Beak is an external anatomical structure of birds.Its functions are the same as lips and teeth of mammals.Beak deformity，a new skeletal disease，was first reported in birds and gained more and more attention recently.Beak deformity(normally a crossed beak) has also been found in some indigenous chicken breeds in China.The chicken with a deformed beak has problems in eating and drinking，which could cause slow growth and early death.This causes economics loss and is harmful to the animal welfare.Until now，the origin，morphosis，growth and development of beak has less been studied.The mechanism of beak deformity in chickens is not clear yet.The study of beak deformity in Darwin’s finches has a greater progress.This article summarized these related studies in birds，aiming to provide guidance for the study of beak development and molecular mechanism of beak deformity in chickens.

An important role for host cell F-actin polymerization caused the cytoskeleton rearrangement during the pathogen infection.This article mainly illuminate that integrin mediated host cell F-actin polymerization during the protozoan parasite invasion，the results can help to analyze the invasive mechanism and develop new drugs and vaccines of parasitic disease，so this research has very important significance.

The objective of this study was to estimate genetic parameters for total number born(TNB) of Yorkshire，Landrace and Duroc pigs in different genetic evaluation groups，which could establish foundation for the national swine joint genetic evaluation.Estimation of the degree of connectedness among 74 national nucleus farms were calculated by the module DMU4 in DMU software，based on the principle of each herd in group with a minimum average connectedness rating of 3% and all herds in group connected with each other，3 groups for each breed were screened(i.e.group1，group2 and group3).Animal model and DMU software were used to estimate variances of animal genetic and permanent environmental for total number born(TNB) of Yorkshire，Landrace and Duroc pigs in 3 groups，fixed effects of contemporary groups were also included in the analysis，records on 61 161，19 974 and 18 002 for Yorkshire，28 239，11 918 and 3 064 for Landrace，and 7 174，6 512 and 2 417 for Duroc in the group1，2 and 3，respectively，were analyzed.As a result，in general，the degree of connectedness was low(the average connectedness rating was 0.20%) and large-scale across-herd genetic evaluation in China was not practicable at present，but it might carry out across-herd genetic evaluation in certain regions.Estimated values of permanent environmental effects ranged from 0.05 to 0.11，estimated values of heritabilities for TNB were approximately 0.10 for all breeds and groups，the results were within the range recommended by relevant literature.The results indicate that genetic parameters of group 1 were better than other groups，therefore，it would expect to be used in the national swine joint genetic evaluation in the future.

In order to establish scientific basis for farm animal models associated with coat color mechanism and human diseases in OCA2 gene，we carried out this experiment.The exons 20-24 of porcine OCA2 gene(NC_010457.4) in absence in GenBank were cloned and mapped.The mapping result was confirmed at the genomic and cDNA levels using PCR amplification and Sanger sequencing simultaneously.Bioinformatics analysis was performed for OCA2 gene.Polymorphic loci of all exons for OCA2 gene were screened in Guangxi Bama Miniature pigs.Meanwhile，the mRNA expression in different tissues was detected by real-time fluorescence quantitative PCR(Q-PCR).A full-length skin cDNA was used as a template to construct recombinant expression plasmid，and then XhoⅠand SacⅡrestriction enzyme double digestion was carried out.We mapped and cloned the porcine OCA2 gene in GenBank successfully.A total of 10 SNP loci were found in all exons，including 7 synonymous mutations and 3 missense mutations（R124H，G509D，R573H）.Porcine OCA2 gene was widely expressed in various tissues.The expression in lung，brain and cerebellum was at relatively higher level compared to that in spleen，stomach，caecum and skin，which exhibited moderate expression level，and the expression level in heart and liver was lower.pEGFP-N1-OCA2 recombinant plasmid was constructed successfully after confirmation of restriction enzyme double digestion and sequencing.This study explored porcine OCA2 gene sequence information and mRNA expression preliminarily，and recombinant expression plasmid of OCA2 gene was constructed，which provide the foundation for the study of disease model and gene biological function in future.

The aim of the present study was to explore the effects of mtDNA on litter size in sheep.Genome sequencing and genotyping technologies were used for DNA variation analyses on mitochondrial coding genes in Dorper，Dorset and Suffolk sheep.In Dorper sheep，twenty-five mtDNA variations were detected，including 2 mutations in rRNA and tRNA genes，respectively，and 21 variations in polypeptide-coding genes(including 9 missense mutations and 12 synonymous mutations).For Dorset sheep，totally 20 variations，including 5 mutations in rRNA genes，1 mutation in tRNA genes，and 14 substitutions in polypeptide-coding genes(including 3 missense mutations and 11 synonymous mutations) were detected.In Suffolk sheep，there were 77 mutations in mtDNA coding genes，including 7 and 3 substitutions in rRNA and tRNA genes，and 67 mutations in polypeptide-coding genes which contained 7 missense mutations and 60 synonymous mutations.The mtDNA mutations presented poor associations with litter size in Dorper and Dorset sheep.However，in Suffolk sheep，14 mtDNA mutations were significantly associated with litter size(P＜0.05)，which both of the maximal values for the effects of the single mutation(T7759C) and haplotype(ATTCATTAAACTTT) were 0.22 per parity.The mtDNA had poor effects on litter size in Dorper and Dorset sheep.Mitochondrial DNA variations were significantly associated with litter size in Suffolk sheep.

The aim of this study was to clone and analyze the entire coding sequence of goat STAT3 gene as well as to detect the STAT3 gene methylation modification and analyze its association with goat body weight using reverse transcription-polymerase chain reaction(RT-PCR)，TA cloning，bioinformatics and bisulfite sequencing(BSP) methods.The results showed that the entire coding sequence of goat STAT3 gene was 2 313 bp in length，and encoded 770 amino acids.The shared amino acid sequence consistency between goat(capra hircus) and sheep(Ovis aries)，bovine(Bos Taurus)，boar(Sus scrofa)，mouse(Mus musculus），rat(Rattus norvegicus)，human(Homo sapiens) were 99.7%，99.6%，99.4%，99.2%，99.4%，99.5%，respectively.The analysis of the methylation difference in the promoter region of goat STAT3 gene between high body weight group and low body weight group demonstrated that the goats with high body weight had higher methylation level than those goats with low body weight(P=0.020).This finding indicated that the methylation modification of STAT3 gene had significant effect on body weight in goat，which could become the epigenetic marker in marker-assisted selection(MAS).These results would provide scientific data for studying genetic and epigenetic mechanism for meat production in goat.

The present study aimed to clone DLK1 gene of goat，predict the structure and function of peptone encoded by DLK1 gene，further investigate its expression regulation in organs or tissues of goat and analyze the correlation between DLK1 mRNA expression and intramuscular fat(IMF) content in muscles.Jianzhou Da’er goat was used as experimental animals and the RT-PCR technique was employed to amplify the DLK1 gene.The biological characteristics of this gene were analyzed by bioinformatics，and the tissue expression specificity as well as the development stage expression specificity of this gene were analyzed using the fluorescence quantitative PCR.The results showed that the DLK1 cDNA was 1 137 bp in length（GenBank accession No.：KP686197），927 bp for ORF and 308 amino acids for protein encoded.The nucleotide sequence and the deduced amino acids sequence of goat DLK1 shared 97% homology with the DLK1 of cattle.Accordingly，the close genetic relationship between goat and cattle was indicated by the phylogenetic tree analysis.The DLK1 protein was an unstable，acidity and hydrophobicity protein.There were 8 phosphorylation sites and 1 N-glycosylation site within the DLK1 protein.This protein was located in endoplasmic reticulum(44.4%)，golgi(33.3%) and plasma membrane(22.2%)，respectively through the sub-cellular level prediction.It was a secretory transmembrance protein.It had 5 domains of the EGF family，which had 2 EGF-CA of EGF family conservative domains.Moreover，the secondary structure of the DLK1 protein was composed of 87.01% random coil and 12.99% β-sheet，indicating an unconventional protein.The results of real-time PCR exhibited that the DLK1 gene was expressed in various tissues at different levels.The mRNA expression level of the DLK1 gene was highest in kidney and lowest in fat.The mRNA expression level of the DLK1 gene in longissimus dorsi of lamb was not significant higher than that in longissimus dorsi of youth and adult goat (P＞0.05).The results of correlation analysis showed that the mRNA expression level of the DLK1 gene in longissimus dorsi was correlated negatively with IMF content(R=-0.6，P＜0.05)，in leg was not correlated positively with IMF content(R=0.2，P＞0.05)，in arm triceps was not correlated negatively with IMF content(R=-0.2，P＞0.05).The results suggest that the DLK1 gene may play an important role in the IMF deposition in goat，which would lay a foundation for further studies about the function of DLK1 gene in IMF deposition.

The purpose of this study was to screen the most stable reference genes in different development periods and tissues in New Zealand White rabbit.Quantitative real-time Polymerase Chain Reaction(RT-qPCR)，a latest online reference genes stability assessment tool RefFinder and Minimum Information for Publication of Quantitative Real-time PCR Experiments(MIQE) guideline were used to compare the expression levels of 6 commonly used reference genes(GAPDH，HPRT1，18S rRNA，B2M，Sdha，β-actin) mRNA in 3 tissues(heart，liver，kidney) of rabbit at embryo(E28.5)，juvenile(10 d) and adult stages(A).The result showed that the most stable reference genes in liver and kidney of New Zealand White rabbit in 3 development periods were β-actin， HPRT1 and GAPDH，successively.But for heart，they were β-actin，HPRT1， Sdha.The most stable reference genes in embryo period of New Zealand White rabbit in 3 tissues were GAPDH，β-actin and HPRT1，successively.For juvenile and adult stages，they were HPRT1，GAPDH and β-actin. To all 3 periods and all 3 tissues of New Zealand White rabbit，the most stable reference genes were successively β-actin，HPRT1 and GAPDH.This study confirmed that the most stable reference genes were different in different periods and tissues of New Zealand White rabbit.To ensure minimal variation between individuals in an experimental group and the accurate statistical analysis of small fold changes，we recommend normalizing RT-qPCR data to the geometric mean of at least 3 validated reference genes as above when related New Zealand White rabbit trials were performed.

To study the effect of vascular endothelial growth factor (VEGF) on DNMT3a expression during ovine oocytes maturation in vitro. In this research, in order to test DNMT3a mRNA and protein expression of oocytes and granule cells (GCs) at different time points during cumulus-oocytes complexes (COCs) cultured in vitro, RNAi and microinjection were used to import 2 pairs of effective double chain small interfering RNA (dsRNA) fragments which were designed for VEGF receptors FLT1 and KDR/Flk-1 into COCs. The results indicated that, during the culturing process of COCs maturation in vitro, the expression level of DNMT3a mRNA of oocytes would be decreased regardless of the presence of VEGF. DNMT3a mRNA expression quantity fell sharply at the 8 h, then, it decreased slowly and tended to be similar to each other groups at the 24 h. DNMT3a mRNA without dsRNA of control group significantly declined faster than that of the other treatment groups (P＜0.01). The expression level of DNMT3amRNA of VEGF groups in oocytes showed a faster decrease than the groups without VEGF. After dsRNA fragments microinjection, interfering effect of FLT1-siRNA & KDR-siRNA group was better than that of the KDR-siRNA group and that of FLT1-siRNA group was the lowest (P＜0.01). During the maturation process, DNMT3a mRNA of GCs would be decreased regardless of the presence of VEGF, and the difference between each group were tiny. At the mid maturity of COCs, DNMT3a mRNA without dsRNA of control group decreased faster than that of the other treatment groups (P＜0.05 or P＜0.01), and the effect of RNAi on FLT1-siRNA group was the best. Whether adding VEGF or not, the protein expression level of DNMT3a in oocytes had been increasing from initial time until the 4 h, then it started to decrease at a constant speed. A noteworthy decrease of DNMT3a protein expression was then observed at the 16 h and was completely unexpressed at the 24 h. The protein expression level of DNMT3a in control group without dsRNA had the fastest decreasing speed. At the 16 h, DNMT3a protein levels of the control group, FLT1-siRNA group, KDR-siRNA group and FLT1-siRNA & KDR-siRNA group were decreased to 23.12%, 31.07%, 41.47% and 37.90% respectively with VEGF comparing to that of the 0 h. At the same time, that of the non-VEGF groups had decreased to 37.38%, 54.60%, 57.58% and 82.75%. In GCs, DNMT3a protein expression levels of groups with VEGF decreased gradually since 0 h and could not be tested at the 20 h, and that of groups without VEGF increased slightly from 0 to 4 h, then presented the average decline gradually until the 20 h where a rapid drop appeared and went completely undetected at the 24 h. In addition, dsRNA had negligible effect on DNMT3a protein expression levels, while the original DNMT3a protein expression level in the GCs were low. In conclusion, during the process of COCs maturation in vitro, VEGF could accelerate the decreasing speed of both mRNA and protein expression levels of DNMT3a. dsRNA fragments microinjection could slow down the decreasing speed of both mRNA and protein levels of DNMT3a. VEGF and dsRNA had no obvious influences on both DNMT3a mRNA and protein expression of GCs.

To understand the velvet antler development mechanism in the level molecular biology，proteome differences of the sika deer velvet antlers aged 10，40，60 and 130 d were compared using 2-DE，mass spectrometry and bioinformatics.Therefore，46 proteins were altered their expressions in the sika deer velvet antlers aged 10，40，60 and 130 d，which were involved in cytoskeleton，transportation，signal transduction，apoptotic process，bone development，protein synthesis，nucleotide biosynthesis，immunity，energy metabolism，cell proliferation and antioxidant activity and protein folding.We focused on the proteins involved in bone development，antioxidant activity and apoptotic process due to the special growth process with rapidly growth and ossification.Therefore，The results showed that several proteins(P4HB，SPARC，peroxiredoxin 2，peroxiredoxin 4，galectin 1 and retinoic acid binding protein 1) might play vital role in the velvet antler growth and ossification process.Taken together，the result provided a basis for further research on the molecular mechanisms involved in the accelerated growth and ossification of deer velvet antler.

To explore the regulatory mechanisms of active immunization against GnRH in female SD rats.24 adult female SD rats were allocated to 3 groups of 8 animals each randomly.8 rats were immunized against GnRH at 12 week of age，4 weeks later an equal dose booster was administered.8 rats were surgically ovarictomy at 11 week of age while 8 intact rats without any treatments.Blood samples were collected at day 0(0 wpv) until just before sacrificed at 56 wpv.Serum levels of FSH，LH，E₂ and P₄ were determined using RIA.After sacrificed，hypothalamus and pituitary were removed，the mRNA expressions of reproduction-related genes were determined by real-time fluorescence quantitative PCR(RT-PCR)；Ovaries were excised，adipose tissues were removed，then the mRNA expressions for reproduction-related genes were determined by RT-PCR.After immunization twice against GnRH，the concentrations of E₂，P₄，FSH and LH in immunized rats showed a significantly lower than that in Control group (P＜0.05).Moreover，active immunization against GnRH significantly down-regulated the mRNA levels of GnRH，GPR54，KiSS1and ER-α in the hypothalamus，GnRH-R，FSH-β and LH-β in the pituitary(P＜0.05)，as well as FSHR，LHR and ER-α in ovaries(P＜0.05).This study indicated GnRH had excellent immunogenic and was a good alternative of surgical castration.Active immunization against GnRH cause animals in a long-term infertility or sterility by the feedback loop of ER-α- GPR54/KiSS-1，and the fertility of immunocastrated rats show a tendancy to recover.

The objectives of the study were to identify the expression and distribution of EphA2 and EphrinA2 gene in hair follicles development and morphogenesis of Fine Wool sheep. The results might lay a theoretical basis for understanding molecular regulation mechanism of hair follicle development. Skins of Aohan Fine Wool sheep in the different period (prenatal stage, E90 d and E120 d; after birth, 1 d and 30 d) were selected. The qRT-PCR and immunohistochemical technologies were used to study their levels of mRNA and protein. The qRT-PCR results showed that mRNA expression level of EphA2 and EphrinA2 gene in E120 d were significantly higher than that of E90 d and 1 d (P＜0.01)，but 1 d and 30 d were not significantly difference (P＞0.05). Immunohistochemical detection results showed that, the level and localization of EphA2 and EphrinA2 protein were spatial and temporal specificity among E90 d, E120 d, 1 d and 30 d of sheep skin body side. And they mainly expressed in hair follicle, hair matrix outer root sheath and epidermis. In conclusion, EphA2 and EphrinA2 may play important roles in hair follicle development and morphogenesis. These results would provide fundamental information for the related research fields.

To investigate the effect of double-stranded RNA(dsRNA) on cocaine-and amphetamine-regulated transcript(CART) gene expression and estrodiol(E₂) and progestin(P) secretion in follicles of Highland Brown Laying hen.A total of 60 healthy layers at 20-week-old were selected and randomly divided into 3 experimental groups(A group：control group；B group：with treatment of dsRNA intravenous injection every 5 d；and C group：with treatment of dsRNA one initial injection).After 30 d of feeding，the different size of 3-5，6-8，9-12 mm follicles of 10 layers from each group were collected for analyzing mRNA expression of CART and determining the concentration of E₂ and P.The results showed that mRNA expression of CART was decreased in group B and C，compared with the control group.Especially，mRNA expression of CART in 6-8 mm follicle was lower than that in the control group with significant difference（P＜0.05）;The levels of E₂ was increased in the follicles with the size of 6-8 and 9-12 mm in group B with significant difference（P＜0.05）；However，there was no significant difference in the level of P among different treatment groups.The results suggested that intravenous injection of dsRNA could affect the follicular development through inhibiting the mRNA expression of CART and promoting the E₂ production in layer.

The aim of this study was to research the effects of constant temperature and diurnal cyclic temperature on nitrogen metabolism and performance of chicken.One hundred and ninety-two 22-day-old Arbor Acres broilers with the same health condition were randomly allocated to 4 treatments in artificial climate（environment-controled） chambers(at the temperature of 21，26，31 and 21/31 ℃).Each treatment contains 6 replicates and 8 birds for each replicate(4 females and 4 males).The experimental lasted 14 d.All limosis birds in each replicates were weighted on 1，7，14 d of the study，respectively.Feed intake was recorded daily，fresh faeces on 1，7 d were collected and subsequently analysed by Kjeldah method in order to analyse the effects of constant temperature and diurnal cyclic changing temperature on growth performance，nitrogen utilization and emission of chicken.The results showed that：1) The average daily weight gain(ADG) and average daily feed intake(ADFI) of birds in the treatment of 31 ℃ were significantly lower than birds in treatments of 21 and 26 ℃(P＜0.05),while the feedgain(F/G) was significantly higher that of the 2 treatments(P＜0.05)；Chickens in the treatment of 26 ℃ had significant lower ADG and ADFI when compared to the treatments of 21 ℃(P＜0.05) while there was no significant difference in F/G observed between the 2 groups(P＞0.05) on 1-7 and 1-14 d；Compared to the birds in constant temperature at 31 ℃，the birds in diurnal cyclic changing temperature at 21/31 ℃ had a significantly higher ADFI(P＜0.05) without significant changes in ADG and F/G(P＞0.05).2) On 7 d of this study，compared to the group of 21 ℃，STP of birds in group of 31 ℃ was significantly decreased(P＜0.05)，while SUN was significantly increased(P＜0.05)；STP of birds in diurnal cyclic changing temperature at 21/31 ℃ was significantly decreased(P＜0.05) when compared to the constant temperature at 26 ℃；On 14 d of experiment，compared to constant temperature at 21 and 26 ℃，STP of birds in treatment of constant temperature at 31 ℃ was significantly decreased(P＜0.05).3) Compared to the group of 21 and 26 ℃，birds in group of 31 ℃ had a significant decrease of NU(P＜0.05) and a significant increase of NEDG and NEFI(P＜0.05);On 7 d of the study，compared to constant temperature at 26 ℃，NU of birds in diurnal cyclic changing temperature at 21/31 ℃ was significantly decreased(P＜0.05);On 14 d of the study，compared to the group of 21 ℃，NU of birds in group of 26 ℃ had a significant decrease(P＜0.05) and a significant increase of NEFI(P＜0.05)；NU of birds diurnal cyclic changing temperature at 21/31 ℃ was significantly decreased(P＜0.05)，NEDG and NEFI significantly increased(P＜0.05) when compared to the constant temperature at 26 ℃.The results indicate that compared to 21 ℃，the treatment of 26 and 31 ℃ have significant effects on nitrogen metabolism and growth performance of birds；diurnal cyclic changing temperature at 21/31 ℃ significantly decreased nitrogen utilization and increased nitrogen emission of birds than constant temperature at 26 ℃.

The objective of this study was to investigate the effects of intra-arterial infusion of amino acids on milk performance and milk composition of fatty acids in dairy cows.8 Chinese Holstein cows，fitted with intra-arterial catheters that was terminated in right external pudendal，were used in this study.Cows were fed with TMR based on corn stover basal diet.The experiment lasted for 27 days，including 21-day preliminary period followed by 3-day carrier infusion(CSc) and 3-day amino acids infusion(CSa).Milk was collected twice daily at the last 2 days of each infusion period，and mixed according to the milk production of each cow.The composition of milk fatty acids was determined by GC-FID.The results showed as follows：1) The milk fat percentage of CSa was significantly higher with 5.92% than that of CSc(P＜0.05)，whereas the remaining milk components showed no significant difference between CSa and CSc(P＞0.05)；2) The concentration of C4∶0，C6∶0，C8∶0，C10∶0，C12∶0，C14∶0 and C16∶0 in milk were significantly reduced after amino acids infusion(P＜0.05)，and the concentration of C18∶1cis-9 in milk tended to decrease(P＜0.10)；3) Compared with CSc，AA infusion significantly decreased the total fatty acids concentration by 27.24% in milk(P＜0.05) in CSa group.These results indicate that intra-arterial infusion of amino acids can decrease the content of short- and medium-chain fatty acids in milk，which did not affect long-chain fatty acids content in milk.

The objective of this study was to investigate the influence of BVDV on the cell maturation and cytokine level of Th1 and Th2 type in dendritic cells (DC) derived from bovine peripheral blood，and to further reveal the immune mechanism of BVDV infection.The monocytes from bovine peripheral blood were separated and induced into DCs，and then were co-cultured with BVDV.The cell morphology and the expression of MHCⅡ，CD80 and CD40 on DCs were observed by microscopy and flow cytometry respectively，the semi-quantitative RT-PCR was used to analyze IL-12p40 of Th1 type cytokines and IL-10 of Th2 at mRNA levels.The results showed that DCs had typical morphology and immunophenotype with 93.81% purity.Post BVDV infection，the MHCII molecules，CD40 molecules and CD80 molecules were greatly lower than that of the control groups，the differences were statistically significant (P＜0.01). IL-12p40 mRNA level was down-regulated，while IL-10 mRNA level was up-regulated，the differences were significant (P＜0.05) compared with the control groups.These results indicated that BVDV infection to DC plays an important role on maturation and regulating the balance of Th1/Th2.

This experiment was conducted to explore the effect of PB2 K627E mutation on the replication competence of classical H1N1 subtype swine influenza virus(A/Swine/Guangdong/1/2011).The rescued and mutant strains of classical H1N1 subtype swine influenza virus were obtained by reverse genetics and point mutation technology，and researches were taken on MDCK cells and mice.The results on MDCK cells showed that，compared with the rescued strain，the mutant virus had a slight degree of CPE；the morphology of plaque of mutant virus was much smaller；the growth curve showed that it had a significant distinct difference.The results of experiments in mice showed that the mutant strain had a low pathogenicity and could not lead the mice loss weight，and virus titer in lung tissues on the 3rd and 5th day post challenge showed that there was a significant distinct difference between the two strains and the pathological damage caused by mutant one was not serious.The results on MDCK and mice both showed that the mutant strain’s replication competence was lower than the rescued one.This conclusion demonstrates that the amino acid of PB2 627 is not only one of the important virulence markers，but also provided the condition to further clarify its pathogenic mechanism.

According to reports，VP60 P2 subdomain of RHDV may play an important role in the viral infection． In this study，we used co-immunoprecipitation(Co-IP) with RHDV VP60 P2 expressed in E.coli as bait protein and mass spectrometry analysis to screen the RHDV interacting hepatocellular proteins.Laminin receptor （LamR），as receptors of many other viruses，was identified and verified as potential interacting partner of P2.LamR gene was amplified by RT-PCR from rabbit liver cells．The recombinant LamR protein was expressed in E.coli and used in GST pull-down to verify the interaction of LamR and P2．Here we screened 26 proteins as potential P2 interacting proteins.After verification，the protein LamR could interact with P2 directly.We found that RHDV VP60 P2 interacted with LamR which would contributed to further study of the infectious process and pathogenesis of RHDV．

A T cell epitope candidate peptide Sp6 of the avian infectious bronchitis virus S1 was predicted with biological structures technology.To identify this peptide exist in S1 protein and verify validity of Sp6，the recombinant plasmid pCAGGS-S1 was constructed.While it can be expressed in eukaryotic cells after determined by indirect immunofluorescence and Western blot，using the recombinant plasmid immune SPF chickens，and the antibodies against S1 were detected by ELISA.Splenic cells were collected from inoculated chickens and stimulated with the peptides Sp6，NP_89-97(PKKTGGPIY) and negative control.By SBRY Green qPCR detecting IFN-γ production of spleen lymphocytes and flow cytometry detecting the multiplication of CD8⁺ T lymphocytes，preliminarily make sure that the candidate peptide is correct.The IFA Western blot and ELISA results showed that the expression of S1 was confirmed in 293T and can induced the humoral immune，indicate that S1 protein is reaction to the original and lied a foundation for further validation of T cell epitope；the results of SBRY Green qPCR showed that spleen lymphocytes of chickens stimulated with the peptide Sp6 secreted significantly-increased IFN-γ.Flow cytometry assay results showed that the CD8⁺T lymphocytes were proliferated by 34.8%，2.6%，and 0，respectively，which indicated that the peptide Sp6 was a T cell epitope of IBV S1 and it can induce activation of chicken lymphocytes to produce cytotoxic T lymphocyte (CTL) reaction in vivo.All above results indicate that the peptide Sp6 was a T cell epitope of IBV S1 and it can induce activation of chicken lymphocytes to produce CTL reaction in vivo.The results possesses significance to mechanism of protective immunity against avian infectious bronchitis virus and the research towards universal IBV vaccine.

Gluten exorphin B5 (GE-B5) was a food-derived opioid peptide identified in vitro in enzymatic digests of wheat gluten，and it was a good candidate of functional feed additives and adjuvant for vaccine development because of its immune function.In this study，we chemically synthesized GE-B5，and then added it to vaccine against infectious bronchitis virus (IBV) H120 strain to immunize chickens for evaluating their growth performance，immunogenicity and protective efficacy.On 0，14，and 21 dpi，the changes in proliferation of peripheral blood mononuclear cells (PBMC) and serum antibody titer were evaluated respectively by MTT method and hemagglutination inhibition assay.The results showed that administration of GE-B5 significantly raised anti-IB antibody titer in the chicken serum and also markedly promoted PBMCs proliferation.Following challenge with IBV Mass 41 strain，chickens inoculated with GE-B5 showed fewer and less severe clinical signs，lower death rate，lower mortality and showed higher protection，as compared to those of the control groups.The result of pathological tissue section showed that the lung and kidney became shorter pathological phenomenon than the control group.This study confirmed that in addition to its immunoregulatory activity and immunopromoting effects，GE-B5 can also promote weight gain.These data have important implications for functional feed additives and novel adjuvants designed for future vaccines.

In order to study the effect of CYP1A1 expression change in Mycoplasma hyopneuminiae infection and to investigate the association betweenCYP1A1and peroxisome proliferator-activated receptors associated-γ (PPAR-γ)，we obtained the full-length coding sequence of CYP1A1 by means of cloning and eukaryotic expression vector construction method，established PAMs cell lines that stably expressed CYP1A1 by method of cell-transfection and resistance screening techniques，constructed cell experimental model infection with Mycoplasma hyopneuminiae and analyzed the relationship of the expression level of PPAR-γ and CYP1A1.The results show that the expression level of PPAR-γ is positively correlated with CYP1A1 and is involved in the regulation of inflammation，which suggest that CYP1A1 may play a role in suppressing inflammation by regulating PPAR-γ.

The aim of this study was to identify the presence of villous M cells in the ileum of newborn piglets and determine the distribution pattern，morphological characteristics and the association with PEDV infection by immunofluorescence assay(IFA)，immunohistochemistry(IHC) and morphological methods.IFA showed a gradually decreased density of villous M cells from lower crypt epithelium to upper villous epithelium.The villous M cells showed typical morphological characteristics of M cells by electron microscope.After infected with PEDV，villous M cells in the ileum exhibited pathological changes obviously.The virus particles of PEDV were observed on the apical membrane，in the cytoplasm，around the nucleus and subepithelial of villous M cells by immunohistochemical staining of PEDV S protein，which were not observed in its adjacent epithelial cells.This study demonstrated the presence of villous M cells in the ileum of newborn piglets and determine the distribution pattern，morphological characteristics and the association with PEDV infection，suggesting the villous M cells in the ileum may be an important entry for PEDV invasion.

To understand the mechanism of PCV2 replication and pathogenesis，change and regulation of interferon in alveolar macrophages of piglets subclinically infected with procine circovirus type 2(PCV2) were studied.Fifteen 30-day-old piglets were selected as experiment animals，which were free of PCV2 and porcine reproductive and respiratory syndrome virus(PRRSV) antibody and antigen.The piglets were divided into three groups：control group(0 dpi)，PCV2 infection group(14 dpi) and PCV2 infection group(28 dpi).After infection，the mRNA transcription of interferon，pattern recognition receptors(PRRs) and pathogen associated molecular proteins(PAMPs) in AMs were detected by real-time PCR.The results showed that the mRNA levels of IFN-β and IFN-γ were significantly higher at 14 dpi and 28 dpi(P＜0.05)；the mRNA expression of RIG-1，MDA-5，TLR4 and TLR9 were significantly increased at 14 dpi and 28 dpi(P＜0.05)；the mRNA level of DAI was significantly elevated at 14 dpi(P＜0.05)，and then decreased at 28 dpi；while there were no significant change in mRNA expression of TLR3，TLR7 and TLR8.Compared with control，the mRNA transcription of MAVS，MyD88，IRF3 and IRF7 were significantly increased at 14 dpi and 28 dpi(P＜0.05).These results demonstrate that the up-regulations of IFN-β and IFN-γ in alveolar macrophages of PCV2 subclinical infection are relative to TLR4/TLR9/MyD88 and RIG-1/MDA-5/DAI/MAVS/IRF3 signaling pathways.

The aim of this study was to explore the role of cpxR gene in the multidrug resistance of Salmonella enterica Serovar Typhimurium and exclude a possibility that acrB masks the effect of cpxR on the other efflux pumps or outer membrane proteins.A acrB gene deletion strain JSΔacrB was constructed by the method of one-step inactivation of chromosomal genes on basis of Red homologous recombination system.A double gene deletion strain JSΔacrBΔcpxR∷kan was constructed by phage P22-mediated transduction with strain JSΔcpxR∷kan constructed in the previous study as the donor and strain JSΔacrB as the recipient.Meanwhile，the cpxR complemental strain JSΔacrBΔcpxR∷kan/pcpxR was prepared through introducting the expression plasmid pBAD-CpxR into the double gene mutant.At last the minimal inhibitory concentrations(MICs) of 10 kinds of commonly used representative antibacterial agents for JS and the above strains constructed in this study were determined with broth microdilution method.The double gene deletion strain and related cpxR complemental strain were constructed successfully.Strain JSΔacrB showed 32-，4-，4-，16-，32-，4-，2- and 128-fold decrease in the MICs of doxycycline，gentamycin，amikacin，ciprofloxacin，enrofloxacin，florfenicol，ceftriaxone and ceftiofur respectively，compared to the parent strain JS.Strain JSΔacrBΔcpxR∷kan showed 2-，8-，2- and 2-fold decrease in the MICs of doxycycline，gentamicin，amikacin，and ceftiofur compared to strain JSΔacrB.While the complemental strain JSΔacrBΔcpxR∷kan/pcpxR showed 4-，16-，2- and 4-fold increase in the MICs of the above four kinds of antibacterial agents compared to strain JSΔacrBΔcpxR∷kan.In conclusion，cpxR is able to regulate the antimicrobial susceptibility of S.enterica Serovar Typhimurium to doxycycline，gentamicin，amikacin，and ceftiofur.

The purpose of the study was to explore the effects of Palmitic acid (PA) and Stearic acid (SA) on genes and protein expression of heme oxygenase 1 (HO-1) and Nuclear Transcription Factor Nrf2 in HepG2 Cells.The HepG2 cells were treated with PA or SA of 0.25，0.5，0.75，1.0 mmol•L^-1 for 24 h，the mRNA and protein expression levels of HO-1 and Nrf2 were detected by quantitative reverse transcription PCR and western blotting，separately.The experimental results were as follows，compared with the control group，the mRNA and protein expressions of HO-1 and Nrf2 were significantly increased with the increasing of PA concentration (P＜0.01)；While the mRNA and protein expressions of HO-1 and Nrf2 were significantly increased with SA of the concentrations of 0.25，0.5，0.75 mmol•L^-1 (P＜0.01)，the increased amount of the mRNA and protein expressions of HO-1 and Nrf2 were relatively decreased when the concentration of SA was 1.0 mmol•L^-1.In conclusion，a relatively higher concentration of PA and a relatively lower concentration of SA can elevate the mRNA and protein expressions of HO-1 and Nrf2 significantly within 1 mmol•L^-1 in HepG2 cells.

The study was conducted to investigate the effects of Jin-Ying-Tang （JYT） on correlated molecules of TLR4NF-κB signaling of LPS-stimulated mouse mammary epithelial cells(MECs) in vitro and explore its underlying molecular mechanisms.ELISA was performed to detect changes of the content of IL-8 and TNF-α in the culture supernatants.The mRNA transcription of TLR4-NF-κB signaling molecules such as IL-1 receptor-associated kinase-1(IRAK-1)，tumour necrosis factor receptor associated factor 6(TRAF-6)，Transforming Growth Factor activated kinase 1(TAK-1)，NF-κB inducing kinase(NIK)，Inhibitor of κB(IκB) were determined by qRT-PCR.The results showed that JYT could significantly decrease the expression of IL-8 and TNF-α in LPS-stimulated MECs(P＜0.05，P＜0.01)；what’s more，JYT could down-regulate the expression of IRAK-1，TRAF-6，TAK-1，IκB，NIK mRNA activated by LPS(P＜0.05，P＜0.01).It is concluded that JYT attenuates LPS-induced inflammatory response in MECs by inhibiting the activitation of IRAK-1/TRAF-6/TAK-1/NIK pathway，but not of IRAK-1/TRAF-6/TAK-1/IκB pathway.

An indirect ELISA which can detect EBOV antibody was established using the purified the envelope glycoprotein as coating antigen.We use the HRP labeled antibody as the secondary antibody，the positive and negative horse sera as controls.After the reaction conditions were optimized，we analyzed the specificity，sensitivity of the indirect ELISA using serum of horse which immunized with EBOV virus like particles.For further evaluation of application of the ELISA，the results were compared to the neutralization assay based on pseudo-type EBOV.The indirect ELISA could detect the antibody against EBOV specifically and showed no cross-reaction with the positive sera of West Nile Fever et al.The indirect ELISA also showed high coincidence with the neutralization assay based on pseudo-type EBOV in evaluation of the immune response after immunization.The coefficient of variation of inter-batch and intra-batch duplicative tests was less than 8％.The indirect ELISA was successfully developed.It provides an effective and convenient method to kinetic detection of antibody against EBOV after immunization.

The objective of this study was to investigate the ambient parameters in dairy cowsheds and their correlation.Four styles of cowsheds in the chill region of Hebei province were used and the continuous record method，fixed spot and time determination method，plate count method were applied to investigate the ambient temperature and relative humidity，airborne dust concentration and airborne bacteria count in 4 seasons，respectively.The results indicated that the indoor temperature was high at noon and low in both the morning and evening，while the opposite tendency in the relative humidity was observed.The temperature-humidity index(THI) was above 72 in all cowsheds in summer，which suggested that heat prevention would be paid more attention in the cowsheds of chill regions.The dust concentrations in all cowsheds ranged from 28.5 to 211.5 μg•m^-3 of PM₁₀ concentrations and from 1.9 to 44.2 μg•m^-3 of PM_2.5 concentrations.In each season，a significant difference was observed on the dust concentrations(PM₁₀ and PM_2.5) among 4 styles of cowsheds(P＜0.05).The PM₁₀ concentrations depended on seasons，showing the highest value in summer and the lowest in winter.Moreover，the PM₁₀ were significantly positively correlated with the temperature(P＜0.05，r=0.60)，while not significantly correlated with the relative humidity(P＞0.05).In addition，the aerobic bacteria counts ranged from 1 804 to 4 944 CFU•m^-3 and the bacteria among all cowsheds showed the significant difference in each season(P＜0.05)，and the bacteria counts were significantly positively correlated with temperature(P＜0.05，r=0.49) and the relative humidity(P＜0.05，r=0.56)，respectively.There was significantly positive correlation between bacteria counts and PM₁₀ concentrations(P＜0.01，r=0.80).In conclusion，the correlation among ambient parameters，including temperature，relative humidity，dust，airborne bacteria，and so on，would be considered to improve the environment in dairy cowsheds.

To reveal the morphological structure characteristics of carotid body of Yak in different hypoxia.In this study,the morphological structures of carotid body observed by histologial，immunohistochemical methods and electron transmission microscopy techniques in health adult yaks at different altidudes in Qinghai Province，and the volume density(Vv)，surface density(Sv)，numerical density on area(N_A) and specific surface(δ) of type Ⅰ cells(light and dark)，type Ⅱ cells and microvessels in carotid body of yaks at different altitudes were compared by light microscope and microstereology.The results showed that there were many more light cells than dark cells.The type I cells contain mitochondria and electron dense-cored vesicles in their cytoplasm，in size and number of mitochondria and electron dense-cored vesicles，no significant differences between 2 of the groups were found with height of sea level.The type Ⅱ cells surrounding type Ⅰ cells were small and little.There were rich capillaries in mesenchyme，increasing diameter of the inner lumen of the capillaries with the elevation increasing.The numerical density on area and surface density of light cells were increased with height of sea level，and there were the significant difference between any 2 of the groups.The volume density，surface density and numerical density on area of dark cells gradually increased with altitude，there was the significant difference between altitude 4 600 m and altitude 2 800 m.The surface density and numerical density on area of type Ⅱ cells gradually increased with the elevation rise，and there were the significant difference between 4 600 m and 2 800 m，3 800 m respectively.The surface density and specific surface of microvessels decreased with altitude.In surface density，there was significantly different between altitude 4 600 m and 2 800 m，in specific surface，there were significant difference between any 2 of the groups.The results suggested that these type Ⅰ cells and type Ⅱ cells increased markedly in number，and the microvessels lumina dilated obviously with height of sea level.