Tuberculosis (TB) is a severe pulmonary disease and a public health burden caused by the infection of Mycobacterium tuberculosis (Mtb). Every year, millions of people die of TB worldwide. Unfortunately, so far, little breakthrough progress has been made in the prevention and treatment of TB. Exosomes are small membrane vesicles that can be secreted by most cells in the body; it plays important roles in intercellular communication and other physiological processes by shuttling various molecules from donor to recipient cells. Recent studies have found that exosomes also play important roles in the host defense against Mtb infection. In this review, we summarized the role of exosomes in anti-Mtb infection and its application.

Testis maturation and development are a prerequisite for spermatogenesis and effective reproduction in mammals, the process of maturation is extremely complex, which was affected by multiple factors, especially genetic factors. Testis development process was strictly regulated by lots of protein-coding genes, while growing evidences showed that non-coding RNAs(miRNAs, lncRNAs and piRNAs)also played an important roles in testicular development and spermatogenesis. In recent years, the study of transcriptome based on mRNA, miRNA, lncRNA and piRNA have been successfully applied in exploring molecular mechanism of mammalian testicular development and spermatogenesis, and have made great progress. In this review, we summarized the current status of testis development research in mammals from the aspect of testicular developmental-related genes, miRNAs, lncRNAs and piRNA in order to provide reference and theoretical basis for understanding global genome expression change in testicular development and better guide in animal breeding in the livestock industry.

The aim of this study was to clone the coding region of phosphorylase kinase gamma 2(PHKG2) gene in Congjiang pig and to explore the function of PHKG2 gene. In this study, the complete CDS region of PHKG2 gene in Congjiang pig was amplified by RT-PCR. By constructing the overexpression vector pEGFP-N3-PHKG2, four pairs of RNAi expression vectors targeting the Congjiang pig PHKG2 gene were designed and synthesized, and transiently transfected into C2C12 cell line and Congjiang pig kidney cells with normal growing cells as a blank control, the expression of green fluorescent protein of each recombinant vector was detected after 24 h. RNA was extracted from cells after 48 h. The expression levels of PHKG2 and glycogen metabolism related genes(glycogen phosphorylase (PYGM), glycogen synthase 1 (muscle)(GYS1), phosphoglyce rate mutase(PGAM2)) were detected by qRT-PCR, and the glycogen content of each group in cells was determined. The results showed that, after double enzyme digestion, sequencing detection and transient transfection of liposomes into C2C12 cells, it was verified that the overexpression vectors pEGFP-N3-PHKG2 and 4 RNAi expression vectors were successfully constructed. After transfecting into Congjiang pig kidney cells, compared with the blank and negative control (pEGFP-N3), the overexpression vector pEGFP-N3-PHKG2 transfected into cells extremely significantly increased the expression level of PHKG2 gene (P<0.01), and PGAM2 and PYGM genes expression were significantly up-regulated (P<0.05), and the glycogen content in cells was significantly reduced (P<0.05). In each RNAi vector, the interference efficiency of shRNA-1 was higher than that of the blank and negative control (NC). The expression levels of PHKG2 gene was extremely significantly down-regulated (P<0.01), and the expression levels of PGAM2, PYGM and GYS1 genes were significantly down-regulated (P<0.05), and significantly increased the glycogen content in the cells (P<0.05). shRNA-2 extremely significantly down-regulated the expression of PHKG2 gene (P<0.01); shRNA-3 also significantly interfered with the expression of PHKG2 (P<0.05); shRNA-4 did not interfere with the expression of the 4 genes detected obviously. The glycogen contents in cells didn't significantly increase after shRNA-2, shRNA-3, shRNA-4 transfected into the cells. In this study, the overexpression vectors and RNAi vectors of PHKG2 gene of Congjiang pig were successfully constructed. The expression of PHKG2, PYGM, PGAM2 and GYS1 genes and glycogen content were significantly affected. PHKG2 gene may be an important candidate gene affecting meat quality of Congjiang pig and to lay the foundation for further studying the role of PHKG2 gene in glycogen metabolism pathway.

To explore the role of lncRNA-ENSSSCT00000018610 in the development of porcine follicles, the full length RNA sequence of lncRNA-ENSSSCT00000018610 was amplified by RACE technique, it's tissue expression profile and expression pattern in ovarian follicles of Meishan and Duroc sows were analyzed by Real-time PCR, and it's target genes were predicted by using the cis and trans methods. A 1 731 bp-length sequence was obtained as the full length RNA sequence of lncRNA-ENSSSCT00000018610. lncRNA-ENSSSCT00000018610 was expressed differentially in variety of tissues(muscle, heart, liver, spleen, lung, kidney, duodenum, ovary, fallopian tube, uterus, pituitary, corpus luteum, hypothalamus), and expressed relatively higher in kidney, lung, uterus and ovary. The expression level of lncRNA-ENSSSCT00000018610 in S and L follicles of Duroc were significantly higher than that of Meishan (P<0.05). The expression level of lncRNA-ENSSSCT00000018610 in M1 and M2 follicles of Duroc were significantly higher than that of Meishan (P<0.01). The potential target genes of lncRNA-ENSSSCT00000018610 including TNIP1, CYP2J2, SCARB1 and IBSP were related to the reproduction traits and participated in follicular development of pig. This result suggests that lncRNA-ENSSSCT00000018610 may participate in follicular development indirectly by regulating its target genes expression.

The purpose of this study was to investigate the biological function of Smad3 gene during Qinchuan cattle preadipocytes differentiation, and provide a theoretical basis for clarifying the role of TGF-β signaling pathway in the growth and development of cattle adipose tissue by overexpressing and interfering Smad3 gene in Qinchuan cattle preadipocytes. In this study, overexpression adenovirus pDC316-EGFP-bSMAD3 and interference adenovirus pDC316-EGFP-ShRNA-bSMAD3-1405 were successfully obtained by using AdMaxTM recombinant adenovirus packaging system reconstructed with Ad5 adenovirus. Then we detected the mRNA expression of Smad3 gene and adipogenesis-related genes by real-time fluorescence quantitative PCR after the adenovirus infected bovine preadipocytes. The results showed that, compared to the control group, the expression of Smad3 gene in Qinchuan cattle preadipocytes were significantly increased about 1 031, 1 286, 633 times, respectively(P<0.001) when bovine preadipocytes were infected by pDC316-EGFP-bSMAD3 for 3, 6, 9 d. Meanwhile, the interference group's (pDC316-EGFP-ShRNA-bSMAD3-1405) expression of Smad3 gene in cattle preadipocytes reduced by 70%, 55% and 57%, respectively (P<0.01) compared with the control group. Moreover, both Oil Red O staining assay and qRT-PCR results demonstrated that Smad3 gene suppressed the lipid accumulation in preadipocytes and markedly down-regulated the expression of adipogenesis-related gene PPARγ, FABP4, C/EBPα and C/EBPβ. In conclusion, Smad3 gene is successfully overexpressed and interfered in Qinchuan cattle preadipocytes mediated by adenovirus, and plays an important role in adipogenic differentiation process in Qinchuan cattle preadipocytes.

This study aimed to explore the expression and function of TLRs gene family in duck bursa of Fabricius, therefore, the mRNA expressions of TLRs genes in duck bursa of Fabricius during the postembryonic development stage were detected by qRT-PCR. The expression patterns were analyzed by clustering and the phylogenetic trees based on the TLRs promoters and coding regions were constructed, respectively. The results showed that the expression level of TLR2a in duck bursa of Fabricius was significantly higher in W2 and W6 than in W4(P<0.05). The expressions of other TLRs in the different postembryonic development stages of duck bursa of Fabricius were not significantly different (P>0.05). TLR1a, TLR2a and TLR2b had similar expression patterns, as well as TLR3, TLR5 and TLR15. In the phylogenetic tree of the coding region, TLR1a, TLR1b, TLR2a and TLR2b were clustered together, and they were closer to TLR15. TLR3 and TLR5 were on the same branch, TLR4 and TLR21 were on two separate branches, respectively, and TLR21 was far away from the other TLRs. However, there was no obvious rules in the phylogenetic tree of TLRs gene family promoter regions. The results indicate that TLRs family members may not be involved in the regulation of postembryonic development in duck bursa of Fabricius; TLR2a and TLR2b may have similar function, as well as TLR3, TLR5 and TLR15. Although TLR1a and TLR1b have high sequence similarity, functional differentiation may occur during the long process of evolution. There was no significant relationship between TLRs expression patterns and its promoter region sequence evolution.

The aim of this study was to analyze the phylogenetic tree of Mongolian horses using the speedy double bootstrap method. The mitochondrial DNA D-loop base sequences of 30 modern horses were used, which included 5 Mongolian horse subspecies (Baerhu, Sanhe, Wushen, Ujimqin and Xinihe horses) 6 for each subspecies and 6 Przewalski's horse as the outgroup, and all 105 possible phylogenetic trees were analyzed. Mega6 and PAMAL4.9 were used, and maximum likelihood phylogenetic tree of Mongolian horse were estimated by maximum likelihood method. Finally, the 3^rd order accurate P-value (speedy double bootstrap P-value, SDBP) of the 105 phylogenetic trees was calculated with the CONSEL, SDBP packages in R3.0. The phylogenetic tree of Mongolian horse with the largest SDBP value had 3 branches; Baerhu and Wushen horses were clustered together, Ujimqin and Xinihe horses were clustered together, and Sanhe horse formed an independent branch which was sister to all other Mongolian subspecies. The Mongolian horse phylogenetic tree with the largest SDBP value was consistent with the predicted relationship between Sanhe and the other 4 Mongolian horse subspecies, and this topological tree also had the highest likelihood value. The results of this study also showed that it was more effective to analyze the topological relationships of Mongolian horses using the maximum likelihood method combined with the SDBP value than using the maximum likelihood method combined with the approximately unbiased (AU) value and the bootstrap P-value (BP) value. Additionally, this approach was more effective than the unweighted pair-group method with arithmetic means (UPGMA), and had similar results to the neighbor joining (NJ) method. The results of this study provided robust genetic data which would produce a more complete picture of Mongolian horse evolution.

To study the effects of shearing on the behavioral and physiological patterns of Small-tail Han sheep, 9 rams and 13 ewes which had similar body condition and needed the first shearing were selected to study the behavioral patterns (before, during and after shearing) and the physical indexes (before and after shearing) from September 1^st to 10^th, 2015. The behavior ethogram were lying, ruminating, standing, walking, foraging, drinking before and after shearing, and rising, blinking, bleating, ear shaking, tail wagging, excreting during shearing. The physical indexes measured were heart rate(HR), respiratory frequency(RF) and rectal temperature(RT). Animal were drawed and K⁺,Na⁺,Mg²⁺ and the blood metabolites of β-endorphin (β-EP), adrenocorticotrophic hormone (ACTH), prolactin (PRL), catecholamine(CA), triiodothyronine (T3), Triiodothyronine (T4), antidiuretic hormone (ADH), cortisol (CORT), growth hormone (GH), glucose (GLU) were measured before,during and after shearing. The results showed that shearing produced acute intensive stress to Small-tail Han sheep. Shearing increased the behavior of blinking, tail wagging, rising in ewes and rams,and the plasma ACTH and GH of ewes first increased and then decreased with the process of shearing and showed significant changes (P<0.05), the other blood ions and metabolites showed no significant changes (P>0.05). There were some differences in these parameters between male and female sheep:The more blinking and bleating occurred in ewes than that in rams during shearing(P=0.006,0.022).The significant differences in ACTH,CA,T4 between rams and ewes at the ending of shearing (P=0.050,0.004,0.029) were detected, and the contents of plasma ACTH and T3 in rams were higher than that in ewes 10 minutes after the ending of shearing (P=0.037,0.031). In comparison, the stress tolerance of rams was stronger than that of ewes. The shearing also significantly changed the behavioral patterns and physical indexes of the Small-tail Han sheep. The increasing of foraging, drinking, ruminating, walking and the decreasing of standing, rectal temperature and respiratory frequency after shearing all accounted for the improvement of the adaptability of Small-tail Han sheep in high temperature environment.

Embryo implantation is one of the important factors affecting litter size in sows. In order to find the key genes regulating porcine embryo implantation. 2 Meishan sows selected and slaughtered on 18^th day and 24^th day of pregnancy,respectively followed by ovary tissues collection. And these tissues would be used in RNA sequencing (RNA-seq) analysis, GO analysis and pathway analysis.The results showed that:1) the most of sequences detected (reads) were gene exons in both tissues (ovary_18 d and ovary_24 d) during embryo implantation in pigs.The most reads were located in chromosome 6 (Chr 6) for ovary_18 d, followed by Chr 14, Chr 1 and Chr 7. For ovary_24 d, the most reads were located in Chr 1, followed by Chr 7, Chr14 and Chr M. In addition, there were more genes expressed and higher expression level in ovary_24 d than that in ovary_18 d. More than 60% genes of top 50 were located in mitochondria according to gene expression level, suggesting that mitochondria genes might play an important role in the regulation of porcine embryo implantation.2)581 genes were up-regulated and 103 genes were down-regulated in ovary_24 d compared with that in ovary_18 d. Among these genes, the expression difference of EN19684 was was highest, which was located in mitochondria, the expression difference of EN11278 was highest, which was located in Chr 14. 3)GO analysis results showed that these differentially expressed genes were significantly enriched in 38 biological processes such as endothelial cell activation, 19 cellular compartments such as host cytoplasm, and 16 molecular functions such as FAD-AMP lyase activity. Pathway analysis results indicated that these differentially expressed genes differed among 185 biological pathways, and the top 3 pathways were PI3K-Akt signaling pathway, Hippo signaling pathway and circadian cycle. In conclusion, genes expressed in ovary are mainly involved in the regulation of post-implantation, and high-expression genes are mostly located in mitochondria. The main function of differentially expressed genes is endothelial cell activation, and the main pathway of differentially expressed genes is PI3K-Akt signaling pathway.

The purpose of this research was to investigate the effect of exogenously added VEGF on the expression of the FLT-1 during the process of ovine oocytes maturation in vitro. Three different in vitro maturation culture methods were adopted:naked oocytes separate cultivation, granule cells with naked oocytes co-cultivation and cumulus-oocytes complexes (COCs) cultivation. Treatment groups were adding exogenous 5 ng·mL^-1 vascular endothelial growth factor (VEGF) while control groups were not. The FLT-1 mRNA and protein expression levels in both ovine oocytes and granule cells were detected by qPCR and Western blot, respectively. The results indicated that:1)Adding VEGF could extremely significantly reduce the FLT-1 mRNA expression in granule cells in the co-cultivation treatment group at 12, 16 and 20 h, and the FLT-1 mRNA expression in COCs treatment group at 4, 8, 12, 16 and 24 h (P<0.01), and extremely significantly reduce the FLT-1 mRNA expression in oocytes in the co-cultivation treatment group at 12, 20 and 24 h, and the FLT-1 mRNA expression in COCs treatment group at 4, 8 and 12 h (P<0.01), but extremely significantly increase the FLT-1 mRNA expression in oocytes in the naked oocytes separate cultivation treatment group at 4-24 h (P<0.01). 2)Adding VEGF could extremely significantly reduce the expression of the FLT-1 protein in granule cells in both co-cultivation treatment group and COCs cultivation treatment group at 4-12 h (P<0.01). The expression of the granule cells FLT-1 protein in both naked oocytes co-cultivation treatment group and COCs cultivation treatment group showed a fluctuating downward trend. The granule cells FLT-1 protein expression in both COCs cultivation control group and COCs cultivation treatment group were higher than that of granule cells with naked oocytes co-cultivation treatment group at all time points.3)Among the 3 in vitro maturation culture methods, except for a sharp increase in the naked oocytes separate cultivation control group at 20-24 h, the oocytes FLT-1 protein expression in the other groups showed a downward trend of fluctuation. In conclusion, the FLT-1 has autocrine and paracrine effects in oocytes and granule cells. The expression of the FLT-1 mRNA and protein in oocyte are closely related to the presence or absence of peripheral granule cells, the number and the way of encapsulation. During in vitro culture environment, the addition of VEGF can effectively activate the FLT-1 mRNA expression, but at the same time combine the FLT-1 protein in order to play a biological effect promoting oocytes maturation, thus reducing the FLT-1 protein expression.

The aim of this study was to investigate the specific contribution of follicle-stimulating hormone (FSH) on maturation of yak oocytes in vitro, the effect of FSH on the expression of epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) in yak oocytes, and the relationship between FSH and apoptosis. In this experiment, different concentrations of FSH (final concentration:0,2,5,10 μg·mL^-1) were added into the medium of the yak oocytes in vitro. The mRNA and protein expression of EGF and EGFR were detected by real-time PCR (qRT-RCR) and immunofluorescence staining, respectively. Meanwhile, the expression of Bax and Bcl-2 were detected by qRT-RCR and Western blotting. The results showed that:1) It was noteworthy that FSH promoted the maturation of oocytes, and the optimal concentration of FSH was 5 μg·mL^-1, and the rate of maturation oocytes was 76.84%, which was significantly higher than other groups (P<0.05). 2) The expression levels of EGF and EGFR depended on the doses of FSH. The expressions of EGF and EGFR were upregulated during the doses of FSH raised from 0 to 5 μg·mL^-1, which was significantly higher in 5 μg·mL^-1 FSH group than other groups (P<0.05). When the concentration of FSH was 10 μg·mL^-1, the expression level of EGF and EGFR was reduced. Immunofluorescence assay showed that EGF was mainly located in cumulus cells, while EGFR was expressed in cumulus cells and oocytes. 3) With the increase of FSH concentration, the expression of Bax and Bcl-2 showed the obvious reverse pattern, the expression level of Bcl-2 increased gradually, while the expression level of Bax decreased gradually. The expression of Bax was the lowest, and the expression of Bcl-2 was the highest when the dose of FSH was 5 μg·mL^-1, but the expression level of Bax increased in 10 μg·mL^-1 FSH group. The results showed that FSH increased the ability of oocyte development and induced the expression of EGF and EGFR during the process of yak oocyte maturation in vitro, and inhibited the apoptosis of oocytes by regulating the expression of apoptosis related factors such as Bax and Bcl-2.

This study was conducted to investigate the molecular mechanisms of muscle metabolism regulated by conjugated linoleic acid (CLA) through affecting the expression of miRNA in porcine muscle. Twelve purebred Rongchang gilts with similar body weight were randomly divided into control and CLA groups. The pregnant gilts in CLA group were fed with 1.5% CLA diet from the beginning of pregnancy to weaning at 28 days old; After weaning, 30 piglets were respectively selected from the control and CLA groups, and the diet of piglets in original CLA group was also added 1.5% CLA. When the body weight of piglets was about 30 kg, 6 piglets from each group were slaughtered to collect the back muscle and leg muscle samples. After RNA extraction, cDNA libraries were constructed by the back muscle and leg muscle samples of the control and CLA groups; The Solexa deep sequencing was performed to analyze the effect of CLA on the miRNA expression profiles in porcine muscle tissue, the target genes prediction and function analysis for differentially expressed miRNA were carried out through the bioinformatics method. The results showed that:1)There were 44 869 982 and 45 105 806 clean reads in the back muscle and leg muscle tissues, respectively, and most of the small RNA sequence were 20-23 nt. 2) 306 and 304 known miRNAs were respectively found in the back muscle and leg muscle tissues, 295 known miRNAs were co-expressed in two tissues. 3) The expression of miRNA in porcine muscle was changed by CLA supplementation and the effect of CLA on miRNA expression in leg muscle was stronger than that in back muscle; 5 miRNAs in the back muscle and 12 miRNAs in the leg muscle were differentially expressed in corresponding CLA treatment (P<0.05), of which only the ssc-miR-224 was differentially expressed in both two tissues, thus 16 differentially expressed miRNAs were identified. 4) KEGG analysis revealed that the target genes of 16 differentially expressed miRNAs were involved in 292 biological pathways, of which 24 pathways were significantly enriched (P<0.05), including the MAPK and Notch signaling pathways, which were known to regulate muscle metabolism. 5) Six differentially expressed miRNAs were randomly selected to validate the sequencing results by qRT-PCR, the qRT-PCR expression results corresponded well with the results from the sequencing. This study suggests that CLA may regulate muscle metabolic pathways by affecting the expression of miRNA.

The study was conducted to investigate the effect of folic acid on metabolism of chicken primary hepatocytes, aiming to provide a reference for exploring regulation mechanism of folic acid on hepatic metabolism and also provide theoretical and scientific basis for its application in poultry. New-born chickens were used for separating primary liver cells. When the confluence reached to about 80%, hepatocytes were exposed to treatment medium. The control group and folic acid addition group were contained in the study. There were 6 replicates for each group. After 12 h treatment, cells were collected to detect triglyceride, NEFA and cholesterols contents. Metabolomics analysis was carried out by GC-MS method. The OPLS-DA model was established to identify differential metabolites, and pathways enriched based on differential metabolites were analyzed through KEGG system. The results showed that folic acid significantly decreased triglyceride and cholesterol contents in hepatocytes compared with the control group (P<0.05). NEFA level was higher in folic acid addition group (P<0.05). Twenty three up-regulation and 9 down-regulation metabolites were found between two groups, which mainly included amino acid, small molecule sugar and acid. Thirty one pathways were enriched based on differential metabolites containing amino acid metabolism, carbohydrate metabolism, lipid metabolism, and so on. The results indicated that folic acid could promote lipid hydrolysis in chicken primary hepatocytes, and was involved in regulating sugar, lipid and amino acid metabolism, which would provide a reference for exploring regulatory mechanism of folic acid on hepatic metabolism in future.

The aim of this study was to investigate the effects of different forms of linseed oil on the growth performance, meat quality, fatty acid content in muscle and mRNA abundance of lipid metabolism key regulation factors and lipid metabolism related enzymes in sheep. A single-factor experimental design was used. Twenty-four healthy Dorper(♂)×Small-Tailed Han sheep(♀) F₂ male lambs with similar days of age((100±10) d) and body weight((27±2.17) kg) were allocated into 4 groups with 6 sheep in each group. The lambs in control(CON) group were fed with basal diet, the lambs in experimental groups were fed the diets with the same fat content(4%) of linseed oil(LO), linseed(L), linseed oil microcapsule(LOM), respectively. The growth performance indexes such as body weight and dry matter intake of sheep were recorded and the meat quality indexes such as pH and shear force were measured after slaughter. The contents of dry matter, protein, fat and ash in meat were measured by drying, Kjeldahl nitrogen method, Soxhlet extraction and high temperature ash, in addition, the content of fatty acids and the expression of mRNA of lipid metabolism related regulation factors and enzymes in meat were determined by gas chromatography and real-time quantitative PCR. The results showed that:1)The different forms of linseed oil had an effect on sheep ADG, and the ADG of LO group was significantly lower than that of CON, LOM and L groups(P<0.05), the fat content in muscle of the test groups were significantly higher than that of the control group(P<0.05), there was no significant change in other nutritional components in muscle(P>0.05), compared with CON group, muscle shearing force was significantly decreased and tenderness was significantly increased in LOM group (P<0.05), however, the different forms of linseed oil had no significant effect on pH, dripping loss, cooking loss and water loss rate of muscle(P>0.05). 2) Compared with CON group, the addition of different forms of linseed oil significantly increased the content of ALA, CLA and AA in muscle, and significantly improved the ratio of PUFA n-6/n-3(P<0.05), the CLA content of LOM group was significantly increased by 375%, 171% and 58% compared with CON, LO and L groups (P<0.05), and the n-3 PUFA content of L and LOM groups were significantly increased by 151% and 178% compared with CON group(P<0.05). 3) The addition of different forms of linseed oil in diets significantly affected the PPARγ and SREBP1 mRNA expression, compared with group CON, LO, LOM and L groups could increase the expression of PPARγ mRNA by 53%, 68% and 36%, respectively, and down-regulated the expression of SREBP1 mRNA by 47%, 49%, 53%(P<0.05). The addition of linseed oil in each group inhibited the expression of FAS and SCD genes, the expression levels of SCD in LOM group were significantly higher than that in L group(P<0.05), and significantly increased the expression of mRNA of LPL gene, which was 2.7 times higher than that in CON group(P<0.05), but had no significant effect on ACC expression(P>0.05). The addition of linseed oil is beneficial to the deposition of PUFA, promote the expression of PPARγ, LPL genes, and inhibit the expression of SREBP-1, SCO and FAS genes. Linseed oil microcapsule can increase the content of PUFA in muscle, especially the content of CLA, improve the meat quality, increase muscle tenderness, its effect is better in increasing the content of PUFA in muscle of mutton sheep.

This study was conducted to investigate the effects of resveratrol (RES) and sanguinarine (SAG) on the growth performance and serum biochemical indexes of 2-6 months old calves. Fifty-four female Holstein calves of 5 days old with average body weight of 42 kg were randomly assigned to 3 groups with 18 calves in each group. Calves were fed one of the following diets:a milk replacer (MR) as the basal diet (MR group), a basal diets supplemented with SAG (0.05 mg·(kg BW)^-1, SAG group) or a basal diet supplemented with RES (4 mg·(kg BW)^-1, RES group). Calves were measured for body weight and body size on 56, 90, 120, 150 and 180 days of age. Blood was collected on 60 and 180 days of age for detecting serum biochemical indexes. The results showed that:1) Compared with MR group, RES supplementation significantly increased the body weight of 180 days old calves (P<0.05). The body weight of 180 days old calves in SAG treatment had a trend to increase (P=0.081 2); 2) In body size, SAG supplementation significantly increased the body height of 150 and 180 days old calves (P<0.05), and hip height of 180 days old calves(P<0.05); 3) Compared with MR group, RES and SAG supplementation significantly decreased serum FFA and INS concentrations of 60 days old calves (P<0.05); RES supplementation significantly increased serum GH, EGF and IGF-Ⅰconcentrations of 60 days old calves (P<0.05); SAG and RES supplementation significantly increased IgA concentrations of 60 days old calves (P<0.05); SAG and RES supplementation had no significant effect on IgG (P>0.05); Serum MDA concentration of 60 days old calves in SAG treatment group were higher than that in MR group (P<0.05). RES and SAG supplementation had no significant effect on serum SOD, GSH-PX and T-AOC concentrations of calves (P>0.05). In conclusion, the addition of RES and SAG in diets increased the body weight and body height of 180 days old calves, respectively, and improved the serum antioxidant indexes of calves.

To explore the host factors that contribute to the pathogenicity of the H5N1 influenza virus, Two H5N1 avian influenza viruses with different pathogenicity mainly determined by PA (CK10 and GS10) were used in this study. A549 cells were infected with the two viruses and immunoprecipitation (IP) experiments were performed by using PA antibodies subsequently to screen host proteins that interact with PA. The host proteins were further identified by LC-MS/MS. As a result, compared with the low pathogenic GS10, there were 160 host proteins interacting with the high pathogenic CK10 specifically. Bioinformatics analysis was employed targeting on these 160 proteins subsequently. Gene Ontology (GO) annotation analysis showed that these proteins mainly involved in the biological processes of translation, gene expression, viral transcription and viral infection. KEGG pathway analysis revealed that these proteins mainly participate in the cell pathways of translation, infectious diseases and signal transduction. Using co-immunoprecipitation (Co-IP), host protein eEF1A1 was identified to interact with CK10 PA protein specifically. Therefore, this study successfully screened out the differential interactome data of PA protein from different pathogenicity H5N1 influenza viruses. It may play pivotal roles in elucidating the complicated pathogenesis of H5N1 influenza virus.

Japanese encephalitis is a mosquito transmitted zoonosis caused by Japanese encephalitis virus (JEV) that seriously harms the health of human and animals, such as pigs and horses. In order to establish a specific method for detection the JEV antibody in various animal serum, a blocking ELISA method was established using recombinant EDⅢ protein and horseradish peroxidase (HRP) conjugated JEV specific Monoclonal antibody 1B10. Results were as follows:The percentage of interruption rate (PI) of 100 JEV antibody negative serum samples were statistically analyzed, the cut-off of blocking ELISA was determined as the sample presenting a calculated PI ≥ 34% were considered positive; while samples with a calculated PI ≤ 25% were rated negative; and those presenting PI between 25% and 34% were considered inconclusive. No cross-reaction with antibody positive serum against PCV2, PRV, CSFV, FMDV and PRRSV was observed in the test. And the intra-and inter-assay variation were less than 5%. Compared with the commercialization JEV antibody detection ELISA kit, the agreement rates of the new established blocking ELISA was 97.2%. The specificity and sensitivity of the blocking ELISA were 98.5% and 94.3%, respectively. The blocking ELISA was used to test against 515 serum samples collected from swine, cattle and sheep, among which 74.5%, 13.3% and 9.2% of samples were JEV specific antibody-positive. The detection results of randomly selected swine, cattle and sheep by the serum neutralization test were agree with blocking ELISA. As a result, a blocking ELISA was successfully established for detecting JEV antibody of swine, cattle and sheep in the present study.

The objective of this study was to investigate the prevalence of serotype Ⅰ Riemerella anatipestifer isolates in Guangdong province and obtain the molecular typing characteristics. R.anatipestifer strains were isolated from brain or liver samples collected from sick and dead ducks or geese during 2007 to 2017, and then these strains were subjected to purification and identification by PCR method. R. anatipestifer serotype Ⅰ were identified by glass aggregation method and tested for pulsed field gel electrophoresis (PFGE) to analyze the homology by clustering analysis with BioNumerics 6.0 software. Results were as follows:A total of 220 R. anatipestifer isolates were collected in our laboratory during 2007 to 2017, and 119 R. anatipestifer isolates were serotype Ⅰ, accounting for 54.09% of all isolated strains. PFGE genotyping showed that R. anatipestifer serotype Ⅰ isolates were differentiated into thirty-three different PFGE types (with similarity percentage of ≥ 85%), including nineteen clusters and fourteen single types. In conclusion, the predominate serotype of R. anatipestifer isolates in Guangdong province was serotype Ⅰ. Serotype Ⅰ R. anatipestifer isolates showed diverse genotyping band patterns. Low degree of homology was observed among these isolates and there was a clonal spread on a small scale.

In this study, we aimed to screen and identify in vivo-induced antigens of Brucella melitensis via in vivo-induced antigen technology (IVIAT), which may provide a scientific basis for selecting of novel virulence molecules, diagnostic targets, vaccine candidate antigens and drug targets for Brucellosis control. The probe was prepared by absorbing the clinical positive serum collected from Brucella-infected animals onto PVDF membrane. Expression library of 16M gene of B. melitensis was constructed for identifying in vivo-induced antigens. The identified genes were confirmed by RT-PCR and the PCR products were sequenced and analyzed using bioinformatic software. The results showed that 14 of in vivo-induced genes were obtained from expression library of 16M gene of B. melitensis by IVIAT using the probe prepared in this study. Further analysis using bioinformatic software demonstrated that the products of these genes are mainly involved in the biological processes of glucose metabolism, tRNA modification, ion transport and transmembrane transport of B. melitensis. Some of these molecules have been identified as drug targets and virulence molecules in Brucella spp, while some may be related to Brucella virulence and immunity. The IVIAT was successfully used to the screening and identification of in vivo-induced antigens of B. melitesis. The in vivo-induced antigens of B. melitesis identified in this study are new candidate antigens and diagnostic markers for vaccine and diagnostic products of B. melitesis.

Schistosomiasis is a globally important helminthic disease of humans and animals,eggs produced by the mature female worms are the main factor that cause pathological damage and disease transmission. The study on controlling female reproductive development has great significance in controlling schistosomiasis. In this study, the full-length of SjELAV-like 2 gene sequence was attained by 5'-RACE and 3'-RACE. The SjELAV-like 2 coding sequence was successfully cloned and expressed, and the polyclonal antibody was produced. Western blotting analysis showed that the protein has good reactionogenicity. RT-qPCR analysis revealed that the gene had the highest expression in schistosomula and tended to be stable after maturation. The expression level of SjELAV-like 2 in the female was significantly higher than that in the male. Its expression in each periods of the male remained maintenance level. Immunohistochemical results indicated that the protein was mainly located in the tegument of Schistosoma japonicum in adult females. RNAi interference technology was employed to study the biological function of SjELAV-like 2 gene. RT-qPCR results showed that RNAi could effectively reduce the expression of SjELAV-like 2 gene in the parasites. The long-term RNAi also reduced the hatching rates about 75.56% (P<0.01), and reduced the liver egg burden about 39.67% (P<0.01). The scanning electron microscope showsed that there were more vesicular protrusions on the tegumental tissues of siRNA interference group worms compared with the negative control. These observations implied that SjELAV-like 2 has important influence on growth and development of Schistosoma japonicum.

To study the effect of monochromatic light on antioxidative capacity of skeletal muscle in broiler during late-embryonic stage, a total of 225 AA broiler fertilized eggs were randomly divided into 5 groups and were incubated under white light, blue light, green light, red light and dark conditions, respectively. The pectoralis major and gastrocnemius muscles were taken at the 15th, 18th and 21st day of embryonic age (abbreviated as E15, E18 and E21, respectively). The activity of antioxidant enzymes (SOD, GSH-Px and CAT), total antioxidant capacity (T-AOC) and the content of malondialdehyde (MDA) were measured. Results were as follows:① At E18 and E21, the antioxidant enzymes activity and T-AOC levels in the white light group were higher than those in the dark group (0.92%-32.79%), but the MDA content was decreased (1.75%-10.32%); ② Compared with the white light group, especially at E18 and E21, the anti-oxidative enzyme activity and T-AOC levels in the green light group were significantly increased (1.72%-51.29%, P<0.01) and the MDA content was significantly decreased (8.78%-18.26%, P<0.05). The anti-oxidative enzyme activity and T-AOC levels under the blue light were increased (0.84%-39.48%) and the MDA content was decreased (2.35%-11.20%). However, the degrees of increase and decrease under the blue light were lower than that of the green light group. In contrast, the red light reduced the anti-oxidative enzyme activity and T-AOC levels (2.29%-28.17%) and increased the MDA content (0.51%-18.08%). Our results suggeste that the monochromatic green light illumination can enhance the antioxidation function of skeletal muscle in the late-embryonic stage of broiler, but monochromatic red light has inhibitory effect.

The experiment was conducted to study the residues of altrenogest in swine tissues, preparing for the determination of withdrawal time. The liver and kidney samples were hydrolyzed by β-glucosidase/arysulfatase and extracted by acetonitrile (the muscle and fat samples were extracted by acetonitrile), then the extracting solution were degreased with n-hexane. After being redissolved with ethyl acetate, the supernatant was purified by 10% sodium carbonate solution. Finally, altrenogest was analyzed by LC-MS/MS, and was calibrated by the external method. The results showed that the standard curve was obtained with the correlation coefficients (R²=0.999) in the concentration range of 1-200 ng·mL^-1. The limit of quantification for altrenogest was 1.0 μg·kg^-1, and the limit of detection for altrenogest was 0.5 μg·kg^-1. The recoveries of altrenogest were 68.9%-78.9% at three spiked levels of 1, 100, 200 μg·kg^-1 with the relative standard deviations less than 11% (n=6). After the pigs were orally administered altrenogest of 0.4 mg·kg^-1 of body weight onetime every day for 18 d, the concentration of in all of the tissues at 6 h was highest; the concentration of altrenogest in fat at 5 d was lower than MRL, while the concentration of altrenogest in muscle and kidney tissues were lower than LOQ; the concentration of altrenogest in all of the tissues at 10 d were lower than LOQ. The order of residue rate was liver > kidney > fat > muscle, and liver was considered as the target organ. The concentration-time dates were analyzed by WT1.4 to get the withdrawal time with 99% upper tolerance limit, the WT advised was 9 d.

In this study, high-throughput sequencing was used to study the effect of Nisin on intestinal microflora in diarrhea mice. The model of diarrhea mice was established by peritoneal perfusion of E. coli O₁, and the best dose of the drug was screened by animal protection test in vivo. The mice were randomly divided into blank control group, negative control group, ampicillin, ciprofloxacin hydrochloride group and Nisin group. Gavage was continued for 15 days, and cecum stool were collected. Five samples were analyzed by high-throughput sequencing. The results showed that:1) E. coli O₁ can duodenal villi in diarrhea mice. Nisin increased the percentage of lymphocytes, to enhance immunity. 2) The best Nisin dose (0.002 g·mL^-1) was screened by mouse protection rate test, the protection rate was 37.5%, lower than that of ampicillin (50%), ciprofloxacin hydrochloride group. 3) Counting of cecal colonies:the number of E. coli and Enterococci in Nisin was lower than that in the negative control group (P<0.05), especially in Nisin group. The number of Lactobacillus in Nisin group was higher than that in other group (P<0.05). 4)The diversity index (ACE=1 417.25, Chao1=1 378.45, Shannon=7.56) in Nisin group was higher than that of negative control group (ACE=969.54, Chao1=340.29, Shannon=6.63). There are significant differences in the flora structure. 5) The Bacteroidetes were the lowest in the Nisin group, with the highest proportion of Firmicutes, Clostridium, Akkermansia and Lactobacillus being higher than Other groups. Therefore, Nisin enhanced the immune function of diarrhea mice, increased the number of beneficial bacteria in the intestine and reduced the number of harmful bacteria; intake of appropriate amount of Nisin can regulate the proportion of intestinal flora.

To explore the species and population distribution of endophytic fungi from Oxytropis trichophora, Oxytropis trichophora samples in Tongwei county of Gansu province were collected, then were sterilized by using the method of surface disinfection. After that, endophytic fungi were insolated, and then the morphology techniques and ITS sequence analysis were used to identify their species, The phylogenetic relationship of endophytic fungi was analyzed by MEGA 5.05 software. The results showed that 29 species of endophytic fungi were isolated from Oxytropis trichophora belonging to 4 classes, 5 orders, 6 families and 10 genera, the isolation rate of stem was the highest (39.13%), followed with root (23.91%) and leaf was the least (11.11%). Alternaria sp. and Fusarium sp. were the dominant species of endophytic fungi, relative frequency (RF) were 27.59% and 17.24%, respectively. Trichoderma sp. was the most widely distributed in roots, stems and leaves of Oxytropis trichophora. The phylogenetic analysis of endophytic fungi isolated from Oxytropis trichophora showed that they were divided into two groups according to their genetic relationship. The result indicated there was an obviously difference in quantity, species and population distribution of endophytic fungi between different parts of Oxytropis trichophora. Stem was the most vulnerable to infection and colonization by endophytic fungi, and the diversity was the maximum in stem of Oxytropis trichophora.

This study was conducted to explore anti-BVDV (bovine viral diarrhea virus) effect of three kinds of Tibetan medicine. BVDV was proliferated by virus propagation technology on bovine kidney cells (MDBK) and the TCID₅₀ was detected. The maximum safe concentration and the CC₅₀ value were measured by CCK8 detection and CPE observation. Antiviral inhibition tests were performed by three different modes, adding virus before adding drugs, adding drugs before adding virus, and adding drugs and virus pre-acted for a while in vitro. The Effective inhibition rate, the EC₅₀ value and the therapeutic index (TI) were calculated by statistical analysis software, which would further clarify the antiviral effection of the extracts. The results showed that TCID₅₀ was 10^-4.67·0.1 mL^-1 by the Reed-Muench method. The maximum safe concentrations of Terminalia chebula, Corydalis hendersonii, Aconitum tanguticum, ribavirin were 1 mg·mL^-1, 1 mg·mL^-1, 8 mg·mL^-1 and 2 μg·mL^-1 respectively. All of the maximum inhibition rates of the drugs were more than 50% in virus-and-drug mode. The most effective inhibitory rate treated with Corydalis hendersonii was 72.86% at the dose of 1 mg·mL^-1, which was significantly higher than that of ribavirin (P<0.01). In addition, all of the maximum inhibitory rates were less than 30% in "virus before drugs" and "virus after drugs" mode, indicating unobvious adsorption block effect and unobvious replication blocking effect. In summary, all three kinds of Tibetan medicine had direct killing effect on BVDV. Corydalis hendersonii had the most significant direct inactivated effect and that was superior to ribavirin, which would expect to be further developed into antiviral drugs.

This study was conducted to elucidate the regulation of Yujin Powder (YJP) on serum immunoglobulin and antioxidance-related factors of Large Intestine Dampness-heat Syndrome (LIDHS) rat. Sixty Wistar rats were randomly divided into 6 groups with 10 rats in each group:normal control group, LIDHS model group, self-healing group, high, middle and low dose of YJP groups. The rat model of LIDHS was established by such complex factors as high-sugar and high-fat diet, improper diet, drinking, high temperature and humidity environment and intrap-eritoneal injection of Escherichia coli, which imitated the inducing conditions of LIDHS. After the model being successfully established, the rats were treated for 5 days with high, middle and low dose of YJP, respectively. And then levels of serum immunoglobulin IgA, IgG, IgM were detected, the spleen and thymus were collected for histopathological observation, and the lipid peroxide (LPO), malondialdehyde (MDA), reduced glutathione (GSH), and oxide nitrogen (NO) levels in serum and liver tissue homogenate were detected. The results showed that compared with the normal control group, the serum levels of IgA, IgG and IgM in the model group were significantly increased (P<0.01). In comparison with the self-healing group, they were all reversed to different degrees after different doses of YJP administration, and the most obviously in the high-dose group. In high dose group, the content of IgA and IgM were significantly decreased (P<0.05) and IgG was extremely significantly decreased (P<0.01). The pathological observation showed that the splenic and thymic morphological structure of model group rat disorder, spleen congestion, and severe thymus atrophy. In the high-dose group of YJP, the spleen structure tends to be normal, and the degree of thymus atrophy was reduced. Compared with the normal control group, the content of MDA and LPO in the serum and liver tissues in the model group were extremely significantly increased (P<0.01), and the content of GSH and NO were extremely significantly decreased (P<0.01). Compared with the self-healing group, they were all reversed to different degrees after treatment with different doses of YJP administration, and the most obviously in the high-dose group. In high dose group, the content of MDA and LPO were significantly or extremely significantly decreased (P<0.05 or P<0.01), and the GSH and NO content were significantly or extremely significantly increased (P<0.05 or P<0.01). There were abnormal immune function and disturbance of oxidation and antioxidant balance in LIDHS model rats. YJP can treat LIDHS through reducing the serum immunoglobulin levels, improving the structure of spleen and thymus and regulating the balance of oxidation and antioxidation. And the effect of high dose was the best.

The purpose of this study was to investigate the effect of glucose on cryopreservation and metabolism of cashmere goats semen. In this study, the mixed semen samples were diluted in extender containing different concentrations of glucose (0, 28, 56, 84 and 112 mmol·L^-1) and frozen-thawed. After thawing, sperm motility, the percentage of sperm with intact DNA, intact acrosome and intact plasm membrane, level of ROS and Ca²⁺ in sperm and concentration of ATP in sperm, and concentrations of lactic acid and pyruvic acid in sperm suspension were assessed by using computer-assisted sperm analysis system (CASAS), flow cytometry and microplate reader, respectively. The results showed that the semen extender supplemented with 56 mmol·L^-1 glucose could increase sperm motility, the percentage of intact acrosome and plasm membrane in frozen-thaw cashmere goats semen (P<0.05), however no significant differences were observed in the percentages of intact DNA among the groups (P>0.05). The group of 56 mmol·L^-1 glucose could extremely significantly increase the level of ROS (P<0.01),while the groups of 28 and 56 mmol·L^-1 glucose could extremely significantly decrease the level of Ca²⁺(P<0.01); the group of 56 and 84 mmol·L^-1 glucose extremely significantly increase the ATP concentration, in comparison to the other concentration groups (P<0.01). After thawing, lactic acid was not detected in the low concentration glucose groups (28-56 mmol·L^-1), but a large amount of lactic acid was detected in the 112 mmol·L^-1glucose group(P<0.01). Glucose had a dose-dependent effect on pyruvic acid production in sperm suspension when semen extender supplemented with low concentration glucose(28-84 mmol·L^-1), but high concentration glucose (112 mmol·L^-1) significantly decreased pyruvic acid production(P<0.05). In summary, the semen extender supplemented with 56 mmol·L^-1 glucose could promote metabolism of cashmere goat sperm during freeze-thawing and improve cryopreservation effect.