CRISPR/Cas (Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated(Cas)) system is a DNA modified gene editing tools regulated by a kind of short RNA.This technology is a novel tool，which is more rapid，efficient and accurate than ZFN (zinc-fingers nuclease) and TALEN (transcription activator-like (TAL) effector nucleases) in genetic engineering.It is easy to be designed and has specificity.This paper reviews the structure and function of CRISPR/Cas9 system and the design strategy of Cas9 and its application in animal genetic engineering.CRISPR/Cas9 is successfully used in a variety of animal genetic editing，it is likely to become a new way to establish animal models and to develop a new feasible approach to prevent disease.

Body temperature and the amount of activities are important indicators evaluating health state and physiological status of livestock，and they are significant to promote the efficiency of dairy farming.Since that reason many studies were focusing on how to detect the body temperature and the amount of activities of cow，and the detecting methods of mechanization，automation and intelligence were gradually developed from traditionally manual operation，and the sensitivity and accuracy of detecting method and the availability of the collected data are improving.The degree of coverage of the data is becoming more and more comprehensive.So the regularities of body temperature and the amount of activities for illness and reproduction were being discovered constantly.However，only the activity information can be automatically collected and applied in large-scale.Although the automatically collecting techniques of reticulo-rumen boluses and vaginal implant for the body temperature have been developed，they can not be applied widely.The automatically collecting method for body temperature is still being explored.This paper reviewed the collection methods of body temperature and the amount of activities，and their regularities in illness，oestrus，pregnancy and calving were analyzed.The prospect of development and application for the automatically collecting technology of body temperature and the amount of activities of dairy cows were discussed，which will lead to a revolution of dairy industry in the future.

To investigate new application of CRISPR/Cas9 technology，we established a method to study genes function in living organism by the application of CRISPR/Cas9 in genetic manipulation.By using in vivo electroporation，immunohistochemistry and PCR analyzing，we first validated the function of DCX and the results indicated that CRISPR/Cas9 could be used in down-regulated gene expression in mouse genetic manipulation.Then this method was also used to study the function of constant exons of Pcdhα cluster. To further clarify the mechanism that led to the phenotype of neuron migration defects by DCX，we designed experiments to identify the genotype of target genes.The results indicated that CRISPR/Cas9 could lead to deletion，inversion and duplication of target genes.In conclusion，CRISPR/Cas9 technology could be used in mouse genetic manipulation and edit the target genes efficiently，which might be an ideal method to research gene function in living organism.

To explore the theoretical basis of the microRNAs in regulating the reproductive process in Small Tail Han sheep，the ovarian microRNAs profiles were constructed and analyzed in estrus and diestrus.One ovary was collected with surgery method at estrus (the 1st day) and diestrus (the 13th day) from the same sheep，respectively.The RNA sequencing data were obtained with illumina Hiseq2000 sequencing platform.The expression profiles and differential expression of microRNAs were analyzed by bioinformatics method.The real-time quantitative PCR (qRT-PCR) was used for validating the differentially expressed microRNAs between the 2 periods in ovary.The microRNAs expression profiles of Small Tailed Han sheep was constructed in the period of estrus and diestrus in ovaries.The highest expression of microRNA was observed for oar-miR-99a and oar-miR-143 in the period of diestrus and estrus，respectively.Three microRNAs，oar-miR-200a，oar-miR-200b and oar-miR-200c，were significantly differentially expressed between estrus and diestrus.The expression level of 2 randomly selected microRNAs by qRT-PCR was the same as the result by RNA-seq data.These microRNAs expression profiles will be used to provide informations for further studying the microRNAs in sheep ovary.The differentially expressed microRNAs may regulate the ovarian periodic activity through the metabolism and immunity pathway by the analysis of predicted target genes and enriched pathways.

The aim of this study was to determine the mRNA expression of genes related to lipid metabolism in muscle tissue of different goat breeds，and to analyze their association with intramuscular fat (IMF) content，which was predicted to provide basic data for revealing the mechanism underlying IMF formation.Longissimus dorsi，biceps femoris muscle and triceps brachii muscle from 6 healthy castrated Jianzhou Big-eared goats and Chengdu Brown goats after slaughter，respectively，were collected for detecting IMF content，and the real-time PCR was used to detect the mRNA expression of genes related to lipid metabolism，and their association was analyzed.The result showed that，except for PPARG，the other lipid metabolism related genes(including SREBP1c， THRSP， INSIG1，ACACA，SCD1，DGAT1 and DGAT2) were expressed higher in Jianzhou Big-eared goat than in Chengdu Brown goat.Among them，the expression of THRSP，SCD1 and DGAT2 were significantly higher in Jianzhou Big-eared goat than in Chengdu Brown goat in different tissues (P<0.05).Except for ACACA，the expression of other lipid metabolism related genes were the highest in longissimus dorsi of Jianzhou Big-eared goat，with no significant difference between biceps femoris muscle and triceps brachii muscle (P>0.05) (except for PPARG).While the mRNA expression of THRSP and SCD1 were the highest in longissimus dorsi (P<0.05)，the lipid metabolism related genes had no significant differences between biceps femoris muscle and triceps brachii muscle in Chengdu Brown goat (P>0.05).Jianzhou Big-eared goat had higher IMF content than Chengdu Brown goat in all the 3 tissues (P<0.05)，longissimus dorsi had higher IMF content compared with the other tissues.THRSP，DGAT2 and SCD1 showed positive association with IMF in various tissues of Jianzhou Big-eared goat and Chengdu Brown goat.These results indicate that THRSP，SCD1 and DGAT2 may play important role in promoting fat deposition in meat goat，and also provide basic data for revealing the mechanism underlying goat IMF formation.

The study aimed to investigate the genetic variation of thyroid stimulating hormone receptor (TSHR) gene in Ujumqin sheep and Hu sheep，which had different reproductive performance and oestrous cycle，and the distribution of different haplotypes caused by the polymorphic site and its effects on mRNA and secondary structure of protein.This study provided a theoretical basis for seeking the influence of the mutation on TSHR gene expression and phenotype.Direct sequencing was taken to study genetic variation of the TSHR gene in 172 sheep of 2 Chinese Mongolian sheep strains.6 mutant sites were found and genotyped.Chi-square test for independence was performed to find the genotypes of the 4 SNPs (SNP1，SNP3，SNP4 and SNP5) which were significantly different between the 2 sheep populations（P＜0.01）.The SNP5 was missense mutation，the other sites were synonymous mutations.Linkage disequilibrium analysis was used to screen the 4 sites，complete linkage happened between SNP3 and SNP4，and the other sites present different degrees of interlocking relationship.Bioinformatics analysis revealed that not only the mutations occured in the TSHR，but also different haplotypes could result in the changes of mRNA and the secondary structure of protein.These results indicate that：(1) The genotypes distribution of 4 sites in TSHR gene have significant differences between the 2 sheep populations.They form 6 haplotypes，among which H1(CGCC) is the advantage haplotype in Ujumqin sheep，and H4(TTTG) is the advantage haplotype in Hu sheep.(2) The SNP5 site affects the stability of mRNA and protein secondary structure of TSHR gene，which may be one of the important functional sites with potential phenotypic effects.

The aims of this study were to evaluate the expression profiles of Protamine 1（PRM1） mRNA in reproductive organs of bucks and to analyze the correlation between expression of PRM1 mRNA in spermatozoa and semen quality index.In this experiment，mRNA expression was detected by real-time fluorescent quantitative PCR.Cellular localization of PRM1 in testis was examined by immunohistochemistry.The correlation was analyzed by GraphPad Prism 5 software.The results showed that：the expression of PRM1 mRNA in testis was the highest in all tissue detected，which was 247 times higher than that in the epididymal caput（P＜0.05）.The expression of PRM1 mRNA in epididymal caput was significantly higher than that in epididymal corpus and cauda（P<0.05）.The expression of PRM1 mRNA in other tissues was very few.The expression of PRM1 mRNA in testis was gradually increased with the increase of ages in one year old.The PRM1 mRNA expression in testis at 12 months old were significantly higher than that at 9 months old（P＜0.05）.The PRM1 mRNA expression in testis at 9 months old was significantly higher than that in other ages.Concentration of PRM1 mRNA in sperm was positively correlated with the sperm motility （R2=0.586 0，P=0.000 5） and sperm concentration （R2=0.442 2，P=0.004 9）.PRM1 was located in elongating spermatids and spermatozoa in testis of bucks.There was no positive signal in other cells of testis.This study indicated that the expression of PRM1 mRNA was spatial and temporal-specific pattern.The sperm PRM1 mRNA content could be index of fertility of bucks.

To elucidate the effects of exogenous glutathione (GSH) on embryos transcriptome，bovine IVF embryos at 8-16-cell and morula stage were collected for high-throughput RNA sequencing (RNA-seq).Quantitative determination of genes expression was used to verify the RNA-seq data.Gene ontology and KEGG pathway enrichment analysis of differential expression genes were performed.The accuracy and validity of RNA-seq data were confirmed with combination of error rate of base，mapping of reads to bovine genome，distribution region analysis and consistency between qPCR and RNA-seq results of ten genes.A total of 4 100 genes were differentially expressed between embryos at two stages and 3 952 genes were identified as expressed in embryos treated with GSH.A high enrichment of genes involved in 84 GO terms，such as ATP synthesis，respiratory chain，DNA synthesis and translation.Pathway analysis revealed 27 pathways involved in cell adhesion，TCA cycle，GSH metabolism and amino acid metabolism.Additionally，2 known conserved domains (S-TKc，MH1) and 2 new domains (LCR，DWB) were found in 5 new transcripts by prediction，compared with known transcripts.To sum up，GSH treatment can result into the comprehensive change at transcriptional level of bovine IVF embryos and improve their development consequently.

To determine the key candidate genes related to semen quality in different seasons，the mRNA profiling of Chinese Betelnut-jiang buffalo were analyzed by RNA-seq.Based on similarities in growth status，5 Betelnut-jiang buffalo bulls were selected and their semen quality were tested (including ejaculate volume，fresh sperm motility，sperm density and abnormality rate).According to the semen quality，the samples of summer (treated as SBL group) and spring (as BL group) were selected for RNA-seq.We collected the whole blood in jugular vein of Betelnut-jiang buffalo bulls，extracted the mRNAs and mixed them within group.Then，the mRNA profiling was used to measure the mRNAs in Betelnut-jiang buffalo bulls.Gene expression levels with >1.5 fold change and P value < 0.05 between the BL and SBL groups were further analyzed.Furthermore，GO and KEGG were used to analyze the genes enrichment.Finally，RT-PCR was used to confirm the RNA-seq result.The results indicated 84 differentially expressed genes.Among these，56 genes were up-regulated and 28 genes were down-regulated in SBL group.The GO search revealed that these differential expression genes were mainly related to stress reaction and immune response.And the KEGG selected the TNF signaling pathway and T cell receptor signaling pathway.Neurotrophin receptor alike death domain（NRADD，also named heat shock factor protein 1(HSF1)），localisation of FOS (FOS) and jun proto-oncogene (JUN)，were strongly associated with semen quality in different seasons which could be used as target genes for further study.

To investigate the effect of miR-324-3p on regulating melanin production by targeting MC1R，miR-324-3p was over-expressed by liposome transfection method in alpaca melanocytes in vitro.The expression of MC1R and hair color genes (Mitf，Tyr and Tyrp2) were detected by quantitative real time PCR and Western blotting and the melanin prodution was analyzed by Microplate Reader.The results showed that miR-324-3p was expressed extremely significant difference in the alpaca skins with 1.64 folds in brown alpaca skins vs white alpaca skins（P<0.01），the gene expression of MC1R，Mitf，Tyr and Tyrp2 were down-regulated and the melanin production was reduced（especially Mitf (P<0.01)）.The results suggested that miR-324-3p regulated melanogenesis by targeting MC1R gene to down-regulate the expression of the downstream genes including Mitf，Tyr and Tyrp2 in the skin of alpaca.

In order to develop a high sensitive method of detecting goose prolactin，luciferase detecting system based on PRLR-JAK-STAT5 signal transduction pathway was designed in the present study.Firstly the CDS region of goose PRLR gene was cloned，the signal transducer and activator of transcription 5（STAT5) was artificially synthesized and inserted into the eukaryotic expression vector pCMV6-Entry and luciferase reporter pGL3-Enhancer plasmid as signal receiver vector and signal responder vector respectively.Secondly，the two recombinant vectors along with puromycin screening vector pEZX-MR03 and internal control pRL-TK vector were transfected into HEK293T cells.After puromycin screening，stable transgenic cell lines were isolated and stimulated by a serial of different concentrations of chicken PRL (0，30，60，90 ng•mL-1).Finally the mRNA relative expression level and enzyme activity of luciferase enzyme were detected by qRT-PCR and dual-luciferase detection system.Results showed that 10 stable transgene cell lines carried the 4 vectors were isolated.After treated with different concentrations of chicken PRL，luciferase gene mRNA expression level was up regulated and luciferase enzyme activity also enhanced in dose dependent manner in one of these 10 cell lines.These results demonstrated that it was feasible of detecting PRL bioactivity using the constructed PRLR-JAK-STAT5 signal transduction system and could be further used in measuring poultry PRL concentrations.

The study was conducted to investigate the effect of dietary lysine (Lys) levels on growth performance and organ development of Jinghong laying hens，and to estimate the optimal dietary lysine requirement of Jinghong laying hens aged from hatch to the age of 4 weeks.A total of 300 1-day-old Jinghong laying hens were randomly allotted to 5 groups with 5 replicates per group and 12 hens per replicates.Lys was added to the basal diet to obtain the Lys level at 1.00%，1.10%，1.20%，1.30% and 1.40%，respectively.The trial lasted 4 weeks.The results showed as follows：The average daily gain in the 1.20% Lys group was significantly higher than that in 1.00%，1.30% and 1.40% Lys groups（P<0.05）；The body weight at 4 weeks in the 1.20% Lys group was significantly higher than that in 1.00%，1.30% and 1.40% Lys groups (P<0.05).The pancreas weight in the 1.20% Lys group was significantly higher than that in the other groups (P<0.05).The jejunum index in the 1.20% Lys group was significantly lower than that in the 1.00% Lys group，and the jejunum relative length in the 1.20% Lys group was significantly higher than that in the other groups (P<0.05).The ileum index in 1.20% and 1.30% Lys groups were significantly lower than that in 1.00%，1.10% and 1.40% Lys groups (P<0.05)，while the ileum relative length in 1.2% Lys group was significantly higher than that in 1.00%，1.10% and 1.40% Lys groups (P<0.05).The body weight，carcass weight and eviscerated carcass weight in the 1.20% Lys group at the age of 4 weeks were significantly higher than that in the other groups(P<0.05)。Birds fed 1.20% Lys had significantly higher heart weight，lung weight than that in the other Lys groups.The kidney weight in the 1.20% Lys group was significantly higher than that in 1.00%，1.30% and 1.40% Lys groups (P<0.05).Liver weight in 1.10%，1.20% and 1.30% Lys groups were significantly higher than that in the other groups (P<0.05)，and the shinbone length were significantly higher than that in 1.00% Lys group (P<0.05).Birds fed 1.10% and 1.20% Lys had significantly higher chest width than that in the other Lys groups (P<0.05).The spleen index（0.290%） and bursa of fabricius index（0.543%） in the 1.30% Lys group were significantly higher than that in the 1.00% Lys group(P<0.05).Hens fed 1.10% and 1.20% Lys had significantly lower serum uric acid content than that in 1.00% and 1.40% Lys groups(P<0.05).According to the quadratic regression analysis based on community evenness，body weight，average daily gain，carcass weight，heart weight，liver weight，ileum index，ileum relative length and uric acid content，the optimal dietary levels of Lys for Jinghong laying hens were 1.145%，1.158%，1.155%，1.154%，1.151%，1.155%，1.189%，1.204% and 1.142%，respectively.The optimal averaged Lys levels was 1.161%.These results indicate that addition of appropriate Lys level in diet can promote growth performance and organ development.The optimal Lys level in diet of Jinghong laying hens from hatch to the age of 4 weeks is 1.16%.

This study aimed to investigate the feasibility and approximate proportion of estimating metabolizable energy value of single grain using substitutional method.Fifty-four castrated Dorper×Thin-tailed Han crossbred rams (body weight，(48.3±1.3) kg) were randomly divided into 9 groups and fed one of the following 9 experimental diets：basal diet and 8 experimental diets with different substitutional ratio of wheat.Digestibility and respirometry trials were conducted to measure digestible and metabolizable energy (DE and ME) and to determine the appropriate substitutional ratio of wheat for daily ration.The results showed as following：1)With the increasing of substitutional ratio of wheat，the nutrient digestibility，urine energy，DE and ME were improved，whereas the fecal energy was decreased.2) The digestibility of gross energy，dry matter，organic matter and crude protein in wheat that was calculated by the substitutional method were not affected by treatments (P>0.05).3) When the substitutional ratio of wheat was in the range of 28.37%-45.95%，the DE and ME of wheat were 14.55 MJ•kg-1 and 11.86 MJ•kg-1.They were respectively similar to 14.52 MJ•kg-1 recommended by NRC (2007) and 11.91 MJ•kg-1 calculated from experimential equation (DE×0.82).In conclusion，the appropriate substitutional ratio of single concentrate in mutton sheep diet is in range of 28.37%-45.95% and the optimal substitutional ratio of wheat for daily ration is 28.37% in the current study.

This experiment was conducted to investigate the effects of caponization and ovariectomy on comb development，slaughter performance and fat metabolism in male and female chickens at different weeks，and to provide the reference for industry development of castrated chicken and research on lipid metabolism.Eighty Beijing-you female chickens and eighty Beijing-you male chickens with similar weight were selected at 3 weeks，and divided into control group and gonadectomy group randomly with sham-operated as controls，caponization and ovariectomy as treatments，totally for 4 groups.Comb development，slaughter performance and fat metabolism indexes were examined after slaughter at 13，17 and 22 weeks.After blood and liver samples collected，automatic biochemical analyzer was used to determine triglyceride content in serum，whilst soxhlet extraction methods were used to determine total fat content in liver.Compared with controls，caponization significantly decreased comb length，comb height and comb weight at 13，17 and 22 weeks of male chickens (P<0.05)，meanwhile ovariectomy significantly increased comb length，comb height and comb weight at 17 and 22 weeks of female chickens (P<0.05).Caponization and ovariectomy had no significant effect on live weight，carcass weight，dressing percentage，percentage of breast muscle and thigh muscle at 13 weeks of chickens (P>0.05).Additionally，caponization had no significant effect on live weight at 17 and 22 weeks of male chickens (P>0.05)，but capons had lower percentage of thigh muscle at 17 and 22 weeks and exhibited higher percentage of breast muscle at 17 weeks and higher percentage of abdominal fat at 13，17 and 22 weeks than those in control (P<0.05).Ovariectomy significantly increased live weight，carcass weight and dressing percentage at 17 weeks and percentage of abdominal fat at 17 and 22 weeks than those in control (P<0.05).The triglyceride content in serum and total fat content in liver of capons were significantly higher than those in control at 13，17 and 22 weeks (P<0.05).Ovariectomized group exhibited significantly higher content of triglyceride in serum at 17 weeks and total fat content in liver at 17 and 22 weeks of female chickens than those in control (P<0.05).The results indicate that caponization significantly inhibited comb developing of male chickens and ovariectomy significantly promoted comb development.Caponization had no influence on live weight at different weeks but significantly reduced percentage of thigh muscle，and increased percentage of abdominal fat.Ovariectomy increased live weight and percentage of abdominal fat to some extent.Finally，gonadectomy might promote abdominal fat deposition of chickens by increasing the content of serum TG and total fat in liver.

To investigate the effects of cecal fermentation products on donkey milk synthesis，4 donkeys installed permanently cecal fistula were selected (body weight (182±9) kg) at late lactation stage ((155±15) d).Through continuous infusion method via cecum，using 4×4 Latin square design，3 different proportions and combinations of short chain fatty acids (SCFA) were continuous infused for 3 h：AP group (acetic acid of 150 mmol•h-1 + propionic acid of 50 mmol•h-1)，AB group (acetic acid of 170 mmol•h-1 + butyric acid of 30 mmol•h-1)，APB group (acetic acid of 130 mmol•h-1 + propionic acid of 50 mmol•h-1 + butyric acid of 20 mmol•h-1)，and the control group was perfused equivalent volume of buffer solution，to study the effects of different compositions and proportions of SCFA on donkey milk production and milk fatty acids.The results showed that there was no significant difference (P>0.10) in milk yield among different treatments.Milk fat，non-fat solid，lactose and protein contents were significantly higher in AB group than in APB group (P<0.10)；In AP and AB groups，milk protein contents were significantly higher than that in the APB group (P<0.10)，the other indices of milk constituents showed no significant difference among different groups (P>0.10).Compared to control group，percent of middle-chain fatty acids in AP and APB groups were significantly increased (P<0.05)，while percent of the long-chain fatty acids in AP，AB and APB groups were lower than that in control group(P <0.05).It was concluded that infusion of acetic and butyric acids through cecum helped to improve contents of milk constituents，and infusion of acetic and propionic acids increased percent of middle-chain fatty acids of milk fat，SCFAs from cecum directly affected donkey milk synthesis.

The aim of this study was to explore the role of out membrane protein P2 (OmpP2) in Haemophilus parasuis serovar 11 reference H465 strain to induce pro-inflammatory cytokine mRNA transcription，and NF-κB and MAPKs signaling pathways in porcine alveolar macrophages (PAMs).The OmpP2 was extracted from the H.parasuis H465 and stimulated in PAMs with 5 and 10 μg•mL－1 for 6 and 12 hours.The total RNA and cells protein were extracted and the RNA was reversed into cDNA.The mRNA transcription levels of IL-1α，IL-β，IL-6，IL-8 and TNF-α were detected by Real-time PCR，and the protein of NF-κB P65，IκBα，ERK，JNK and P38 were detected by Western blot.The result showed that the mRNA transcription of IL-1α，IL-β，IL-6，IL-8 and TNF-α in PAMs induced by the OmpP2 from H.parasuis were significantly up-regulated (P<0.05)，and the protein of JNK，P65 in PAMs induced by the OmpP2 from H.parasuisis significantly increased and the protein of IκBα was significantly declined(P<0.05).The above results exhibit that the OmpP2 could effectively activate the JNK and P65 proteins and increase the degradation of IκBα in NF-κB and MAPKs signaling pathways to promote the pro-inflammatory cytokines expression.

The objective of our research was to evaluate the growth performance and pathogenicity of piglets concurrent infection with porcine encepholomyocarditis virus (EMCV) under immunosuppression condition caused by porcine circovirus type 2 (PCV-2).Sixteen piglets were chosen for three challenge groups including EMCV，PCV-2 and concurrent infection with PCV-2 and EMCV，and control group.The effect of concurrent infection on piglets was conducted by the relevant indexes including clinical symptoms，pathological examinations，growth performances，immune index，detoxification and so on.The results indicated that the groups infection only with EMCV or PCV-2 showed their own classical clinical characteristic and minimal injury.However，the mortality rate of concurrent infection increased rapidly，the average daily weight gain decreased significantly，degeneration and necrosis in myocardium and brain tissues was more severe，the lymphocyte ratio in the blood still remained low and the neutraliz-ation antibody of EMCV maintained at a low level in a long time.The piglets shed virus lasted 12 days after infection，longer than the group infected only with EMCV for 9 days.The results showed that PCV-2 early infection can promote more severe pathogenicity and lethality，and lower immunity in piglets induced by EMCV，and the ability of virus replication and detoxification in piglets enhanced.

To discuss the sequencing and analysis of avian influenza virus (AIV) genome by high-throughput sequencing，genomes of the 20 AIV strains isolated from eastern China were sequenced by Ion Torrent PGM.The advantage and disadvantage of the method were evaluated.The characteristic of genome and evolution were analyzed.The molecular evolution relationships of the other 6 internal genes were also analyzed.The results showed that the whole genomes of all the 20 strains were completely sequenced by Ion Torrent PGM.All the 20 viruses are restricted to h9.4.2.5 clade of H9 subtype low pathogenic strains.NA genes of all the viruses are N2 subtype.High-throughput sequencing can be used in the genome analysis of AIV strains.

The aim of the present study was to find out whether ARV activates the phosphatidylinositol 3-Kinase-dependent Akt (PI3K/Akt) pathway according to the PXXP or YXXXM motif of σA and σNS protein.Gene splicing by overlap extension PCR was used to change the PXXP or YXXXM motif of σA and σNS gene.Recombined plasmids that contain mutant σA and σNS genes were generated.The Akt phosphorylation profile of transfected cells were examined by flow cytometry and Western blot.The results showed that σA and σNS genes were expressed in the Vero cells，and the expression of P-Akt of the σA mutant groups (Amino acid 110-114 and 114-117) decreased markedly.The results indicated that the σA protein of ARV activate the PI3K/Akt pathway by the PXXP motif.The results of this study reveal the mechanisms by which ARV manipulate the cellular signal transduction pathways，which may provide new ideas for novel drug targets.

 To detect the contamination of Chicken infectious anemia virus (CIAV) in attenuated vaccines for poultry，primers and TaqMan probes were designed and synthesized based on the published CIAV genome sequence，and a real-time quantitative PCR method for detecting the CIAV was established by optimizing the reaction conditions.Results analysis showed that the sensitivity of the qPCR may increase detection to 10 copies•μL－1，and the method was approximately 100-fold higher than the sensitivity of the conventional PCR.This detection method had good specificity，and showed no cross-reactions with ALV，REV and MDV genes，the CV (coefficient of variation) of intra-assay and inter-assay were less than 2%，and also showed that the method had the characteristics of better repeatability.In our experiment，a low dosage of 1 EID50•1 000－1 plumes and in 2 positive (5.1%) of 39 samples were detected for CIAV detection from commercial poultry vaccines by qPCR method.In this study，suggesting that this method provides a high sensitivity，strong specificity and better repeatability system for detection of a low dosage CIAV contamination in attenuated vaccines.

 Rotavirus (RV) VP6 gene is the main target gene of molecular detection，but the gene point mutations affects the molecular detection of RV.The purpose of this experiment was to establish a detection method of yak RV by RT-PCR method which applied in clinical samples based on the study of genetic variation yak RV VP6.The fragments of yak RV VP6 ranged in 931-1 338 bp were obtained with the primers designed by Prime 5.0 software from 30 samples of yak diarrhea.Sequence analysis showed that，yak RV VP6 genes represented multiple point mutations in the section of 931-1110 bp compared with bovine rotavirus (BRV)，while the section between 1 100-1 338bp was highly conserved.According to this result，the assay has successfully established a method with good specificity and stability.The primer could amplify the specific fragment of yak RV and BRV，with no amplification of other unrelated pathogens.The detection limit of viral nucleic acid of the assays was 0.45 pg•μL-1.With a remarkable detection rate in yak RV，the RT-PCR method provided a useful tool for the diagnosis and epidemiological investigation of yak RV disease.Additionally，this method can also be used in BRV detection.The RV detection rate in 234 samples of yak diarrhea from different provinces were 60.00% in Tibet (36/60)，95.00% in Qinghai (57/60)，85.19% in Sichuan(46/54) and 90.00% in Yunnan (54/60)，respectively.The result shows that RV infection is an important cause of yak diarrhea.

The aim of the present study was to compare the stereology characteristics of mitochondria and electron dense-cored vesicles (EDCVs) of carotid body(CB) type Ⅰ cells between yaks and Qaidam yellow cattle.In this study，the ultrastructures of CB in health adult yaks (n=5) and Qaidam yellow cattle (n=5) at altitude of 3 200 m were observed by electron transmission microscopy technique，and the volume density (Vν)，surface density (Sν)，numerical density on area (NA) and specific surface (δ) of mitochondria and EDCV of CB type Ⅰ cells between yaks and Qaidam yellow cattle were compared by stereology.The results showed that the CB parenchyma cells were composed of type Ⅰ cells with many mitochondria and EDCVs in the cytoplasm and type Ⅱ cells in yaks and Qaidam yellow cattle，The Vν of mitochondria of CB type Ⅰ cells in yaks were greater than that of in Qaidam yellow cattle (P>0.05)，the Sν，NA，δ were less than that of in Qaidam yellow cattles，with no significant difference in Sν and δ between yaks and Qaidam yellow cattle (P>0.05) and significant difference in NA between 2 gropes（P<0.05）；The Vν of EDCV of CB type Ⅰ cells in yaks were less than that of in Qaidam yellow cattle (P>0.05)，and the Sν，NA，δ were slightly greater than that of in Qaidam yellow cattle (P>0.05)，respectively.The results suggest that the structure characteristics may make the yak was more insensive to perceive hypoxia environment than the Qaidam yellow cattle.

 The objective of the present study was to examine the expression and location of chick melanoma differentiation associated gene 5 (chMDA5) in chick immune organs.Fifty health Highland white chicks were euthanized to collect bursa of Fabricius，thymus and spleen at 1，7，14，21，28，31，35，42，45，49-day-old，separately.Then，real-time fluorescence quantitative PCR，Western blot and IFA techniques were used to detect the expression of chMDA5 in chick immune organs.The results showed that the chMDA5 mRNA were examined in bursa of Fabricius，thymus and spleen at each age，chMDA5+ cells were detected in the cortex of bursal lymphoid follicles，the cortex and medulla of thymus lobule，the white pulp and red pulp of spleen.The expression pattern of chMDA5 protein in bursa of Fabricius and thymus tissue was consistent，which showed an upward trend in 1 to 7-day-old chicks and reached peak at 7-day-old，and then went down in 14 to 35-day-old with a fluctuation trend，then gradually increase and stabilized.In spleen，the protein expression of chMDA5 was increased with the increasing age in a period of about 14 days.The results indicated that the chMDA5 were expressed in chick immune organs，and there were differences in expression of chMDA5 with different ages.This study provide a scientific test basis for further exploration about the correlation between chMDA5 and chick immune function.

This study was designed to compare the pharmacokinetic parameters of enrofloxacin following intravenous and intrauterine administrations to investigate their bioavailability in sows.16 cross-bred (Landrace×Yorkshire) healthy，adult，non-lactating sows，were used.The sows were randomly divided into two groups with eight animals each.The sows of first group were administered with enrofloxacin injection by intravenous route，at a single dose of 2.5 mg•kg^-1；whereas those of second group were administered with enrofloxacin uterine perfusate by intrauterine administration at a single dose of 50 mL per sow.Enrofloxacin concentrations were determined by reverse phase-high performance liquid chromatography (RP-HPLC) with fluorescence detection.A non-compartmental analysis of drug plasma concentration vs.time profiles was performed with pharmacokinetic modeling using Winnonlin software.Following intravenous administration，the main pharmacokinetic parameters were as follows：t _1/2（21.71±1.66）h，Vd（1.89±0.33）L•kg^-1， Cl（60.18±8.92）mL•h^-1•kg^-1，AUC_0-t（39.94±5.75）mg•h•L^-1，AUC_0-∞（42.32±6.03）mg•h•L^-1.The absorption of enrofloxacin was rapid and well absorbed after intrauterine administration，with t _1/2 of （28.55±2.31）h，T_max of （3.19±1.46）h，C_max of （1.61±0.20）mg•L^-1，AUC _0-t of （49.06±5.18）mg•h•L^-1，AUC _0-∞（53.55±5.84）mg•h•L^-1.The absolute bioavailability (F) was 63.15%.Concentrations of the active metabolite ciprofloxacin were not detected in any samples.The results showed that the sows can absorb enrofloxacin well by way of intrauterine administration，with slow elimination and high bioavailability.And the effect on body after intrauterine administration should be considered.

This study aimed to investigate the effect of Staphylococcus aureus (S.aureus) on TGF-β1/Smad signaling pathway and transdifferentiation in bovine mammary fibroblasts (BMFB).BMFB were stimulated with heat-killed S.aureus (HKSA) at 10 ⁴，10 ⁵，10 ⁶，10 ⁷，10 ⁸ CFU•mL^-1.The transcription of TGF-β1，α-SMA and collagen-Ⅰ mRNA were detected by Real-time PCR after 24 h.The expression of α-SMA，collagen-Ⅰ and p-Smad2/3 protein were detected by Western blot.BMFB were pretreated with TGF-β1 receptor-specific inhibitor SB-431542，BMFB were stimulated with heat-killed S.aureus (HKSA) at 105 CFU•mL^-1.The transcription of α-SMA and collagen-Ⅰ mRNA were detected by Real-time PCR after 24 h.The expression of α-SMA，collagen-Ⅰ and p-Smad2/3 protein were detected by Western blot.Immunofluorescence was used to detect collagen-Ⅰ expression.The results showed that the transcription level of TGF-β1 mRNA，expression of α-SMA，collagen-Ⅰ and p-Smad2/3 increased significantly at different concentration after 24 h stimulation compared with unstimulated BMFB (P<0.05).The expression of TGF-β1，α-SMA，collagen-Ⅰ and p-Smad2/3 reached the maximal level at 105 CFU•mL^-1.The expression of α-SMA，collagen-Ⅰ and p-Smad2/3 were significantly reduced by pretreating BMFB with SB-431542 (P<0.01).The results indicate that TGF-β1/Smad signaling pathway plays an important role in S.aureus induced BMFB transdifferentiation.

This study aimed to study the effect of Aspergillus niger-fermented Atractylodes macrocephala Koidz on the immune function of mice.An immunosuppression mice model (cyclophosphamide) was utilize to study the effect of fermented Atractylodes macrocephala Koidz on spleen index，thymus index and T lymphocyte proliferation.High performance liquid chromatography (HPLC) was used to analyze the changes in the active omponents of Atractylodes macrocephala Koidz with or without fermentation by Aspergillus niger.Orthogonal experiment was conducted to select the best conditions.The results showed that：1)The low，middle and high dose groups (0.5，1，2 g•kg^-1) of fermented Atractylodes macrocephala Koidz can effectively increase thymus index (P<0.05).In addition，middle dosage group of fermented Atractylodes macrocephala Koidz can effectively increase spleen index and T lymphocyte proliferation (P<0.05)；2) The results of HPLC showed that the content of atractylenolide Ⅰ was effectively increased in the Atractylodes macrocephala Koidz processed by Aspergillus niger compared with unfermented Atractylodes macrocephala Koidz；3) The best fermentation conditions were as follows：inoculum concentration was 8%；water content was 40%；fermentation time was 96 h，under the optimized conditions，the content of atractylenolide Ⅰ was 1.4 times of those in raw Atractylodes macrocephala Koidz.These results indicated that fermented Atractylodes macrocephala Koidz could improve immune function of mice by increasing the content of atractylenolide Ⅰ.

The aim of this study was to explore the sequence and the immunogenicity of LSm14A gene of the Frizzle chicken.Total cellular RNA was isolated from the livers of frizzle chickens hepatic tissue，and full length CDS region and prokaryotic expression fragment of LSm14A gene were amplified by reverse transcription polymerase chain reaction RT-PCR.The recombinant plasimd was transfected into BL21 competent cells，then His-tag Ni-NTA spin columns were applied to purify the recombinant protein which were produced by IPTG induced BL21.To generate the corresponding hyperimmune serum，rabbits and mice were immunized with the purified recombinant protein rHis-cLSm14A.Sequence analysis revealed that the full length of LSm14A CDS region transcripts from the frizzle chicken was amplified by RT-PCR.The amplified LSm14A CDS region had 1 368 bp and was 100% identical at the base sequence level to the sequence in GenBank.It has a close similarity to the duck source which reaches to 95.1%，on the contrary，the similarity to LSm14A from the mouse source，Xenopus，monkey source，human source，pig source，anser source were distant.The titers of serum mouse-anti-cLSm14A and rabbit-anti-cLSm14A antibodies were both higher than 1∶3 200.Western blotting assay showed that the recombinant protein displayed an immune response with the His-antibody，mouse-anti-cLSm14A hyperimmune serum and rabbit-anti-cLSm14A hyperimmune serum，The band weighted about 19.6 kDa in the western blotting.All the results indicated that the LSm14A molecule of frizzle chicken had a high conservative property and good immunogenicity，which laid a solid foundation for the further study of biological characteristics of frizzle chickens and antiviral natural immune mechanism of poultry.

The objective of this study was to evaluate effects of force-feeding stress on growth performance，meat characteristics of legs，serum hormone and blood gas parameters of Peking ducks.A total of 160 thirty-nine-day-old Peking ducks with average body weight (2.573±0.096) kg were randomly divided into 2 groups （force-feeding group and control group） with 80 ducks per treatment.After 6 days，8 ducks from each treatment were selected randomly to be weighted for calculating average body weight and average daily gain，and meat characteristics of leg(pH and drip loss rate)，serum hormone (ACTH，CORT，T3 and T4) and blood gas parameters were tested.The results showed that the average body weight and average daily gain in force-feeding group was significantly higher than those in control group (P<0.05).And the CORT，AnGap，Hct and Hb in force-feeding group was significantly higher than that in control group (P<0.01).The pH at 45 min and 24 h of tibialis anterior in force-feeding group were significantly lower than those in control group (P<0.01).It was concluded that force-feeding could enhance gaining weight significantly.However，force-feeding could affect the meat characteristics of leg significantly and induce physiological stress of Peking ducks by increasing serum CORT.At last，force-feeding could also influence the blood circulation system of Peking ducks.