In order to clarify the effect of 17 beta-hydroxysteroid dehydrogenase 1 (HSD17B1) gene on the reproductive performance of buffalo,in this study,buffalo HSD17B1 gene was cloned by 3'-RACE,analyzed by bioinformatics,and studied with eukaryotic vector construction and cell transfection technology.The results showed that the coding region of buffalo HSD17B1 was 954 bp,3'-UTR was 58 bp,encoded 317 amino acids.The buffalo HSD17B1 gene shared 100%,100%,92%,94%,87%,87% and 87% of similar nucleotide sequence with that of Bos taurus,Ovis aries,Sus scrofa,Equusca ballus,Canis lupus,Loxodonta africana and Homo sapiens,respectively.Phylogenetic tree analysis showed that HSD17B1 gene was highly conserved in different species and evolution.HSD17B1 protein was weakly acidic,without signal peptide,located inthe cytoplasmic,and with the presence of type1_17beta-HSD-like_SDR_c,PRK05993,LPOR and FabG domains.The buffalo HSD17B1 eukaryotic expression vector was successfully constructed,after transfected into 293T cell lines,HSD17B1-EGFP fusion protein was detectable.In a word,the success cloning and construction of eukaryotic expression vector of buffalo HSD17B1 gene,provided an important reference for surveying the regulation mechanism of HSD17B1 gene during buffalo folliculogenesis and embryogenesis.

The specific primers were designed according to Ovis aries DRA gene sequence deposited in GenBank and the multiple cloning site of the plasmid pYD1,which was a vector used for protein surface display on Saccharomyces cerevisiae.The gene encoding DRA was amplified by PCR using the genomic RNA of Ovis aries.The 762 bp fragment was cloned and released in GenBank and registration number was KR422362.The PCR product was inserted into the yeast surface display plasmid vector pYD1 by double enzyme digestion.It was indicated that DRA gene was successfully integrated into the genome.Dot mutation was made at both ends of exon 2 in DRA gene for making restriction enzyme cutting site and design the exon 2 specific primers according to mutated Ovis aries DRA gene sequence.Sequenced exon 2 amplification products based on DNA pooling of sheep large sample template was analyzed the polymorphic loci.The polymorphic exon 2 246 bp fragment was obtained by double enzyme digestion and connected to surface display restructuring mutation carriers pYD1-DRA by the same double enzyme digestion,and then we successfully constructed yeast surface display libraries.We transformed it into Saccharomyces cerevisiae EBY100 cell.Yeast monoclone was identified by PCR amplification and sequencing,and we confirmed that DRA gene had been integrated into Saccharomyces cerevisiae genome.After galactose induced,it was detected that DRA gene library had been successfully demonstrated on the yeast cell surface under the fluorescence microscope by immunofluorescence method.

To establish a rapid and accurate multiplex PCR method for the clinical detection of three main Gram-negative bacilli from livestock and poultry in Guizhou province,three pairs of specific primers were designed and synthetized according to 23S rRNA gene of Escherichia coli(E.coli),KMT gene of Pasteurella and invA gene of Salmonella.In this study,A mutiplex PCR reaction system was established for detecting the three pathogens at the same time,and the reaction system was optimized and it's performance was evaluated.The results showed that the best concentration of primers was 1.0,1.5and 1.0 μmol/L,respectively,and the best annealing temperature was 56 ℃.The results of performance evaluation showed that the method had good specificity and sensitivity,the sensitivity of Salmonella could reach to 1.44 pg/μL,was the highest.70 Gram-negative clinical isolated strains were detected by the multiplex PCR and it was proofed that the method could identify the three pathogens rapidly and accurately,and it could provide effective technical for the rapid clinical diagnosis and epidemiological survey.

The aim of this study was to investigate the differential expression genes induced by ApoCⅢ,and study the function of ApoCⅢ.Porcine aortic vascular endothelial cells were successfully isolated using enzyme digestion,and then screened the differential expression genes induced by ApoCⅢ using the Solexa high-throughput sequencing technology.The results showed 647 differential expression genes,including 390 up-regulated genes and 257 down-regulated genes.The qRT-PCR results verified that the gene expression results from Solexa sequencing data were reliable.GO and Pathway analysis showed that the function of differential expression genes were related to immune response,cell apoptosis and death.These findings suggested that ApoCⅢ affected the physiological function of porcine aortic endothelial cells by the molecular pathways of inflammation,cell adhesion and apoptosis,which provided a theoretical basis for further understanding the molecular mechanisms of atherosclerosis caused by ApoCⅢ.

By the infection of Brucella virulent strain and attenuated strain in mice macrophage RAW264.7,the assay was aimed to explore the relationship between NF-κB signaling pathways and Brucella virulent strain and attenuated strain in intracellular survival.Use different MOI Brucella (2308,RB51,16M and M5) to infect mice macrophage RAW264.7,after 0,4,8 and 24 h infected,cracking cell and collecting supernatant,we detected the effect of Brucella on activation of NF-κB signaling pathway by Western blotting.Different concentrations of NF-κB signaling pathway inhibitor were incubated with mice macrophage RAW264.7,with different multiplicities of infection (MOI) of Brucella infecting cells,ELISA kits to detect the expressions of TNF-α,IL-1β and IL-6 cytokine;At the same time,count the number of intracellular bacteria of CFU.The results showed that rough cattle Brucella strains RB51 could strongly activate NF-κB signaling pathway,smooth cattle Brucella strains 2308 was weak in the activation;At the same time,the activation of NF-κB signaling pathway was concentration dependent.When the MOI was 80,infection time was 8 h,NF-κB activation degrees of rough cattle Brucella strains RB51 and smooth cattle Brucella strains 2308 were the strongest,and this pathway was involved in producing TNF-α and IL-6;NF-κB signaling pathway inhibitor BAY11-7082 affected Brucella intracellular survival.So rough cattle Brucella strains RB51 intracellular survival and NF-κB signaling pathway activity were closely related.The results laid the foundation for the further study of Brucella intracellular pathogenesis,also provided scientific basis for the research of new drugs to Brucella,and prevention and treatment of brucellosis.

In order to understand the differential expression among tissues or breeds of grazed goat,β-actin gene was used as control,the expression levels of melanocortin-4 receptor (MC4R) mRNA in 7 tissues of Qiandongnan small Xiang goat,Guizhou White goat,Guizhou Black goat,Qianbei Ma goat and Nanjiang Yellow goat were detected by Real-time fluorescence quantitative PCR technique with double standard curve method.The results showed that MC4R mRNA was expressed in all 7 tissues of 5 grazed goat breeds,the expression levels of subcutaneous fat and kidney were the highest,the expression levels of liver was the second,the expression levels of lung and heart were the third,and the expression levels of semimembranosus muscle and longissimus muscle were the lowest.There were no differences of the expression level of MC4R mRNA in liver among different breeds.In subcutaneous fat,kidney,lung,heart,semimembranosus muscle and longissimus muscle,the highest expression levels of MC4R mRNA were all observed in Guizhou White goat.The results suggested that for grazed goats,the expression levels of MC4R mRNA existed tissue differences,and except for the liver,it existed breed differences,too.This research laid a basis for molecular marker assisted selection of growth traits,carcass traits,and meat quality traits of grazed goat.

In this study,through a series of screening and optimization of reactions condition,an indirect ELISA method was developed for detections the antibodies against classical swine fever virus (CSFV) with the purified recombinant CSFV E2 protein.The results showed that the optimal concentration for coating antigen was 1.0 μg/mL and incubated at 37 ℃ for 1 h.The proper serum sample was diluted by 1:200 and the sealing time was 37 ℃ for 1 h.Enzyme labeled second antibody was diluted by 1:10 000 and the reaction time was 45 min incubating at 37 ℃.The D450 nm<0.30 of sample defined as negative of CSFV antibody in the serum sample.The intra-batch and inter-batch variation coefficients were 4.8% and 6.9%,respectively.It indicated that the method in this study had a high stability.Specific test showed that the indirect ELISA had no crossing reactions with the antibodies against PRV,PRRSV,PCV and PPV.80 serum samples were detected in this study,the positive coincidence rate of indirect ELISA was 83.58%,the negative coincidence rate was 76.92%,and the total coincidence rate was 80.25% compared to the results of import blocking ELISA kit.The results suggested that the established indirect ELISA method in this study had an excellent clinical application.

The aim of this study was to determine the effect of vitamin E on Cx43,the mechanism and function of vitamin E on bovine granulosa cells apoptosis and proliferation.In this study,granulosa cells were isolated from bovine ovary and cultivated in vitro by adding different concentration of vitamin E (0,25,50,100,200 and 500 μmol/L) for 24 h.After cultured,apoptotic cells were detected by FCM,mRNA expression levels of BCL2/BAX、P53 and Cx43 genes were determined by Real-time PCR and cell proliferation was detected by CCK8.The results showed that compared to control group,100 μmol/L vitamin E could significantly inhibit the apoptosis of granulosa cells (P<0.05).Real-time PCR detection results showed that vitamin E significantly changed the mRNA expression levels of BCL2/BAX,P53,Cx43 genes (P<0.05).Vitamin E could significantly improve granulosa cells proliferation when granulosa cells were treated for 24 and 36 h (P<0.05).The results provided a theoretical basis on further analysis for studing the influence mechanism of vitamin E on oocytes development and maturity,and improvement of female animal reproduction by influencing granulosa cells proliferation and apoptosis.

To screen applicable PCR diagnostic primers of Neosporacaninum based on Nc2 and Nc5 genes,two pairs of primer were designed according to the Nc2 and Nc5 genes sequences conserved region and"Quarantine Protocol For Neosporosis"(SN/T 3499-2013)were applied to verify the characteristic of primers.14 Holstein and 19 Simmental cattles blood DNA were detected using these primers by PCR to select specific primers which would be used to establish the new detection method and understand the neosporiasis infection rate of different local cattle farms.The results showed that 105,128 and 231 bp gene fragments were amplified using three pairs of primers,which were consistent with the expected fragment size.The F1/R1 and SN/T 3499 F/SN/T 3499 R minimum detectable amount both were 19.9 fg/ μL and the minimum amount of F2/R2 was 199 fg/ μL,indicating that F1/R1 and SN/T 3499 F/SN/T 3499 R were more sensitive than F2/R2.The bands were bright when used F1/R1,F2/R2 primers to amplify 19.9 pg/μL and 199 fg/μL DNA samples,proving two primers had good repeatability.6 positive samples were detected among 33 blood samples,and the positive rate was 21.43% and 15.79%,the recombination rate was 100%.It showed that F1/R1 and F2/R2 primer were both suitable to detect eneosporosis by PCR and the method was preliminary established.The condition of local cattle Neosporacaninum infections were learned.This study could provide a scientific basis for the effective prevention and control of neosporosis.

The experiment was carried out to establish a method for preparation and identification of N-glycolylneuramic acid IgY antibody.Neu5Gc was linked to carrier proteins by carbodiimide(EDC)method.After the immunization of immunogen to laying hens,the IgY antibody was gained.Neu5Gc-IgY was analyzed and identified by ELISA.The results of UV and agarose gel electrophoresis showed that the artificial antigens of Neu5Gc were successfully synthesized.The development of the IgY antibody titer was monitored by indirect ELISA.The result showed that the IgY antibody was generated at 7th day after the first immunization.The titer was gradually increased and reached its peak (1:10 000) at 63th day after the first immunization.The high titer of the IgY antibody maintained through the whole observation period.The sensitivity of anti-Neu5AGc IgY antibody was detected by the competitive blocking ELISA.The linearity about the first chicken of the standard curve showed good,the liner regression equation was y=30.28x+16.923,R²=0.9581.The IgY antibody gained in this study laid the foundation for the establishment of indirect ELISA immunoassay method for detecting Neu5Gc in red meat,milk and tumor tissues.

This experiment was aimed to study the antigenicity of prokaryotic expression of the N gene fragment of porcine epidemic diarrhea virus (PEDV),and lay a foundation for establishing an indirect ELISA method of PEDV.The N gene segment was amplified by RT-PCR,then the recombinant plasmid with the vector pET-30a(+) was constructed,which was induced by 0.5 mmol/L IPTG at 37 ℃ for 4 h.Furthermore,the expressed product was analyzed by SDS-PAGE and Western blotting.Sequencing results proved that recombinant plasmid was correctly constructed.The result showed that the PEDV polyclonal antibody could specifically bind to PEDV N protein,which indicated that the recombinant fusion protein had excellent immunogenicity.A prokaryotic expression vector for the fragment of N protein was successfully constructed in this study,which laid a foundation for the development of diagnosis of PEDV.

In order to produce antimicrobial peptide PBD-1 with bioactivity in Pichia pastoris,according to published amino acid sequence of PBD-1 and the partiality codon of yeast,the PBD-1 gene was amplified by SOE-PCR and cloned into pPIC9K to construct a recombinant expression vector pPIC9K-PBD-1.The recombinant vector was linearized by SacⅠ,and then transformed into SMD1168 by electroporation.Positive yeast expression strain was obtained by PCR.The antimicrobial peptide PBD-1 (approximately 4.5 ku) was expressed by methanol induction.Antibacterial activity assay showed that the antimicrobial peptide PBD-1 had better antibacterial activity against E.coli,S.aureus and B.subtilis.

In this study,sequence variation of GRA25 genes among 22 Toxoplasma gondii (T.gondii) strains from different hosts and geographical locations were examined.The complete GRA25 genes from 22 T.gondii isolates were amplified,sequenced,and nucleotide variations were determined.Phylogenetic analysis among the different T.gondii isolates were conducted using maximum parsimony (MP) and maximum likelihood (ML) methods.The biological characteristics of the protein GRA25 of the T.gondii RH strain were predicted using bioinformatics software.The sequences of all the examined T.gondii strains were 939 or 948 bp in length.Sequence comparison of all 22 GRA25 sequences identified 82 variable nucleotide positions (0 to 4.4%).The results of phylogenetic analysis showed that strains belonging to the classical typeⅠand Ⅱ could not group into their own branches based on the GRA25 sequences.Bioinformatics analysis revealed that the protein GRA25 contained 7 hydrophobicity regions,10 alpha regions,3 beta sheets,8 random coils and 8 linear B-cell epitopes.These results suggested that the GRA25 gene was not an ideal genetic marker for population genetic study of T.gondii strains,but it might represent a good vaccine candidate against toxoplasmosis.

This experiment was conducted to study the effects of supplemental different levels of sugar beet pulp on the nutrients of digestion and metabolism in Yanqi horse.12 Yanqi horses were randomly divided into three groups according to performance,body weight,body measurement and age.The groups were referred to as the control group,group Ⅰ and group Ⅱ,each group with 4 Yanqi horses.Yanqi horses in the control group,group Ⅰ and group Ⅱ were supplied with 0,0.6,1.2 kg/d of sugar beet pulp on the condition of feeding the same basal diet and roughage (oat straw).The total duration of the experiment was 20 days,the first 15 days of the experiment was the preliminary period,and the formal experiment lasted for 5 days.The results showed that compared with the control group,the apparent digestibility of OM,NDF,ADF and digestibility amount of NDF,ADF in group Ⅱ was extremely significantly improved (P<0.01),the digestibility amount of crude protein,calcium and the apparent digestibility of OM in group Ⅱ was significant difference (P<0.05);The digestibility amount of NDF,ADF of group Ⅰ was increased by 8.19% (P<0.05) and 9.43% (P<0.05),respectively,and the apparent digestibility (amount) of OM,crude protein,calcium were no significant difference (P>0.05).Compared with group Ⅰ,the apparent digestibility (amount) of OM,NDF,ADF of group Ⅱ were significantly or extremely significant difference (P<0.05;P<0.01),but the apparent digestibility (amount) of crude protein,calcium were no significant difference (P>0.05).Compared with the control group and group Ⅰ,the digestible engergy of group Ⅱ was increased by 14.92% (P<0.01) and 11.23% (P<0.01),but no significant difference was observed between group Ⅰ and control group (P>0.05);The metabolic engergy of group Ⅱ was increased by 11.73% (P<0.05) and 14.83% (P<0.05),respectively.Therefore,the digestion amount of OM,CP of Yanqi horse and the digestion amount and the apparent digestibility of NDF,ADF could be significantly or extremely significantly improved by the supplementation of 1.2 kg/d of sugar beet pulp,and the ability to use the nutrients were obviously improved for Yanqi horse.

The paper was to study the correlation between body weight and body size indexes of 11 to 14 months old growing dairy buffalo,which could provide a scientific reference to estimate the weight of dairy buffalo.Seventy six dairy buffalo with the age of 11 to 14 months were selected from the buffalo breeding farm in Guangxi buffalo research institute,body weight and body size indexes were measured according to normal means.Multivariate regression and polynomial regression were analyzed by the function of LINEST in Excel and correlation analysis and principal component analysis were used to analyzed the body weight and body size indexes by SPSS 17.0.The results showed that all the indexes of the body size traits factors affected the buffalo's body weight in different degree.There were extremely significant positive correlation between body weight and body height,body slanting length,high cross height,heart girth,abdominal circumference,chest width,chest depth,rump length,rump width and cannon circumference (P<0.01),in which heart girth was the most relevant (P=0.823),followed by the abdominal circumference,body slanting length,chest depth,cannon circumference and so on.The complex correlation coefficient of multiple regression equations between the body weight and the body size indexes were obtained by polynomial regression,and there was a strong linear relationship between body weight and body size indexes of 11 to 14 months old dairy buffalo,and body size indexes could be used to estimate the body weight of dairy buffalo.From the first principal component with the characteristic roots and the cumulative contribution ratio,we could know body weight,heart girth,body slanting length,abdominal circumference and cannon circumference had a big feature vector,and also had a big contribution to the first principal component,which basically reflected dairy buffalo's body structure of 11 to 14 months old,and this could be called body factor,the second principal component was called rump width factor and the third principal component was called body height factor.

Two experiments were conducted to investigate the effect of different dietary concentrate to forage ratios (C/F) on the growth performance,serum biochemical indexes and economic benefits of Dorper sheep.TestⅠ:40 weaned lambs with the 20 to 35 kg body weight were divided into 4 groups with 10 sheep in each group and provided 4 different C/F diets:group A (40:60),group B (50:50),group C (60:40),group D (70:30) and the experiment lasted for 70 d;TestⅡ:40 growing sheep with the 30 to 45 kg body weight were divided into 4 groups with 10 sheep in each group and provided 4 different C/F diets:group E (35:65),group F (45:55),group G (55:45),group H (65:35) and the experiment lasted for 70 d.The results showed as follows:In the testⅠ:①With the increase of C/F,the ADG of rams increased,but that of ewes was increased initially and then decreased,the ADG of group A in male and female were significantly lower than other three groups (P<0.05),and there were no significant differences in group B,C and D (P>0.05);ADFI of male and female were reduced significantly (P<0.05);F/G was decreased significantly of ram (P<0.05),while that of group B was significantly lower than the other three groups of ewe (P<0.05).②There were no significant differences of TP,TG and Glu in 4 groups (P>0.05),but C/F had different effects on other serum components.③The average net profit in group D of rams was 53.03% higher than group B,and 35.33% higher than group C,while that in group B of ewes was 61.76% higher than group C,and 54.49% higher than group D.In the testⅡ:①With the increasing of the C/F,the ADG of the growing rams was increased gradually,but group G of ewe was significantly higher than group E (P<0.05),and there were no significant differences in group F,G and H (P>0.05).ADFI of male and female was reduced significantly (P<0.05);F/G was decreased significantly of ram (P<0.05),while that of ewes was decreased initially and then increased,group G was significantly lower than the other three groups (P<0.05).②There were no significant differences in TP,TG and Glu in 4 groups (P>0.05),but C/F had different effects on other serum components.③The average net profit in group H of ram was 69.49% higher than group F and 52.13% higher than group G.While that in group G of ewe was 1 282.61% higher than group F.Under the conditions of this experiment,the appropriate C/F was 60:40 to weaned male lambs,while 50:50 to weaned female lambs,and the appropriate C/F was 50:50 if they were mixed fed.TestⅡ showed that the appropriate C/F was 55:45 to both male and female growing sheep.

This experiment was aimed to analyze the amino acids composition and nutrient value of Black Angus raised in Ujinmqin grassland and provide basic data for feeding,cross breeding,and meat quality evaluation.Highrib,tenderloin,ribeye,nates meat,topside,shoulder clod and brisket samples were collected from seven Australian imported Black Angus raised in Ujinmqin grassland with 500 kg?38 kg body weight.The content and composition of amino acids were determined and then nutritent value were analyzed using FAO/WHO amino acids pattern.The results showed that the moisture and crude fat of samples were 68.73% and 30.56%.There were total 17 kinds of amino acids were determined in the samples and UAA,SAA and BAA were abundant.The ratios of these three abundant amino acids to total amino acid (TAA) were 25.8%,32.7% and 41.6%,respectively.The EAA content,EAA/TAA,EAA/NEAA and the EAA scores were all higher than FAO/WHO amino acids pattern.The amino acids content of ribeye,tenderloin and highrib were higher than that of other parts of muscle.In conclusion,the meat of Black Angus raised in Ujinmqin grassland had abundant composition and high content of amino acids,which were able to provide enough nutrient elements for human beings.

In the absence of clinical symptoms,dairy cows with subclinical endometritis are defined as sustainably extensive infiltration with the polymorphonuclear cells (PMN) in endometrial samples,and it often lead to dairy cow with repeated infertility.In China,the infertility proportion of dairy cows is 50.62% caused by endometritis,in which 90% is caused by subclinical endometritis.It is difficult to be observed during the early stage of subclinical endometritis,and the best treatment time is delayed in the most cases causing huge economic losses.So it has become a research hotspot to make correctly and timely diagnosis of this disease.At present,the main diagnosis methods of dairy cows subclinical endometritis are vagina endoscope method,cell brush,biopsy technology,ultrasonic diagnosis technology,leukocyte esterase method and so on.However there is no uniform criterion.Different diagnosis methods of subclinical endometritis were summarized,analyzed and discussed in this paper to provide the references for the subclinical endometritis diagnosis more accurately and earlier,and reduce the losses of dairy farms.

This study investigated the effects of replacing partial basal diet with wild buckwheat on fatty acids and amino acids in longissimus dorsi muscles of Congjiang Xiang pigs.Forty Congjiang Xiang pigs with 20 kg initial body weight,were randomly assigned to 4 groups,10 heads per group.The pigs in control group were fed the basal diet.In the other groups (groupⅠ,Ⅱ and Ⅲ),diets were substituted with fresh wild buckwheat at 10%,20% and 30% on the early fattening stage and 20%,30% and 40% on the late fattening stage,respectively.After feeding trial,three pigs from each dietary treatment were slaughtered to determine the fatty acids and amino acids in longissimus dorsi muscle.The results showed that:①The partial substitution of basal diet with wild buckwheat had some effects on fatty acids in porcine muscle.Compared with the control group,the content of linoleic acid in the group Ⅰ,the content of linolenic acid in group Ⅰ and Ⅱ,the content of peanut diene acid of group Ⅱ and arachidonic acid of group Ⅲ were increased by 54.69%,43.57%,62.50%,25.00% and 127.10%,respectively.The saturated fatty acid (SFA),polyunsaturated fatty acid (PUFA)and essential fatty acid (EFA)of all testing groups were higher than that of control group (P>0.05).②The amino acids were affected in some degree by the substitution rate of wild buckwheat.The content of asparaginic acid in control group was significant lower than that in group Ⅰ(P<0.05) and extremely significant lower than that of group Ⅲ (P<0.01).The content of glycine in group Ⅲ was significantly higher than that of groupⅠ and Ⅱ (P<0.05),higher than that of control group (P>0.05).The content of methionine and lysine in group Ⅲ were significantly higher than that in control group and group Ⅰ (P<0.05),respectively.The EAA/TAA in group Ⅰ was significantly lower than the other groups(P<0.05).In conclusion,partial substitution of basal diet with wild buckwheat could increase the contents of PUFA and EFA,and improve the amino acid profile of Congjiang Xiang pigs.

Yak mainly lived in the Qinghai-Tibetan plateau in Qinghai province of China and lived on grasses.Many studies had shown that yak's ability to digest cellulose was far higher than Yellow cattle and dairy cows.Because there were significant differences in the rumen microorganisms among these three kinds of animals.There was a large microbial population in the yak rumen that could degrade cellulose.In this paper,the characteristics of microorganism,influence factor and the mechanism of cellulose degradation in yak were summarized.In addition,the brief introduction of the yak rumen microbial's possible applications had carried on in order to provide theoretical basis and strengthen the use of cellulose degradation bacteria in the areas such as modernization of traditional Chinese medicine fermentation and so on.

Epoxyeicosatrienoic acids (EETs) are synthesized from arachidonic acid by cytochrome P450 epoxygenases.They modulate ion transport and gene expression,producing vasorelaxation as well as antiinflammatory and cell proliferation effects.Many of the functional effects of EETs occur through activation of signal transduction pathways and modulation of gene expression,events probably initiated by binding to a putative cell surface EETs receptor.However,EETs are rapidly taken up by cells and are incorporated into and released from phospholipids,it suggests that some functional effects may occur through a direct interaction between the EETs and an intracellular effector system.EETs synthesis,metabolism,mechanism of action are reviewed in this paper.

The study was aimed to extract stachydrine hydrochloride in leonurus by HPLC-ELSD,and establish hydrochloride stachydrine determination methods for providing theoretical basis of leonurus extraction process optimization.The chromatographic conditions:venusil HILIC hydrophilic column;Mobile phase was acetonitrile -0.2% acetic acid solution (volume ratio 80:20);The flow rate was 1.0 mL/min;And the column temperature was 30 ℃;The detector was ELSD and drift tube temperature was 80 ℃ with the atomization chamber temperature 50 ℃ and carrier gas volume flow 1.0000 mL/min.The results showed that there was a good linear relationship when the stachydrine hydrochloride content was 0.49 to 4.91 μg (R²=0.9993);There was the highest rate of active ingredients extraction of when the extraction time was 1 h,material to water ratio was 20:1 and extracted for 2 times.Combined with production practice,the final optimization scheme was that the extraction time was 1.5 h,material to water ratio was 20:1 and extracted for 2 times.In summary,the determination of stachydrine hydrochloride in leonurus should use HPLC-ELSD method which had high specificity,good stability,response and reproducibility and easy to handle.This method could provide the theoretical basis for optimizing the leonurus extraction process.

With the deepening of modern biological science,obstetrics,pharmacology and toxicology intersect penetration with each other,thus gradually make the development of reproductive toxicology.Reproductive toxicology include two parts:Reproductive toxicology and developmental toxicology,it is a method focused on exploring whether there are influences of environmental and drugs factors on parental reproductive functions and the development of offspring.In this paper,the injury effects of test drugs on the reproductive cytogenesis,fertilization of egg cell,embryogenesis,pregnancy,childbirth,breast-feeding and development of newborn pups were summarized.

In order to investigate skeletal muscle development mechanism,the expression of follistatin (FST) and fibromodulin (FMOD) gene were comparatively analyzed in fetus different skeletal muscles and different stages between Ujumqin sheep and Texel sheep during early stage.Two gene expression were analyzed including longissimusdorsi,semimembranosus,semitendinosus quadricepsfemoris and biceps femoris muscle were compared by variance analysis methods of four stages (85,100,120 and 135 d).The results of quantitative Real-time PCR showed that the expression of FST gene showed a tendency to increase with the addition of stage,and the expression of FST gene in Texel sheep was higher than that in Ujumqin sheep in semitendinosus (100,120 d),semimembranosus (120 d),and biceps femoris (120 d) (P<0.05).The expression of FST gene in Texel sheep was extremly higher than that of Ujumqin sheep in semitendinosus (100,135 d),longissimusdorsi (85 d),quadricepsfemoris (100 d) and biceps femoris (100 d) (P<0.01).As a whole,the expression of FMOD gene was the highest in quadriceps femoris,and was lower in other four muscles,it's expression in Ujumqin sheep was higher than that in Texel sheep.It was inferred that FST and FMOD genes might be related to muscle development during early stage.

The paper was aimed to investigate the polymorphism of copy number variation (CNV) in different pig breeds.Three CNV regions of CNVR91,CNVR92 and CNVR143 were chosen from the porcine SNP60 chip genotyping results.The polymorphisms of three CNVs were determined by Real-time quantitative PCR method,taking five pig breeds as samples,including Yorkshire pig,Xiang pig,Kele pig,Nuogu pig and Rongchang pig breeds.The results showed that the dominant status of CNVR91 was loss in Xiang pig,while it was normal in other four pig breeds.The major type of CNVR92 was deletion in Xiang pig,Yorkshire pig,Kele pig and Rongchang pig breeds,with a high normal percent in Nuogu pig.For CNVR143,the dominant event was gain in Xiang pig and Nuogu pig breeds,but it was not diverse in other three pig breeds.These results indicated that three CNV regions emerged with polymorphism in five pig breeds,which might have effects on gene expression in CNV regions and physiological function by dosage effect especially in Xiang pig,Nuogu pig and Kele pig breeds.

The aim of this study was to investigate the effects of MSTN and MyoG genes on goose skeletal muscle growth.In this study,MSTN and MyoG genes expression were detected in breast and leg muscle of Zi and Rhine goose by Real-time PCR,and the correlations between genes expression levels and carcass traits were investigated.The results showed that the breast muscle weight and breast muscle rate of Rhine goose were extremely significant higher than Zi goose (P<0.01).MSTN and MyoG mRNA expression in breast muscle of Zi goose were significantly higher than that of Rhine goose,and the mRNA level of MSTN in leg muscle of Rhine was extremely significant higher than that of Zi goose (P<0.01),there was no significant difference of MyoG mRNA between Zi goose and Rhine goose (P>0.05).There was extremely significant difference between MSTN mRNA expression in breast muscle and leg muscle of Zi goose (P<0.01).MSTN mRNA expression in leg muscle was significantly higher than that of breast muscle of Rhine goose (P<0.05).There was extremely significant difference between MyoG mRNA expression in breast muscle and leg muscle of Zi goose and Rhine goose (P<0.01).There was a extremely significant negative correlation between MSTN mRNA expression in breast muscle with body weight,breast muscle weight and breast muscle percentage (P<0.01).There was a extremely significant positive correlation between MyoG mRNA expression in breast muscle and leg muscle with body weight (P<0.01).MSTN and MyoG gene might have positive and negative regulation effect on muscle growth.

In this study,CDS sequence of DRB3 gene exon 2 in Tianzhu White yak and Gannan yak were analyzed,a clean single chain was obtained to get the accurate haplotype by direct sequencing and amplification refractory mutation system (ARMS) method,the double chain of high heterozygosity was typed.The results showed that 65 SNPs and a codon insertion and deletion were discovered,all together separated to 30 haplotypes,in which seven haplotypes were found firstly,it should be peculiar to Tianzhu White yak and Gannan yak.Compared with published bovine DRB3 gene exon 2 haplotypes,the analysis results showed Tianzhu White yak and Gannan yak shared haplotypes of cattle (Bos taurus) and zebu (Bos indicus),which meaned they might crossed with cattle (Bos taurus) and zebu (Bos indicus).PAML analysis showed that 4,25,30,53,59,62,63,66,78 amino acid sites were affected by positive selection,and mainly occurred in Tianzhu yak,it had a certain relation with breeding history of Tianzhu White yak.

This experiment aimed to study the effect of brilliant cresyl blue (BCB) on in vitro maturation of pig oocytes and the developmental capacity of pig SCNT embryos.The cumulus-oocyte complexes (COCs) were stained with different concentrations of BCB (13,26,39 and 52 μmol/L) for 90 min,and then we divided the COCs into BCB⁺ and BCB^- for in vitro culture 42 to 44 h.The results showed that,with the concentration of BCB increased,the staining rate (20.00%,46.39%,51.66% and 59.03%) raised gradually while the maturation rate of oocytes (74.03%,72.16%,70.53% and 48.61%) reduced,the percentages of oocytes staining by 26 μmol/L BCB for 90 min were higher than that of other groups in staining rate and maturation rate.However,the nuclear maturation rate of BCB⁺ groups were higher than that of BCB^- group.Therefore,26 μmol/L BCB was selected as the most effective concentration dying the oocytes (BCB⁺),which were used as parthenogenetic activation and nuclear transfer embryos.The cleavage and blastocyst rates of parthenogenetic activation and SCNT embryos in BCB⁺ group were significantly higher than that of BCB^- group (P<0.05),but there were no significant differences between the cleavage and blastocyst rates in the groups of BCB⁺ and control (P>0.05).Reconstructed embryos derived from the COCs stained with BCB were transferred to five surrogates,and six cloned piglets were obtained from one of the two pregnant pigs.These results showed that COCs stained with BCB was an effective method to select high-quality oocytes,which could improve the efficiency of in vitro embryo production.

In order to determine the genetic diversity in Cervus elaphus using AMELY gene in Y chromosome,200 blood samples from Cervus elaphus yarkandensis,Cervus elaphus asiaticus,Cervus elaphus xanthopygus,Cervus elaphus songaricus and Cervus elaphus kansuensis populations were collected and AMELY genes were sequenced in this study.The haplotype diversity of Y chromosome was analyed,phylogenetic tree was built to explore the genetic diversity and the paternal origins about Cervus elaphus.The results showed that:Cervus elaphus yarkandensis had the most variation sites and the highest nucleotide diversity.The genetic distance between Cervus elaphus yarkandensis and other Cervus elaphus were far.6 haplotypes were identified in this study,named as A1,A2,A3,A4,A5 and A6,respectively.Cervu elaphus yarkandensis,Cervus elaphus asiaticus and Cervus elaphus kansuensis had separate haplotype.The NJ and ML phylogenetic trees showed that Cervus elaphus songaricus,Cervus elaphus asiaticus,Cervus elaphus xanthopygus and Cervus elaphus kansuensis clustered together which Cervus elaphus yarkandensis and Cervus elaphus kansuensis were form a department,separately.Cervus elaphus asiaticus,Cervus elaphus xanthopygus and Cervus elaphus yarkandensis clustered into one branches and there might be gene exchange among Cervus elaphus yarkandensis,Cervus elaphus kansuensis and other Cervus elaphus.

Oocyte cryopreservation is an important part of embryo biotechnologies (e.g.in vitro fertilization,intracytoplasmic sperm injection and somatic cell nuclear transfer),and is of a great significance in conserving the germplasm resources of excellent and endangered animals,accelerating the process of livestock breed improvement.Compared with the conventional freezing method,vitrification has many advantages,such as fast cooling rate and high efficiency,and is widely used in oocyte cryopreservation.However,fertilization and developmental competence of vitrified oocytes are still not ideal,which seriously affects the application of vitrified oocytes.Vitrification leads to increased calcium ion (Ca²⁺) concentration and abnormal Ca²⁺ oscillation pattern in oocytes,which hardens their zona pellucida and disorders their fertilization signal.By integrating previous findings,present paperanalyzed the influence and mechanism of vitrification on intracellular Ca²⁺ concentration and Ca²⁺ oscillation pattern,and proposed that extracellular Ca²⁺ influx and intracellular Ca²⁺ release induced by vitrification led to Ca²⁺ increasing in vitrified oocytes,and inositol 1,4,5-triphosphate (IP3) Ⅰ receptor maldistribution and mitochondrial damage led to abnormal Ca²⁺ oscillation pattern in vitrified oocytes.Above information may provide technical reference for researchers to regulate Ca²⁺ concentration and Ca²⁺ oscillation pattern positively in vitrified oocyte,and further improve their fertilization and subsequent developmental competence.

In order to meet the requirement of the development of cashmere goat industry,and improve the matching accuracy and scientificity of breeding sheep selection and the production efficiency,it is needed to use information technology and modern breeding theory in cashmere goats accurate management and scientific breeding.Using MVC software design pattern,B/S three-tier network architecture,database,BLUP breeding and other technologies,a cashmere goat breeding and production management systems based on RFID was designed and developed in this study.The system contained information management,breeding analysis,production management,nutrition regulation and benefit analysis and other functional modules.In the system,WOMBAT genetic evaluation software was used in calculating cultivation and inbreeding coefficient,then based on this,the optimal combination parents were choosn;The system was designed for different breeding objectives,such us accelerating advances in genetic improvement of cashmere goat economic traits,and developing the production technology through the joint breeding.Based on cashmere goats individual information,the system could automatically generated recommendations for sheep grading,breeding,selecting and cluster grouping;It could also generat warnings on materials,diet formulation and other control points in production (such as pre-production period,light control for increase cashmere,grazing duration,prevention,feeding and so on),it would provide technical support for "1396" sheep breeding and efficient feeding,thus made cashmere goat breeding and production informationalized,standardized and scientific.

To detect the association of the biological traits and genetic properties in sheep genome,array comparative genomic hybridization (aCGH) system was used to identify the CNVs in the sheep genome and the CNVs map was constructed in Mongolian sheep,Kazakh sheep and Tibetan sheep.The results showed that 28 CNV regions (CNVRs) were found,containing 11 gains,15 losses and 2 gain-losses.The HBB gene was amplified in Mongolian sheep and Tibetan sheep,which might be attributed to adaptability in low oxygen and high altitude environment.Real-time PCR was performed for CNVRs and CNV genes,83.3% of Real-time RCR results were consistent with the CGH.The study that performed the genome-wide detection of copy number variations of sheep in Northern China,would provid foundation for studying genetic variation in different sheep breeds.

The aim of this study was to clarify the effects of estrogen (E₂) on cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) gene expression in the cow oviduct tissue in vitro.Quantitative Real-time PCR and Western blotting were used to analyze the mRNA and protein expression of COX-1 and COX-2 at different time points after different concentrations of E₂ treatment.The results indicated that the relative expression of COX-1 and COX-2 mRNA reached the highest levels with 10^-11 mol/L E₂ treatment,the relative expression of COX-1 mRNA reached the highest level at 8 h after treatment;The relative expression of COX-2 mRNA reached the highest level at 2 h after treatment.The relative expression of COX-1 protein increased significantly at the range from 8 to 48 h after E₂ treatment.However,we did not detect COX-2 protein expression.

In order to breed high quality pig strain and improve the pork quality,the intramuscular fat content and the differences of UCP3 gene expression were studied in Luchuan pigs with Duroc reciprocal cross F1 and its parents Luchaun pigs.8 head of Luchuan pigs,Luchuan pigs with Duroc reciprocal cross F1 generation were respectively selected to analyze the correlation of UCP3 gene expression with intramuscular fat and eye muscle area.Expression of UCP3 mRNA in muscles of different pig breeds were measured by Real-time RT-PCR,and the eye muscle area and intramuscular fat content were measured too.The results indicated that the eye muscle area,intramuscular fat and the UCP3 gene expression were significantly different in the three groups of pigs (P<0.05).The crossbreds inherited the big body size of Duroc,and the high intramuscular fat content of Luchuan pig.The extremely significantly negative correlations were observed between UCP3 gene expression and the pig eye muscle area,the extremely significantly positive correlations were observed between UCP3 gene expression and intramuscular fat content.UCP3 gene should be the main candidate gene for fat trait research in pigs.

In order to study the best propagation parameters of H1N1 subtype influenza virus in chicken embryo,this study was conducted to research the effects of egg selection and preservation before hatching and hatching parameters setting on production of H1N1 subtype influenza virus.In the egg selection and preservation,we mainly investigated the egg weight,egg shape index,storage period,disinfection time and method and other factors,the results showed that egg weight of 55 to 65 g,egg shape index of 1.30 to 1.35,egg storage period of 1 to 4 d,storage temperature of 16 to 18 ℃,humidity of 70% to 80%,during storage period,eggs formaldehyde fumigation time 30 min,we could provide the best eggs for H1N1 subtype influenza vaccine production.The effect of hatching parameters on production of H1N1 subtype influenza virus,this experiment mainly investigaed incubation temperature,humidity,times of turning over eggs,aeration and other parameters,the results showed that in the production of H1N1 subtype influenza virus,the optimal parameter set to hatch:temperature 1 to 7 d was 38.2 ℃,8 to 9 d was 38.0 ℃,10 d was 37.8 ℃;Humidity was 65% to 70% from 1 to 10 d;Turning over eggs frequency was 1 times/2 h,before and after each dip 45°;1 to 5 d aeration set to 4,6 to 10 d set to 5.The results provided quality chicken hatchery technology for H1N1 subtype influenza vaccine production in order to ensure the quality and yield of allantoic fluid.

To understand the correlation between drug resistance patterns and biofilm phenotype of Escherichia coli isolated from Wenchang chicken to fifteen drugs,broth microdilution method and improved crystal violet method were used for drug resistance and biofilm-forming ability analysis of 65 Escherichia coli isolated from Wenchang chicken to 15 commonly used drugs.The results showed that 89.23% of the strains were multidrug resistant and 64.62% of which revealed cross resistance;81.54% of the strains had different biofilm-forming ability and only 18.46% of which had no biofilm-forming ability.The results suggested that most of the strains with biofilm-forming ability were multidrug resistant,but there was no significant difference between the strains with biofilm-forming ability and those performed no biofilm-forming ability.

To explore the therapeutic effect of Humulus scandens alcohol extract on secretory diarrhea in mice,we used lavage of senna to produce diarrhea mice mode.The mental state,body weight,colon intestinal wall thickness and histopathological changes were observed daily during the experiment period and the disease activity index was detected to evaluated therapeutic action of Humulus scandens alcohol extract on diarrhea.The results showed that compared with model group,the treatment group had significant effects on metal state,body weight and small intestinal villus recoveries;Compared with model group,body weight of treatment group had a rising trend but still couldn't reach the normal level.The DAI of treatment group decreased compared with model grope,there was no significant difference between blank group and treatment group (P>0.05).Compared with model group,intestinal wall thickness of treatment group were significant differences (P<0.05),the treatment effect was remarkable.Histological examination showed that in blank group,small intestinal villus was neatly,the hierarchical structure was clear,and there was no small vessel hyperemia and no villous intestinal edema.While small intestinal villus of model group disordered,most of the villus fell off,there was inflammation exudation on the surface of villi,villous edema and congestion of small blood vessels.Compared with model group,symptoms of treatment group significantly changed,which showed greatly reduced cell inflammatory exudation,there was no obvious edema and congestion in villi interstitial,small intestinal structure was roughly the same as normal tissue,the treatment effect was obvious.Humulus scandens alcohol extract had significant therapeutic effect on diarrhea in mice,and could provide a theoretical basis for the treatment of secretory diarrhea.

The assay was aimed to isolate Toxoplasma gondii (T.gondii) strains from stray cat in Eryuan and Nujiang of Yunnan province.The cat tissues (heart,liver,lung and brain) were digested by acid pepsin solution,intraperitoneally inoculated in Kunming mice,passaged at least 3 generations,and followed by specific PCR amplification of partial B1 gene using species-specific primers.Three T.gondii isolates were isolated from 18 stray cats,PCR result showed that we got the specific target band,and the sequence result of the specific PCR product showed that it was ribosome B1 gene sequence of T.gondii. Homology comparison analysis showed that the isolates was 100.0% homology with T.gondii B1.The method of inoculation into the mice with the tissues that was digested by acid pepsin solution was an effective way to isolate T.gondii strain from animals,and the specific PCR assay was an accurate method for the rapid identification of T.gondii.

The etiology of lamb diarrhea disease is complex,however,bacterial and viral infection are the most critical factors and cause most serious harm.In order to explore the main pathogens of lambdiarrhea,many scholars and experts have undertaken extensive research on different aspects of viral and bacterial etiology of lamb diarrhea with various methods.Recently,the research about the etiology concentrates on the main code genes and functional proteins in the progress of pathogen invading host and molecular epidemiology.This paper reviewed the recent and main pathogens of lamb diarrhea caused by bacteria and viruses from the molecular level,and proposed some ideas about the development of etiology of lamb diarrhea in the future.

The assay was aimed to clarify the reasons why dairy cattle became ill with the symptoms including fever,breathing difficulty,salivation and subcutaneous emphysema at neck or chest in a dairy cow farm at Hubei province,even part of dairy cows were dead for exhaustion.The disease was diagnosed by laboratory methods,and treatment scheme was proposed.The samples from heart,liver,lung or trachea tissues were collected,then M.bovis and pathogenic bacteria were isolated,respectively.Biochemical characteristics,pathogenicity test and drug sensitivity analysis were performed on the isolated pathogenic bacteria.RNA was extracted from bovine serum,then bovine ephemeral fever virus was identified by RT-PCR.The results showed that nothing grew in PPLO medium,and the isolated bacteria could form indole,but did not ferment glycogen and inositol in the biochemical tests.The isolated pathogenic bacteria was conformed as serotype A P.multocida by PCR while bovine ephemeral fever virus of RT-PCR result was negative.The pathogen could kill mice and the pathogenic bacteria could be isolated from heart blood of the dead mice in pathogenicity test.The isolated bacteria was sensitive to cefoperazone or levofloxacin in drug sensitivity analysis.In conclusion,the disease was diagnosed as bovine serotype A P.multocida infection.Some treatments including choosing sensitive antibiotic drugs based on the drug sensitivity tests,holding sick cattle under quarantine treatment and isolating suspected cattle should be taken.Moreover,some complex measures such as keeping the air flowing,cleaning up in time,improving the management,and preventing stress from congestion,cold and long distance transport were very important to prevent and control serotype A P.multocida infection in dairy cows.