Swainsonine，one of indole quinolizidine alkaloids，is a metabolite of locoweed and its parasitic fungi.The swainsonine has good anti-virus，anti-tumor effects and can enhance the role of immunity，has great potential for medicine development.This paper briefly expounds pharmacology，detection method，medicinal value and development potential of swansonine.And the research progress of its source and locoweed endophytic fungus synthesizing spherosin were summarized to enrich swainsonine’s source and provide the supply of abundant raw materials for its scientific and medical application.

Melanocyte，generated from embryonic neural crest stem cells，plays important roles in coat and skin color formation.Melanin，the product of a series of enzyme catalyzed reactions in melanocyte，determines animal coat and skin color.Melanogenesis is a complex process involving coordinated regulation of some transcription factors，hormones and signaling pathway molecules.microRNA (miRNA) is a class of endogenous，non-coding，small RNA molecule (about 22 nt)，which regulates gene expression mainly at the post-transcription level.More and more evidences demonstrated that miRNA involve in the regulation of melanogenesis.Herein，it was reviewed in this paper the studies on the discovery and identification of melanin-associated miRNAs，and the research progress of how miRNAs regulate animal coat and skin color.

 This study aimed to find tissue-specifically expressed miRNAs in ovine adipose tissues.Three small RNA (sRNA) libraries from perirenal，subcutaneous and tail adipose tissues were built for Solexa sequencing.The sequencing results were validated by Stem-loop qRT-PCR technology and further analyzed by bioinformatics methods.The PCR results shown that seven in eight randomly selected miRNAs were consistent with the sequencing results.In perirenal and subcutaneous adipose tissues，128 and 112 conserved miRNAs were obtained，198 and 196 novel miRNAs were found，respectively.Combined with the previous analysis results of miRNAs detected in tail adipose tissue，12 conserved and 66 novel miRNAs were specifically expressed in perirenal adipose tissue；one conserved and 30 novel miRNAs were specifically expressed in subcutaneous adipose tissue；and one conserved and 34 novel miRNAs were specifically expressed in tail adipose tissue.These results provide scientific bases for further research on the regulatory role of miRNA in fat metabolism.

In the present study，PID1 mRNA expression profile in several tissues of Tibetan chicken during different growth phases and the correlation between PID1 mRNA expression and intramuscular fat (IMF) content were investigated.PID1 gene of Tibetan chicken was cloned using RT-PCR and the structure as well as function of PID1 protein was predicted using relevant softwares.Furthermore，the expression profile of PID1 gene was investigated by qRT-PCR and the relationship between PID1 mRNA expression and IMF content in chicken muscles were analyzed.The results showed that PID1 gene was 654 bp encoding a unstable，hydrophilic，acidic protein with 217 amino acids and a PTB (phosphotyrosine binding) domain.There were 14 phosphorylation sites，4 O-glycosylation sites，1 N-glycosylation site，7 protein kinase phosphorylation sites，and 3 disulfide bonds within the PID1 protein.The predicted secondary structure of PID1 protein was composed of alpha helix (26.73%)，beta fold (20.74%) and random coil (52.53%)，belonging to one of mixed proteins in cytoplasm.qRT-PCR results showed that PID1 mRNA could be expressed in various tissues，with the highest expression level in fat (P<0.01).The temporal expression showed that the expression level of PID1 gene was the highest in breast muscle of 1 day Tibetan chicken，and was the highest in leg muscle of the 210^th day cocks (P<0.01)，while the highest in leg muscle of the 119^th day hens (P<0.01).For fat tissues，the highest expression level appeared in the 119^th day and the 154^th day cocks，and the 210^th day hens，respectively.The correlation analysis indicated that there was a significant correlation between PID1 mRNA expression and breast muscle IMF content in Tibetan chicken（P<0.05）.These results suggested that the PID1 gene might play an important role in the IMF deposition of Tibetan chicken.

In order to understand the development pattern of body weight and skeletal muscle of ducks，this experiment was conducted to compare the difference of growth traits about body weight，breast muscle and leg muscle between different duck breeds.The body weight，pectoralis major (PEM) weight and gastrocnemius (GAS) weight for 20 Gaoyou ducks (GYD) and 20 Jinding ducks (JDD) at day 0，4，8，12，16，20，24，28，35，42，49，56，63 and 70 were determined，respectively.The data was used to compute and compare the accumulate values，absolute values and relative values between GYD and JDD.Three models of Gompertz，Logistic and Bertalanffy were used to fit growth curves of body weight，PEM weight and GAS weight for the 2 breeds.The inflection day (ID) and weight gain at inflection (WGI) was calculated from parameters of the models.The results showed that：1) Body weight and GAS weight of GYD were significantly higher than that of JDD at the incubation period，and PEM weight of GYD was remarkably higher than that of JDD from day 4 to 70.The body weight，PEM weight and GAS weight of GYD were 1.41 times，1.50 times and 1.20 times of JDD，respectively from day 28 to 70.2) All the growth speeds of body weight，PEM weight and GAS weight of GYD were faster than that of JDD，but the maximum growth speeds day of the 3 traits are identical between the 2 breeds，which were day 20 for body weight，day 56 for PEM weight，day 20 for GAS weight.3) The body weight growth intensity of GYD was higher than that of JDD，while the growth intensities of PEM weight and GAS weight were less than that of JDD during stage of day 0-16 via analysis of fitting data.From then on，however，the growth intensities of the 3 traits approached to be identical between the 2 breeds.There was no significant difference between the 2 breeds in the varying pattern of growth intensity of body weight，PEM weight and GAS weight.4) The inflection day of PEM weight (52 day both for the 2 breeds) were far latter than that of GAS weight (16.5 day both for the 2 breeds).The growth intensity of PEM weight was less than that of GAS weight before day 16，and from then on，it was higher than that of GAS weight.During the stage of day 0-28，the growth intensity of PEM weight decreased slowly and then rapidly.In contrast，the growth intensity of GAS weight decreased rapidly and slowly from day 0 to 28.At the growth stage of day 28-70，the breast-body index of GYD was higher than that of JDD，while the leg-body index was significantly less than that of JDD.These results showed a stronger growth intensity of PEM than that of GAS during day 28-70.Under the identical growth condition，there is no significant difference between breeds as to the duck growth pattern of body weight，PEM weight and GAS weight.However，there are significant differences between breast muscle and leg muscle，breast muscle growth slower than leg muscle at early while faster at later，and breast muscle contributed more than leg muscle to meat performance.

The research was conducted to clone BMPR-IB gene coding sequence of the Small-tailed Han sheep with BB genotype，construct the recombinant eukaryotic expression vectorwhich was transiently transfected into cashmere goat fibroblast cells，and detect the expression of BMPR-IB and other genes.The BMPR-IB gene was amplified by RT-PCR and the eukaryotic expression vector pEGFP-BMPR-IB was constructed by cloning BMPR-IB gene fragments into the pEGFP-N2 vector frame，which were followed by the transfer of recombinant plasmids into goat fibroblast cells by liposome Lipofectamine LTX&PLUS.After transfection for 48 and 72 h，transfection cells were collected to extract RNA and total protein.Real-time quantitative PCR and Western blot approachs were used to identify the expression level of target genes and other related genes.The results showed that BMPR-IB gene which was amplified including CDS region was about 1 550 bp in length，and highly homologous with the published sequences.Real-time PCR detection results showed that the expression of BMPR-IB gene in transgenic cells were significantly higher than that in control group (P<0.01).The expression level of IGF-I gene in transfected cells were significantly higher than that in control group (P<0.01)，while the expression of TLR4，IFN，MHC，PNRP，GDF5 and INH genes were significantly lower (P<0.01).Western blot detection results showed that the expression of BMPR-IB and IGF-I in transgenic cells was higher than that in control group，although the expression of BMP4 and TLR4 decreased，the difference did not reach significant level (P>0.05).The result showed that expression vector of BMPR-IB was constructed successfully and the expression of BMPR-IB gene in goat fibroblast cells was realized，which provide the basis for the preparation of positive cell strains，cell lines and transgenic animals.It is also concluded that the over-expression of BMPR-IB gene up-regulates the IGF-I expression and down-regulates the TLR4 expression in transfected cells.

The present study was conducted to investigate the correlation between environmental temperatures and the parameters of reproductive performance for the entire year of 2014 in Yancheng City of Jiangsu Province.The performance of sows in a large-scale pig farm in Yancheng City of Jiangsu Province was analyzed including the litter size (LS)，number of dead fetuses (NDF)，number of weaning piglets (NWP)，weaning-to-estrus rate within 7 d (WER_7d)，and return-to-estrus rate (RER).The environmental temperature parameters were recorded including the daily highest temperature (DHT)，daily lowest temperature (DLT)，average values of DHT per month (ADHT)，average values of DLT per month (ADLT)，total number of days with DHT greater than or equal to 30 ℃ per month (TNDHT₃₀)，total number of days with DLT less than or equal to 10 ℃ per month (TNDLT₁₀).The results showed that，the TNDHT₃₀，ADHT and ADLT parameters all attained the peak values (19 d，30.3 ℃，and 23.0 ℃，respectively) in July.Compared to the average levels for the whole year，the values were highest for the RER of October and the NDF of July (P<0.05).In addition，the parameters of the WER_7d of September，LS of July，and NWP of July were decreased to the lowest levels (P<0.05).The correlations between the ADHT and the TNB，NWP，NDF and WER_7d parameters were significant.Furthermore，there were significant correlations between the TNDHT₃₀ and the LS，NWP and NDF parameters.In conclusion，the seasonal infertility rate might be attributable to the negative impact of the high ambient temperature and the increased exposure time on the reproductive endocrinology，oosperm growth，and the fetal development in sows during summer.

The objective of the present study was to investigate the effects of active immunization against GnRH on skatole metabolism in boars.Thirty-six boars at the age of 10 weeks were randomly allocated to 3 groups.Included intact males(not administrated)，Immuno-castrates(immunized against GnRH at 10 and 18 weeks of age)，and Surgical castrates (surgically castrated at the age of one week).The hormone levels of testosterone and estradiol in plasma were determined by ELISA，the androstenone levels in plasma and androstenone，skatole levels in fat were determined by HPLC.The mRNA expression of skatole-metabolism genes in the liver were analyzed by real-time fluorescence quantitative PCR technique，and the protein expression of them were determined by ELISA.Active immunization against GnRH in boars resulted in a atrophy of testes and reduction of plasma testosterone levels (P<0.05).Plasma testosterone，androstenone，estradiol levels and fat androstenone and skatole levels in immunized boars were similar to that of surgical castrated boars (P>0.05)，in which were significantly lower than that of intact controls (P<0.05).In the liver，CAR COUP-TF1，CYP2E1，CYB5A，CYP2C49，GSTO2 mRNA expression in immune-castrated boars were similar to that of surgical castrated boars (P>0.05)，which were significantly higher than that of intact controls (P<0.05)；the CYP2A19 mRNA expression in surgical castrated pigs was higher than in intact controls (P<0.05)，while CYP2A19 mRNA expression in immunized boars was in between，which was significantly different from either surgical castrates or intact controls (P<0.05)，PXR and SULT1A1 mRNA expression were not significantly different among 3 groups (P>0.05).In the liver，the CYP2E1，CYB5A protein expression were highest for surgical castrated pigs and lowest for intact controls，with vaccinated pigs at an intermediate level；CYP2A19，CYP2C49，GSTO2 protein expression in surgical castrated boars were similar to that of surgical castrated boars (P>0.05)，in which both were significantly higher than intact controls(P<0.05)；but SULT1A1 protein expression had no significant difference among 3 groups (P>0.05).Active immunization against GnRH in boars decreased plasma testosterone，estradiol and androstenone levels，increased CYP450s and GSTO2 mRNA and protein expression and to reduce boar-taint by accelerating skatole metabolism in the liver.

This study screened the candidate genes of horn trait which provides scientific basis for the horn developmental mechanism and the polled yak breeding.The horn bud and forehead skin tissue from horn and polled yaks with 90 d gestational age were collected，and the mRNA different expression of OLIG1,OLIG2,IFNAR1,IFNAR2,C1H21orf62,GCFC1,IFNGR2,SYNJ1,IL10RB,GART,RXFP2,FOXL2,TWIST1,TWIST2,ZEB2,E-cadherin,P-cadherin genes were analyzed using real-time quantitative PCR（qRT-PCR）.The results showed that expressions of OLIG1,IFNAR1,C1H21orf62,SYNJ1,IL10RB,GART,RXFP2,TWIST2 and E-cadheringenes showed no significant differences in horn and skin between horned and polled yaks （P>0.05）.There were significant difference at transcript levels of IFNGR2，FOXL2，TWIST1 and P-cadherin genes in horn buds between horned and polled yaks（P<0.05）.IFNAR2 and GCFC1 had significant transcription difference in the expression of horn bud and skin between horned and polled yaks （P<0.05）.OLIG2 and ZEB2 had highly significant transcription difference in horn bud expression between horned and polled yaks （P<0.01）.We concluded that OLIG2 and FOXL2 might be the key candidate genes.ZEB2，TWIST1 and IFNGR2 might also involve in the formation of horn. P-cadherin might participate in the development of horn.

The present study was carried out to verify whether EGF inhibited the apoptosis of cumulus cells of yak by regulating the expression of HIF-1α.The cumulus cells of yak were cultured in vitro with the supplement of different concentration of EGF.The expression levels of HIF-1α，Bax and Bcl-2 in different groups were detected by qRT-PCR and immunofluorescence.TUNEL was used to evaluate apoptosis rate.Results showed that：(1) After the addition of different concentration of EGF to the cumulus cells，there would be found a declined mRNA relative expression of HIF-1α and Bax and an increased mRNA relative expression of Bcl-2，with a characteristic manner of concentration-dependent.When the concentration of EGF was as high as 50 ng•mL-1，the mRNA relative expressions of HIF-1α and Bax were the lowest，while that of Bcl-2 was the highest.(2) The protein relative expressions of HIF-1α and Bax were decreased and that of Bcl-2 was increased，with a characteristic manner of concentration-dependent.When the concentration of EGF was as high as 50 ng•mL-1，the protein relative expressions of HIF-1α and Bax were the lowest，while that of Bcl-2 was the highest.(3) Apoptosis detection shown that the apoptosis rate from control group was the highest.It was extremely declined when the concentration of EGF was 25 ng•mL-1 (P＜0.05)，and down to its lowest level when EGF was 50 ng•mL-1 (P＜0.05)，while rose again when EGF was continues to increase.It is concluded from this study that EGF can inhibit the apoptosis of cumulus cells by regulating HIF-1α and may be related to mitochondrial Bax and Bcl-2 apoptotic pathway.

By expressing fibrolytic enzymes targeting cellulose and xylan through transgenic technology，new breeds of monogastric animals with enhanced feed conversion rates could be developed.Here we described the co-expression of cellulase genes EGXA and BGL4，and the xylanase gene XYNB，under the control of EF1α promoter and linked via 2A peptides，in both HEK293A cells and transgenic mice.Recombinant enzyme expression was detected in gastrointestinal tract as determined by both Western blot and immunohistochemistry，and were functionally active in cellulolytic enzyme assays.Notably，all 3 fibrolytic enzymes were highly expressed in the intestine tissue of transgenic mice.In summary，we have demonstrated that the EF1α promoter in conjunction with 2A peptide sequences could successfully mediate the co-expression of EGXA，BGL4 and XYNB genes in transgenic mice.The study presents a feasible method for generating genetically modified animals with enhanced feed conversion ability.

This study aimed to investigate the effects of low protein level and protein compensation on growth performance，nutrients apparent digestibility and metabolism and serum indices of male Hu twin lamb.Sixteen pairs of male Hu twin lambs weaned at day 15 after birth were selected and randomly divided into 2 treatments by paired design.One group was fed normal protein level milk replacer (CP：25%) and starter (CP：21%) as normal protein(NP) group，the other group were fed equal energy but lower protein milk replacer (CP：19%) and starter (CP：15%) as low protein(LP) level group.Milk replacer and starter were fed from wean to day 60.NP and LP groups were fed starter with 21% protein from day 60 to 90.The results showed as follows：body weight of lambs in LP group was significantly lower than that in NP group from day 30 to 90 (P<0.05).Crude protein apparent digestibility and retain nitrogen in LP group were significantly lower than that in NP group (P<0.05)，but nitrogen metabolism rate was higher than that in NP group (P<0.05) from day 50 to 60.Crude fat apparent digestibility and nitrogen metabolism rate of LP group were obviously higher than NP group (P<0.05) from day 80 to 90.At the day of 60，blood urea nitrogen (BUN) concentration and insulin growth factor-Ⅰ in LP group were lower than that in NP group (P<0.05)，but the activity of alkaline phosphatase (ALP) and glutamic-pyruvic transaminase (ALT) were much higher than that in NP group (P<0.05).At 90 days，ALP and ALT activity and growth hormone (GH) level were higher in LP group than that in NP group (P<0.05).In conclusion，during the lactation period，low protein level ration reduced the body weight of lambs，decreased the level of BUN and IGF-Ⅰand apparent digestibility of CP，whereas the N metabolism rate increased.And the body weight of lambs in LP group can not reach the normal weight after 30 days protein compensation.

This study was designed to investigate the effect of novel bio-bedding under slatted floor (BBSF) system on duck house air quality and Muscovy duck production performance.A total of 6 000 Muscovy ducks with 30 days of age were randomly and equally allocated into 3 types of houses，which were BBSF，conventional floor bio-bedding (CFBB) and slated floor (SF) system houses，respectively.The concentrations of harmful gas，dust，airborne lipopolysaccharide (LPS) and microorganisms in house at 08：00，14：00 and 20：00 every 10 days were tested，and Muscovy duck production performances were determined.During experiment period，the concentrations of NH₃，LPS，total aerobe，Escherichia coli，“Salmonella and Shigella” in BBSF were lower than that in CFBB (P<0.05) or SF (P<0.05) at 36，46，56 and 66 days of age，respectively，and PM10 concentration in BBSF was lower than that in CFBB (P<0.05) at 56 days of age.The concentrations of NH₃，total aerobe，Escherichia coli，“Salmonella and Shigella” in BBSF were lower than that in CFBB (P<0.05) or SF (P<0.05) at 08：00，14：00 and 20：00，respectively，and LPS concentration at 14：00 and CO₂ and LPS concentrations at 20：00 in BBSF were all lower than that in CFBB (P<0.05).Duck daily gain in BBSF were higher than that in CFBB (P<0.05)，while livability and feed efficiency (P<0.05) in BBSF were also higher than that in CFBB and SF.The results indicate that，compared with CFBB or SF，BBSF is better in controlling house air environment and improving growing Muscovy duck health and production performance.

 The study aimed to investigate the effect of two different kinds of pregnant sow husbandry systems，electronic sow feeding (ESF) group housing system and the traditional individual stall system，on reproductive performance，welfare levels (behavioral expression，incidence rate of injury，secretion of eyes and snout) and the average cost for each weaned piglet.100，60，120 pregnant sows from each system in the 2 farms with ESF and individual stall systems were chose to compare their reproductive performance，behavior index，scars and eye nasal secretions between the 2 systems within the same farm，and account the cost of weaning piglets.The results showed that，in reproductive performance，there was no significant difference (P>0.05) in annual fodder consumption，days of pregnancy，number of pigs born alive in each litter，number of weaned piglets in each litter and annual parity for sows bred between the 2 systems，but the ESF system significantly increased the weight of weaned piglets (P<0.05).The result showed that individuals in the 2 systems had similar reproductive performance and the ESF system had an advantage on suckling pig growth.In terms of animal welfare，a significant decline in the frequency of standing，lying，sham-chewing，bar-biting，urination，drinking，genital injury and secretions of the eyes and snout (P<0.05) were recorded in the ESF system.There was also a considerable rise in the occurrence of sleeping，fighting，vocalizing，feeding，defecation and sow’s physical injuries (P<0.05) in the ESF system.The results showed that individuals in the ESF system had better sow welfare than the individuals in stall system in most areas，the increase in vocalizing，fighting and physical injuries was because these were more opportunities for individual interaction among sows in the ESF system.In terms of the costs for producing a weaned piglet，there was a significant increase in water and electricity expenses and the depreciation of plant and equipment (P<0.05) in the ESF system，but at the same time，a significant reduction in wage costs (P<0.05)，which resulted in no significant difference in the total costs for breeding weaned piglet between the 2 systems(P>0.05).The findings of this investigation show that there is no significant difference between the ESF group housing system and individual stall system in terms of average weaned piglet cost and reproductive performance，but that the ESF system has improved animal welfare for the sows and can effectively reduce labor costs.However，it also needs well trained and responsible personnel to manage the system properly.

The objective of this research was to develop a novel，high-throughput Genome Lab Gene Expression Profiler Analyser-based multiplex PCR method (GeXP-multiplex PCR) for simultaneous detection of 6 immunosuppressive chicken viruses.Using chimeric primers，6 immunosuppressive chicken viruses，including Marek’s disease virus (MDV)，three subgroups of avian leucosis virus (ALV-A/B/J)，reticuloendotheliosis virus (REV)，infectious bursal disease virus (IBDV)，chicken infectious anaemia virus (CIAV) and avian reovirus (ARV) were amplified and identified by their respective amplicon sizes.The concentration of the chimeric primers，Mg ²⁺ and Taq，and annealing temperature，and time were optimised according to the amplification efficiency of the GeXP-multiplex PCR assay.The assay specificity for each immunosuppressive viral target was individually and simultaneously tested with a mixture of 8 sets of chimeric primers in a multiplex PCR assay.Plasmid DNA and transcribed ssRNA were diluted to a final concentration ranging from 105 copies•20 μL^-1 to 1 copy•20 μL^-1 and then subjected to the GeXP-multiplex PCR assay with 8 sets of chimeric primers，both individually and in pre-mixed solutions.A total of 300 clinic specimens of disease chicken were detected by using GeXP-multiplex PCR.The results were confirmed using independent real-time PCR and sequencing to determine true positives.The specificity and sensitivity of the optimised GeXP-multiplex PCR assay were evaluated，and the data demonstrated that this technique could selectively amplify these 6 viruses at a sensitivity of 100 copies•20 μL^-1 when all 6 viruses were present.Of the 300 examined clinical specimens，190 were positive for immunosuppressive viruses according to the GeXP-multiplex PCR assay.The GeXP-multiplex PCR assay is a high-throughput，sensitive and specific method for the detection of 6 immunosuppressive viruses that can be used for differential diagnosis and molecular epidemiologic surveys.

In order to evaluate the infectious risk of H3N8 avian influenza viruses(AIV) of duck origin to chickens，four H3N8 AIV viruses with HA genes belonged to different genotypes were selected to challenge SPF chickens.It was demonstrated that the four viruses could infect chickens without pre-adaptation，and the four viruses had different replication capacity in chicken’s organs.After challenge，there weren’t obvious clinical symptoms in chickens，and most challenged chickens could shed viruses through orpharyngeal and cloacal routes.In addition，DK/ZJ/S4088/2013 and DK/GZ/S1245/2013 could transmit from infected chickens to healthy contact chickens.In conclusion，the four duck H3N8 AIV viruses in this study posed the potential risk to infect chickens and even could transmit among chickens，and measures should be taken to avoid the contact between chickens and ducks in the breeding and circulation of poultry.

In order to explore the roles of cytokines responding to attenuated duck hepatitis A virus genotype A (DHAV-A) vaccine immunization，the effects of attenuated DHAV-A MY strain on cytokine transcription in ducklings were analyzed.The mRNA transcription levels of 8 cytokines，such as IFN-α，IFN-β，IFN-γ and IL-1β，IL-2，IL-6，IL-8，and TNF-α，were determined by RRT-PCR in liver，spleen and brain of ducklings at different time points of 0.25 0.5，1，2，3 and 5 days post attenuated DHAV-A MY strain injecting 1-day-old SPF ducklings，and the virus loads of DHAV-A MY strain in liver，spleen and brain were also detected by RRT-PCR.The results indicated that the virus load reached the highest level in liver and brain at 2 d time point，and at 3 d time point in spleen post attenuated DHAV-A MY strain injection.The mRNA transcription levels of 8 cytokines in liver，spleen and brain up-regulated very significantly at the corresponding time points when the virus load reached the highest level post DHAV-A MY strain injection (P<0.01).The results indicated that attenuated DHAV-A MY strain could stimulate the transcription of anti-viral cytokines IFN-α，IFN-β，IFN-γ and TNF-α，as well as proinflammatory cytokines IL-1β，IL-2，IL-6 and IL-8，our results enrich the immunization mechanism of DHAV-A MY strain.

The purpose of our research was to study the differential expression proteins of Porcine reproductive and respiratory syndrome virus (PRRSV) infected porcine alveolar macrophage (PAMs) relative to the un-infected control PAMs.Differential proteins in response to PRRSV infection were analyzed by High Performance Liquid Chromatography coupled with tandem mass spectrometry，and then identified using software Max-Quant 1.0.7.4 combining with the protein database PaxDb.Thirty-nine differential proteins were identified in the PRRSV infected group，including thirty up-regulated proteins and nine down-regulated proteins.The up-regulation of SPP1，IFIT3 and IL-8，and down-regulation of STAT3 in PRRSV infected PAMs were verified by Western blot and ELISA.This study provides new evidences for further studies on the molecular mechanism of PRRSV pathogenesis by using LC-MS/MS.

The aim of the experiment was to clarify the role of the capsular polysaccharide in anti-porcine alveolar macrophage phagocytosis of Haemophilus parasuis.The anti-phagocytosis test was conducted by using indirect immunofluorescence method and flow cytometry with H.parasuis SC096 wild-type strain，and capsular deficient strain.The results showed that the H.parasuis SC096 wild-type strains were observed mainly outside the macrophages，while capsular polysaccharide-deficient were observed in macrophages by indirect immunofluorescence experiments.Flow cytometry results showed that the total macrophage phagocytosis rate was 12.51% and swallowed efficiency was 89.61% in the wild-type strain group；the total macrophage phagocytosis rate was 27.18% and swallowed efficiency was 91.06% in the capsular polysaccharide-deficient group.By adding exogenous capsular polysaccharide，total macrophage phagocytosis rate was reduced to 2.72%，swallowed efficiency was reduced to 32.72% in the wild-type strain group；the total macrophage phagocytosis rate dropped to 3.59%，swallowed efficiency was nearly unchanged at 91.36% in the capsular polysaccharide-deficient group.The ability of anti-macrophage phagocytosis of H.parasuis was reduced through removing capsular polysaccharide，which demonstrated that capsular polysaccharide may inhibit the ability of macrophage phagocytosis.

In order to preliminary study the immunologic functions of porcine TRIM11，the cDNA of the target gene was amplified from the cDNA of porcine submandibular lymph node，the protein structure of porcine TRIM11 was predicted and its tissue expression profile was analysed，the cloned porcine TRIM11 cDNA was connected into PiggyBac (PB) transposon vector，then the recombinant vector (PB-TRIM11) was transfected to PK15 cells to establish stably transfected PK15 cell line.The results showed that the coding length of the cloned porcine TRIM11 was 1 407 bp and the sequence code 468 amino acids.Porcine TRIM11 protein was characterized by the presence of the tripartite motif.Tissue transcription profile analysis showed that，the transcription of TRIM11 was higher in fat，lung，whereas the transcription of porcine TRIM11 was lower in heart，ileum，recutum，duodenum.DNA sequence analysis of TRIM11 showed that the cloned TRIM11 cDNA was consistent with sequence from GenBank and the recombinant vector (PB-TRIM11) was constructed.The transfected PK15 cell line stably expressing TRIM11 was established successfully and the transcription of TRIM11 was identified by qRT-PCR.PK15 cell line that stably express porcine TRIM11 and control cells were infected with pseudorabies virus (PRV)，respectively，then the cell supernatants were harvested at various time points after infection and the virus titers were detected.The results showed that the viral TCID₅₀ of the TRIM11 over-expression group was higher than the control all the time.It indicates that the TRIM11 over-expression cells might be more susceptible to PRV.The study provides the experimental basis for the role of porcine TRIM11 in the innate immunity system in the future.

Canine influenza (CI) is an infectious disease caused by canine influenza virus (CIV) infection，which is mainly manifested as acute respiratory symptoms.In order to explore the effect of let-7a on CIV induced cells apoptosis，MDCK cells were infected by CIV in vitro，and AO/EB staining and flow cytometry were used to detect cells apoptosis，and the transcription of let-7a was also determined by real time PCR，and the effect of let-7a on cell apoptosis was evaluated by the transfection of let-7a.The results showed that CIV could induce apoptosis of MDCK cells and downregulate the transcription of let-7a in infected cells，and over-transcription of let-7a could effectively inhibit the virus induced apoptosis.This study showe that let-7a plays an anti-apoptotic role in CIV infected cells，and the low transcription of let-7a promotes apoptosis，and this mechanism may play a role in the respiratory tract injury of CIV infected dogs.

The experiment was conducted to investigate the effect of oral immunization with H5N1 inactivated avian influenza virus (IAIV) and adjuvant levamisole hydrochloride(LMS)，Poly(I：C)，Polyethyleneimine (PEI) respectively on the number and distribution of antibody secreting cells in chicken jejunum and ileum.Chicken were immunized through the oral route with H5N1 IAIV and adjuvants LMS，Poly(I：C) and PEI respectively，IgA and IgG secreting cells were detected by Immunohistochemistry to evaluate the immune effect.The number of IgA and IgG secreting cells in jejunum and ileum induced by PEI plus H5N1 IAIV were substantially (P<0.05) greater than antigen-alone treatment.The number of IgA secreting cells in jejunum and ileum induced by LMS and Poly(I：C) plus H5N1 IAIV were substantially (P<0.05) increased than antigen-alone treatment，the number of IgG secreting cells have no changes.Our study showed that chicken oral immunization with H5N1 IAIV and adjuvant LMS，Poly(I：C) and PEI respectively strengthen intestinal humoral immune level in intestine，but the cost of PEI was cheapest and most effective oral immune adjuvant.

This experiment was conducted to detect the existence of oxytocin receptors (OTR) in the carotid body (CB) of female goats to discuss effects of oxytocin (OT) on the activity of CB.The distribution characteristics of OTR in the carotid body of female goats were studied by using immunohistochemical SP staining.The results showed that，the parenchyma cells and the interstitial tissue were immunolabelled in different degrees.In the type Ⅰ cells and type Ⅱ cells of CB，uniformly strong immunostaining was found in the whole cell membrane and cytoplasm，whereas the nucleus of type Ⅰ cells were not or weekly immunolabelled，the nucleus of type Ⅱ cells were strong immunolabelled.In the interstitial tissue，moderate immunoreactivity products distribute in the vascular endothelial cells and nerve fibers.Image analysis documented a significant difference of OTR expression between the parenchyma cells and the interstitial tissue.The OTRs were widely distributed in CB of female goat，suggesting that oxytocin may exert potential effects on carotid body.

This study aimed to establish a highly efficient and stable gene editing technique mediated by CRISPR/Cas to knock out the targeted gene，and explore its application in chicken preliminarily，and to provide the operation basis for the subsequent poultry genetic edition.We cloned the full-length of C2EIP (chr2，Expression in PGC) gene according to sequence in NCBI Database，and designed 3 gRNAs named gRNA1，gRNA2 and gRNA3 based on the location of APM in the sequence to construct the cas9/gRNA vector.SSA activity assay，T7E1 digestion method and sequencing of TA cloning were used to detect the knock-out efficiency of the gRNA after the cas9/gRNA vector transfected into DF-1.Results of SSA activity assay showed that only cas9/gRNA3 vector could knock out the gene effectively，fluorescence activity which transfected cas9/gRNA3 was twice higher than the control group；Result of T7E1 digestion showed that activity of cas9/gRNA3 was 27%，sequencing of TA cloning results showed there were 8 mutational samples in 30 samples，and the efficiency of gene-knockout was 26%.In this study we established the technology of gene knockout mediated by CRISPR/Cas in chicken，which can be used to konck out genes in DF-1 stably.

This research was conducted to establish the stable rabbit embryonic fibroblast cell lines and identification methods. Rabbit embryonic fibroblasts were isolated from 14-15 d rabbit fetus by trypsin enzyme digesting skin tissues, and passaged in vitro steadily. Morphology observation, growth curve measurement, immunofluorescence and karyotype analysis were applied to cell lines identification. The sex of 2 cell lines were determined by amplifying core sequence of rabbit SRY gene. The results showed that rabbit embryonic were spindle shaped and fibroblasts grew fast in vitro, conforming to the pattern of  “latent phase-logarithmic growth phase-stagnate phase”. Immunofluorescence assay showed that vimentin and fibronectin staining were positive, while cytokeratin staining was negative, indicated the high purity and activity of rabbit embryonic fibroblast cells. The result of chromosome analysis showed that 80% ploidy of one cell lines maintained normal (2n=44) when cultured up to passage 5. For cell lines sexing, SRY-PCR analysis suggested the 2 determined cell lines were male. These results indicated that our research could be used to establish rabbit embryonic fibroblast cell lines and determine sex, providing adequate and reliable fibroblast cells for transgenesis, cloning and iPS rabbit research.

The objective of this study was to express and purify the nucleocapsid (N) protein of Schmallenberg virus (SBV) with the native conformation，and to prepare its monoclonal antibodies (McAbs).To this end，the SBV-N gene with an inserted coding sequence for an N-terminal hexahistidine (6×His) tag was cloned into the pFastBac^TM 1 vector.The resulting recombinant donor plasmid，pFastBac-His-SBV-N，was then transformed into DH10Bac E.coli competent cells.After selection with the blue/white screen，the recombinant bacmid，rBacmid-His-SBV-N，was transfected into Sf9 insect cells to generate recombinant baculoviruses that express the His-SBV-N fusion protein.After amplifying the recombinant baculoviral stocks on Sf9 cells，His-SBV-N fusion protein was purified using nickel nitrilotriacetic acid (Ni-NTA) agarose.The purified protein was then used to immunize BALB/c mice to prepare SBV-N-specific McAbs，and an indirect double-antibody binding ELISA was applied to detect whether the generated McAbs recognize different antigenic sites.The screened McAbs that recognize different antigenic sites were labeled with horseradish peroxidase (HRP) using the sodium periodate oxdization method.Finally，four McAbs (2A11，2E1，4H11 and 6E12) recognizing different antigenic sites were successfully obtained and labeled with HRP.The results of isotype identification demonstrated that 2A11 belongs to IgG1，and 2E1，4H11 and 6E12 belong to IgG2b.Indirect immunofluorescence assays revealed that all of the four McAbs reacted with the BHK-21 cell line stably expressing the SBV-N protein (BHK-21-EGFP-SBV-N).Western blot analyses further showed that the four HRP-conjugated McAbs reacted positively with the His-SBV-N fusion protein.Taken together，the successful preparation of His-SBV-N fusion protein and its McAbs provide valuable biological materials that can be used in the serological diagnosis of Schmallenberg disease.

Recently，our laboratory identified a novel astrovirus from fecal samples of diarrheic yak，the aim of this study was to develop an RT-PCR assay for detecting the novel yak astrovirus (yak AstV).Firstly，one pair primers were designed to amplify ORF2 gene fragments (630 bp in length) of yak AstV.By sequence analysis，one pair primers of RT-PCR assay were then designed for detecting yak AstV.We obtained 13 ORF2 gene fragments from 20 fecal samples of diarrheic yak，which shared a homology of 99.3%-100% within fragments，but only a homology of 59.5％-80.8％ with bovine astrovirus (BAstV).The RT-PCR assay developed in this study was specific to yak astrovirus，no amplifying for BAstV and other pathogens tested in this study.The detection limit of viral nucleic acid of the assay was 57.3 fg•μL^－1.The assay was significantly better in detecting yak AstV compared to the other two reported assays for detecting bovine astrovirus.The detecting results of clinical samples by this assay showed that the positive rates of yak AstV were 55.5% (76/137) in diarrheic fecal samples and 7.7％ (2/26) in healthy fecal samples.Compared to healthy samples，yak AstV was significantly more prevalent in diarrheic fecal samples (P<0.01).In conclusion，ORF2 gene sequences of yak AstV cloned in this study varied a lot compared with those of BAstV，but were highly conserved within yak AstV species.Thus，the RT-PCR for detecting yak AstV based on the ORF2 sequences of yak AstV greatly improved the detection rate of yak AstV，providing a useful tool for detection of this novel virus and epidemiological investigation.Meanwhile，the results of this study indicated that yak AstV might be associated with diarrhea in yak calves.

In order to clarify the taxonomic status of Babesia spp，the COⅠgene were sequenced from 11 isolates of 7 species reported in China；The phylogenetic tree were constructed using COⅠand 18S rRNA sequences downloaded from GenBank and this study，and also the two phylogenetic trees were compared.As result shown，the length of COⅠgene was 935-999 bp，and the COⅠgene had more variation and informative sites than 18S rRNA.The taxonomic status of bovis Babesia spp.based on COⅠand 18S rRNA shared high similarity.However，there were also some differences from two phylogenetic trees.Such as，the COⅠgene can differentiate Babesia spp and Theileria spp clearly.To 18S rRNA，it was more difficult.The taxonomic status based on COⅠgene of Babesia sp.from Xinjiang was more reasonable.The information of two phylogenetic tree suggested that the Babesia motasi isolated from different region may represent different subspecies.This study supported the candidate gene of molecular taxonomy of Babesia spp.