) The results of CLA and PCA analysis showed that lipid spectral molecular structure were not distinguished among different batches. And the corn germ with pericarp had completely different structure from corn germ cake in CH₃ and CH₂ asymmetric and symmetric stretching region (ca. 2 984-2 781 cm^-1), the unsaturated lipid bands region (ca. 3 016-2 971 cm^-1) and lipid ester C=O carbonyl region (ca. 1 826-1 693 cm^-1). However, the lipid biopolymer conformations were similar among the other samples; 4) Crude fat (EE) had significant correlation with most lipid structure spectral parameters (P<0.05). In conclusion, there is a significant correlation in lipid molecule functional groups and nutritional properties between corn and its by-products, which will provide a novel research foundation for feed analyses by FTIR.

Autophagy is involved in a variety of disease processes and is closely related to viral infection and proliferation. In order to explore the difference and regularity of the mRNA expression of autophagy-related genes Beclin1, ATG5, ATG12 in Marc145 cells infected with porcine reproductive and respiratory syndrome virus (PRRSV), and to correlate PRRSV proliferation with autophagy, the real-time fluorescence quantitative PCR was used. The expressions of Beclin1, ATG5 and ATG12 mRNA were detected by real-time fluorescence quantitative PCR. The proliferation of PRRSV in Marc145 cells was measured by real-time fluorescence. The results showed that the expression of autophagy-related genes in the test group were higher than those in the control group, which means that PRRSV could induce autophagy of Marc145 cells. Meanwhile, the expression of Beclin1, ATG5 and ATG12 mRNA were advanced into the high expression state; the expression principle was positively correlated with the cytopathic effect and PRRSV proliferation.

In order to understand the immunological effect of nonstructural protein of Japanese encephalitis virus (JEV), we analyzed the eukaryotic expression differences of NS1, NS2A and NS1-2A, nonstructural proteins of JEV. CPE was observed on BHK-21 cells by observing JE live attenuated vaccine (SA14-14-2 strain) and JEV Guizhou isolate (GZ strain) respectively. Cell suspensions of virulent, attenuated strains were collected and their total RNA were extracted. The NS1, NS2A and NS1-2A genes were amplified by RT-PCR, and six eukaryotic expression plasmids, pcDNA3.1 (+)-NS1r, pcDNA3.1 (+)-NS2Ar, pcDNA3.1 (+)-NS1-2Ar, pcDNA3.1 (+)-NS1q, pcDNA3.1 (+)-NS2Aq and pcDNA3.1 (+)-NS1-2Aq, were constructed successfully. The healthy mice were immunized with the above plasmids, and the blood and tissues of those mice were collected for JEV antibody detection and cell immunoassay respectively. The results showed that JEV GZ virulent strain and JEV SA14-14-2 virulent strain could proliferate and produce CPE on BHK21 cells; The results of routine blood tests and T lymphocyte subsets detection showed that the 6 eukaryotic expression plasmids immunization in mice increased the levels of CD4⁺ and CD8⁺ cells significantly or extremely significantly. The results indicated that the cellular immunity produced by the non-structural proteins of the attenuated strain JEV was slightly better than that produced by the non-structural proteins of the attenuated strains, but the difference was not significant. These results provide reference for the basic research of encephalitis virus pathogens.

The recombinant adenoviruses of orf virus (ORFV) was constructed by splicing F1L and B2L genes with the self-cut T2A and P2A peptide, and its immune effects were evaluated, which provided the basis for vaccine development of ORFV. The target genes of F1L and B2L from ORFV-F416 strain were amplified by PCR, and the recombinant adenovirus shuttle plasmid was constructed. The shuttle plasmid and backbone plasmid were co-transfected into HEK-293 cells and the ORFV recombinant adenovirus was packaged. The recombinant adenovirus was used to infect MDBK cells and the supernatant was collected to immunize ICR mice. Indirect ELISA, lymphocyte proliferation and ORFV challenge were conducted to evaluate the immunogenicity of mice. The results showed that pAV-CMV-F1L-T2A-B2L-P2A-EGFP recombinant adenovirus was successfully obtained, and its TCID₅₀ was 10^-5.375·mL^-1. PCR and IFA results showed that the target gene was expressed both in gene and protein levels. After immunization with ORFV recombinant adenovirus, the antibody level of mice increased rapidly. ORFV recombinant adenovirus group showed much higher ability of T lymphocyte proliferation, which was significantly higher than that of PBS group (P<0.01). The results of this article showed that the recombinant adenovirus of F1L and B2L genes had positive protective effect in mice, which could stimulate humoral and cellular immune responses, and have no effects on feed intake.

In order to understand the relationship between the disease eradication and the incidence of epidemics disease, the survey data of 297 large-scale chicken farms in China from 2011 to 2015 were collected, avian leukosis and pullorum disease were selected as the specific eradication diseases. At first, the sample mean T-test was performed to determine whether there were significant differences in the incidence of diseases between purified farms and unpurified farms. Based on this, the Tobit model was used for further quantitative analysis. The results showed that the incidence of epidemic diseases in those farms which have carried out the eradication of avian leukosis and pullorum disease had dropped by 1.416% and 1.370% respectively compared with that of unpurified farms. From the points of different growth state, the incidence of avian leucosis of laying hens and breeder cocks in those farms which had eradicated avian leucosis had reduced by 1.837% and 0.949% respectively compared with that of unpurified farms. The incidence of pullorum disease of chicks, adult chickens and breeder cocks in those farms which had eradicated pullorum disease had decreased by 2.360%, 0.904% and 0.591% respectively compared with that of unpurified farms. In addition, the study found that increasing vaccine input and adopting all-in and all-out feeding method can effectively reduce the incidence of diseases. Lastly, it is suggested that the state should increase the publicity of animal disease eradication, so more farms (households) could recognize the importance of animal disease eradication, and combine with their own actual conditions, in order to carry out the eradication of the disease optimistically.

This study aimed to investigate the relationship between the expression of transforming growth factor β1 (TGF-β1), leading to the expression of fibronectin (Fn)/α5 integrin, and the invasion of Haemophilus parasuis (H. parasuis) in porcine alveolar macrophage cell line 3D4/21. The optimal adherence/invasion conditions of H. parasuis to 3D4/21 cells were determined by a series of adherence/invasion experiments. TGF-β1 expression level in the cells infected by H. parasuis was detected using Q-PCR and ELISA. Treatment of cells with recombinant TGF-β1 prior to H. parasuis infection, CFU and flow cytometry were used to confirm the relationship between bacteria variation and the expression of TGF-β1. siRNA interference was used to down regulate the expression of Fn/α5 to confirm the relationship between the expression of Fn/α5 integrin and bacteria invasion. The H. parasuis infection resulted in the elevation of TGF-β1 expression in cells. Treatment of 3D4/21 cells with TGF-β1 prior to infection significantly increased the invasion of H. parasuis. Flow cytometry revealed that the expression of Fn/α5 integrin was increased in 3D4/21 cells treated with recombinant TGF-β1 and H. parasuis. Small interfering RNAs (siRNA) were used to down regulate the expression of Fn and α5 integrin in 3D4/21 cells, which significantly decreased the invasion of H. parasuis. However, H. parasuis attachment was decreased only on cells treated with the α5 integrin siRNA, there was no corresponding decrease in attachment to cells treated with Fn siRNA. These results demonstrate that expression of TGF-β1 and α5 integrin promote the invasion of H. parasuis to 3D4/21 cells and play an important role during this process.

This study was conducted to obtain the recombinant Clostridium septicum α toxin and to evaluate its virulence and immunogenicity. Based on the known sequence, Gcsa, α toxin gene of C. septicum, was optimized and synthesized. Then, Gcsa was cloned into prokaryotic expression vector pET-30a (+) for expression and purification. Reactivity of the purified protein (rCSA) with antiserum of C. septicum was detected by Western blot. Meanwhile, Canine Kidney (MDCK) cell and mice were used to detect the virulence of rCSA. According to the method prescribed in Chinese Veterinary Pharmacopoeia (2015), four rabbits were immunized with rCSA emulsified with oil adjuvant of ISA 201 to prepare antiserum and detect the neutralizing titer. The results showed that rCSA was expressed at a high level as soluble form with a ratio about 50% by gray scale scanning, and it could react with the antiserum of C. septicum. For MDCK cell,the concentration of rCSA to induce apparent cytopathic effect (CPE) was found to be 0.15 ng·mL^-1. At the same time, the Minimum Lethal Dose (MLD) of rCSA to ICR mice has been calculated as 1.5×10⁶ng·kg^-1. After the first immunization, sera from rabbits immunized with rCSA could neutralize 100-130 mice MLD C. septicum toxin per milliliter, and 360-480 mice MLD after twice immunization. Moreover, rabbits in rCSA immunized group fully survived from the challenge of C. septicum toxin at the dose of 1 rabbit MLD, whereas all of the rabbits died (4/4) in the control groups. These data suggest that rCSA has good immunogenicity, and can be used as a potential vaccine candidate for genetic engineering subunit vaccine of C. septicum.

In order to explore the distribution and change rule of HSP70 in yak intestine, six healthy adult yaks (3-6 years old) were selected, and the distribution and change rule of HSP70 in yak intestine were observed and detected by immunohistochemistry(IHC), Western blotting (WB) and real-time fluorescence quantitative PCR (RT-qPCR) methods. Immunohistochemical results showed that HSP70 was expressed in the mucosa, submucosa and muscular layer of yak small intestine, among which the columnar cells of small intestinal mucosa epithelium were strongly positive, but the goblet cells were negative. The intestinal glandular cells of the intestinal glands and the smooth muscle cells of the muscular layer also showed positive expression, and the positive particles were mainly distributed in the cytoplasm and nucleus of the above cells. Similar to the small intestine, HSP70 was also expressed in the mucosa, submucosa and muscular layer of yak large intestine. The positive reaction was mainly located in scattered connective tissue cells of the mucosal epithelium and lamina propria,while positive reaction of colonic gland cells in the lamina propria was found only in the nucleus, but not in the cytoplasm. The statistical analysis of Western blotting and integrated optical density value of immunohistochemistry showed that, from the anterior segment of small intestine to the distal segment of large intestine, the expression of HSP70 increased first and then decreased, and peaked in the cecum. In addition, the relative protein expression of HSP70 in the cecum and colon was significantly higher than that of other bowel segments (P<0.01). Real-time fluorescent quantitative PCR showed that HSP70 mRNA had the highest relative expression in the cecum, which was twice as much as that of the duodenum, 1.68 times of the jejunum, 1.06 times of the ileum, 1.43 times of the colon and 1.52 times of that of the rectum. This study revealed that HSP70 showed a broad positive reaction in yak intestine, and HSP70 gene, HSP70 protein had different expression patterns in the small intestine and the large intestine of yaks. The results revealed the similarities and differences in biological functions played by HSP70 protein between the small intestine and the large intestine and provided information for the study of the expression and function analysis of HSP70 in ruminant tissues.

In order to study the age-related histomorphological changes in the thymus of female Anhui white goats at different stages of growth, 3 female Anhuai white goats in 4 growth periods(infancy, prepuberty, puberty, adulthood) were selected, respectively; and the thymus general indexes were recorded. Hematoxylin-eosin (H.E.) staining was used to observe the morphological structure of the thymus; immunohistochemistry was used to explore the distribution of S100 and Caspase-3 in thymus; in addition, the transcription levels of S100, CASP-3, CASP-8 and TP53 mRNA in thymus were detected using real-time fluorescent PCR (RT-PCR). The results were as follows:1) The length, weight, index and maximum width of the thymus reached their peaks in goats at prepuberty or puberty. 2) The boundaries between the cortex and medulla in the thymus were clear in female goats at prepuberty, puberty and adulthood, but not in infancy. 3) Caspase-3 positive cells were concentrated at the cortex and medulla of the thymus, but S100 was mainly distributed in the junction of the cortex and medulla. 4) No significant difference was found in S100 mRNA levels between different periods (P>0.05); CASP-3 and CASP-8 mRNA levels were highest at prepuberty, and TP53 mRNA was the highest at adulthood of female goats. The results indicated that female goats' thymuses showed age-related deterioration after puberty. CASP-3 and CASP-8 may participate in thymus development, cell renewal and involution processes in the early periods of development. Additionally, CASP-8 and TP53 play important role in thymus involution during adulthood.

To investigate the effect of total flavonoids from the seeds of Ammopiptanthus mongolicus on the immune function and the ultrastructure of thymus and spleen in immunosuppressed mice,100 Kunming mice were randomly divided into five groups(n=20,half male and half female):normal control group, immunosuppression group (cyclophosphamide induced), test group (cyclophosphamide induced, total flavonoids treated) Ⅰ, test group Ⅱ, and test group Ⅲ, and using intragastric administration of medicine to mice. After 28 days, carbon clearance index, thymus index, spleen index, and the content of IL-2, IL-4 in serum were detected. At the same time, the ultrastructure of thymus and spleen were observed through scanning electron microscope. The results showed that the immunosuppressed mice were copied successfully with cyclophosphamide, and the total flavonoids from the seeds of Ammopiptanthus mongolicus could increase the carbon clearance index, thymus index, and spleen index, the content of IL-2 and IL-4 in serum in immunosuppressed mice. The difference were significantly compared with the immunosuppression group, especially in test group Ⅱ[150 mg·(kg·d)^-1total flavonoids] (P<0.01). Compared with the immunosuppression group, the number of mature lymphocytes in immunosuppressed mice thymus and spleen increased significantly in the total flavonoid-treated groups and the lymphocytes connected closely, especially in test groupⅡ[150 mg·(kg·d)^-1total flavonoids] (P<0.01). The results suggest that the total flavonoids from the seeds of Ammopiptanthus mongolicus can reverse and repair obviously the immune function of the immunosuppressed mice and promote obviously the proliferation and activation of lymphocytes,further more, it has does-dependent relationship with the total flavonoids in a certain extent.

In order to investigate the effects of osteoprotegerin (OPG) on autophagy and apoptosis in osteoclasts (OCs) and osteoclast precursors (OCPs), RAW264.7 cells were pretreated with autophagy promoter rapamycin (RAP) for 1 h or autophagy inhibitor chloroquine (CQ) for 30 min, followed by treatment with 80 ng·mL^-1 OPG for another 6 h, and cells were divided into the Control group (Con), the OPG group, the CQ group, the CQ +OPG group, the RAP group and the RAP+OPG group, Western blot was applied to detect the proteins related to autophagy; Flow cytometry was used to observe the apoptosis rate. The results of Western blot showed that in OCPs treated with OPG the expression of Atg5 was increased significantly (P<0.01) and Beclin-1 was increased (P<0.05), while the expression of LC3-Ⅱ showed no significant difference (P>0.05) compared with Con group; Compared with OPG treatment group, the expression of LC3-Ⅱ was significantly increased in the RAP+OPG group. In OCs, compared with Con group, OPG treatment markedly reduced (P<0.01) the expression of LC3-Ⅱ, Atg5 and Beclin-1; Compared with the OPG group, the expression of Atg5 and Beclin-1 were significantly reduced by the combination use of RAP+OPG. The results of flow cytometry showed that in OCPs, OPG and RAP, alone or in combination, markedly increased the apoptosis rate (P<0.01),while there was no difference (P>0.05) in the CQ+OPG group; The effect of RAP+OPG was stronger compared to the OPG treatment group (P<0.01). While in OCs, the apoptosis level was greatly reduced by OPG (P<0.01), both CQ+OPG and RAP+OPG presented significantly decreased apoptosis level (P<0.01). The results of our study demonstrate that OPG induces apoptosis of OCPs by activating autophagy. Nevertheless OPG reduces apoptosis of OCs through restraining the autophagy in OCs. Interestingly, either the inhibition or promotion of autophagy results in the apoptosis of OCs.

The study was conducted to determine whether Astragalus adsurgens Pall is infected by the swainsonine-producing fungi, and the genetic evolution relationship between the Alternaria Section Undifilum species fungal endophytes from locoweeds and A. adsurgens Pall. Fungal isolates were isolated from the tissues of Astragalus adsurgens Pall. To screen the fungi that can produce swainsonine, the hyphal extract of each isolate was detected by means of the α-mannosidase Assay and ULPC-MS respectively. ITS, GPD, and KS genes from the swainsonine-producing fungi were amplified and sequenced for the analysis of homology and genetic evolution. The phylogenetic trees of fungi were built based on fungal ITS and GPD sequences. The taxon of the swainsonine-producing isolates from A. adsurgens Pall was determined according to morphology and the results of genetic evolution analysis. The present study showed that 43 isolates were cultured from the tissues of A. adsurgens Pall. The hyphae of thirteen isolates were confirmed containing swainonine. The results based on the analysis of homology and genetic evolution showed that swainonine-producing isolates were closely related to pathogenic fungus Alternaria gansuense from A. adsurgens Pall and the fungal endophytes Alternaria Section Undifilum sp. from locoweeds. This study determined the presence of swainonine-producing fungi inside A. adsurgens Pall. The isolates producing swainsonine are classified into Alternaria Section Undifilum sp and named Alternaria Section Undifilum gansuense based on the morphology and the results of genetic evolution analysis.

In order to understand the distribution and molecular characteristics of new emerged class Ⅰ Newcastle disease virus in China. We carried out the F gene sequence analysis and phylogenetic analysis of 4 viruses isolated in 2016. Results were as follows:The length of complete F gene of 4 NDV isolates were 1 662 nt, encoding 553 amino acids, and the amino acid identity among all isolates was 97.9%-100%. The amino acid motif at F protein cleavage site of the 4 isolates was ¹¹²ERQERL¹¹⁷, which was consistent with that of lentogenic Newcastle disease virus. The phylogenetic analysis of F gene (47-420 nt) showed that all 4 isolates were newly emerging NDVs in China. Phylogenetic tree constructed by the complete F genes showed 4 Class Ⅰ viruses belonged to sub-genotype 1c, which showed a high level of similarity with isolates obtained from North America, and had the identity of 95.5%-97% with the sub-genotype 1c virus (Duck/Guangxi/1261/2015) isolated in China. Our results indicated that the new emerged class Ⅰ NDVs had the ability to circulate and spread in China.

Domestic poultry in live poultry markets (LPMs), especially waterfowl, play an important role in the evolution, maintenance and spread of the avian influenza viruses (AIVs). Therefore, it is important to conduct a long-term, regular epidemiological surveillance in the LPMs. From October 2016 to September 2017, we collected oropharyngeal and cloacal swabs from the poultry traded in the LPMs in the eastern China monthly. The samples were inoculated into SPF embryonated chicken eggs. Viruses in the allantoic fluid were confirmed by HA test and were divided to different subtypes by HI test with subtype-specific antibodies. Then eight segments of AIVs' genome were amplified by RT-PCR and these segments were sequenced. Sequence alignment and phylogenetic analysis were performed by software such as DNASTAR and MEGA6.0. A total of 1 178 samples from 127 flocks were collected, and 100 H9N2 AIV strains were isolated, among which 7 strains came from waterfowl. Genetic analysis of eight segments showed that all 7 strains belonged to the S genotype H9N2 AIVs. The genotype of H9N2 viruses from waterfowl traded in LPMs was consistent with that of predominant strains which were circulating in chickens in China. Our study highlights the need to constantly monitor the H9N2 viruses in poultry in LPMs to understand the emergence and evolution of strains that may become epidemic.

This study aimed to find the cause of severe respiratory disease in fattened goat herds of many farms in Jiangsu province during 2013 to 2014. In this study, one caprine herpesvirus 1 named JSHA1405 was detected and isolated from nasal swab samples of goat herds with severe respiratory disease, and identified by transmission electron microscopy, PCR and virus pathogenicity test. MDBK cells infected with the third generation of JSHA1405 showed typical cytopathogenic effect (CPE). Under transmission electron microscopy detection, the enveloped virion could be observed with the diameter of 100 nm. Sequence analysis of gB gene indicated that JSHA1405 had some variation compared with E/CH strain, and the homology was 99.2%. Furthermore, the pathogenicity test indicated that JSHA1405 could induce high fever (peaked at 41.8℃) and lasted for about one week, other symptoms included depression, nasal discharge. Virus shedding could be detected from nasal and rectal swabs. Neutralizing antibodies appeared from the 7^th day post infection (dpi) and peaked at 28^th dpi. This is the first report about isolation, identification and pathogenicity study of CpHV-1 in China. The experiments demonstrated that the CpHV-1 strain JSHA1405 was pathogenic to goats. It provides foundation for further study of the viral etiology and the prevention and control of CpHV-1.