Porcine alpha (1,2) fucosyltransferase (FUT2) gene was importance in glycosphingolipid biosynthesis-globo series, potentially played a regulatory role during Escherichia coli (E. coli) F18 infection process in weaned piglets. In order to explore sequence structure of porcine FUT2 gene and its biological function, this test amplified FUT2 gene CDS sequence of Dongchuan pigs by PCR, forecasted and analyzed the protein sequences and functional regions of FUT2 gene, its expression level was detected in 11 tissues of 8 Dongchuan weaned piglets in 35 days old at the meantime. The results showed that the CDS sequence of FUT2 gene was 1 023 bp, which encoded 340 amino acids. FUT2 protein was fat-soluble hydrophilic protein, which the structure was not stable, including a transmembrane helix structure, but without signal peptide that suggested the FUT2 protein was a membrane protein;FUT2 protein included 2 N-glycosylation sites (No. 185 and No. 305 amino acids), without O-glycosylation sites, there were 14 potential phosphorylation sites, included 6 Ser, 2 Thr and 6 Tyr, analyzing the functional regions found that the FUT2 protein had a superfamily of conserved domains:FUT1-FUT2-like (58-319 amino acids). The phylogenetic tree result showed that the relative relationship between swine and cattle was relatively close, but was distant from chimpanzee, human, mouse and rat. FUT2 gene was expressed in all 11 tissues of Dongchuan weaned piglets, there were higher expression in digestive tract and immune tissues. The present results suggested that FUT2 gene might play a role to resistance to E. coli F18 in weaned piglets, and might indirectly against E. coli F18 through the synthesis of fucosyltransferase.

The aim of this experiment was to study the optimum process of Rose laevigata michx fermented by Bacillus subtilis. The process of Bacillus subtilis fermented Rose laevigata michx was optimized by test method combining single factor and response surface.The Rosa laevigata michx polysaccharide content was chosen as indexes and the key factors which had greater impact on ferment effect were selected from carbon source,nitrogen source,water content,inoculation amout and fermentation time for further optimize by response surface. The results showed that water content,inoculation amount and fermentation time were the key factors among the five single factors. When the water content was 53.79%,inoculation amount was 5.74%,fermentation time was 2 days,the average content of Rosa laevigata michx polysaccharide was 15.47% which closed to predicted value (15.36%).Compared with the polysaccharide content in unfermented Rosa laevigata michx,that of fermented Rosa laevigata michx was increased 47.47%. The fermentation process was simple and easy to operate with remarkable fermentation effect,and it could provide theoretical basis for the development of the green feed additive.

In order to study the differences in the structure of the mature peptide of leptin gene before and after the third exon site directed mutation, and speculated its functional differences, the mature peptide of leptin based on GenBank was amplified using specific primers. And the mature peptide of leptin was cloned and sequenced. The physical and chemical properties, structure and function of encoding products of leptin were compared and analyzed by bioinformatics methods. Leptin single base mutations could change the encoding of amino acids, and result in a significant difference in the primary structure and secondary structure, and the tertiary structure was relatively stable. Single nucleotide mutations at the E3-125-C>A of leptin gene, caused structural changes that might cause functional changes.

In order to obtain high purity of interleukin 4 (IL-4) protein,the specific primers were designed and synthesized according to the mink IL-4 gene sequence in GenBank. The IL-4 gene was amplified by RT-PCR using total RNA of the lymphocyte isolated from the peripheral blood that induced by phytohemagglutinin (PHA). The length of IL-4 gene sequence was 399 bp which encoded 132 amino acids. Phylogenetic analysis revealed that the amino acid sequences homology between mink and ferret was 99.2%,90.0% with Ailuropoda melanoleuca and Canis familiaris. Prokaryotic expression vector pProEX-HTb-IL-4 was constructed and induced by IPTG. The recombinant protein of 15 ku was isolated by SDS-PAGE and detected by Western blotting. Highly purified recombinant protein of mink IL-4 was obtained by His-Trap HP affinity columns method. This research laid the foundation for the further studies on the biological function of mink IL-4 gene.

The objective of this study was to investigate the expression level of PPARγ gene mRNA in different tissues from Weining cattle, Sinan cattle, Guanling cattle and Liping cattle in Guizhou province. Total RNA was extracted from the heart, liver, spleen, lung, kidney, adipose and longissimus dorsi of the cattle resources. Primers of PPARγ gene were designed. The developmental changes of PPARγ gene mRNA relative expression in different tissues from 4 kinds of cattle breeds were detected by Real-time PCR, taking GAPDH gene as the internal reference. The results showed that PPARγ gene was expressed in all tissues. The mRNA expression in adipose tissue was extremely significantly higher than that in other tissues (P<0.01), with a higher expression in the spleen and lung as well, while a lower expression in the kidney, liver, heart and longissimus dorsi.The expression of PPARγ gene in the different cattle resouces was different in partial tissues. The results suggested that PPARγ gene mRNA expression existed differences in the tissues,which might be due to its function in different tissues, but different cattle resouces did not basically influence the PPARγ gene mRNA expression.

As an important component of the antigen, peptide epitopes can be recognized by T cell receptor (TCR) and antibodies which induced protective immunity by the host immune system. Traditional research methods, such as overlapping peptide synthesis is long time-consuming which greatly limits the study of epitopes. With the development of bioinformatics, high-throughput sequencing, CRISPR/Cas9 technology, MS and other technologies and explaining the processing mechanism of TAP-dificient protein constantly, the research for epitopes is being perfect continually. In this article, some new methods to study epitopes were introduced, including computer prediction of B cell or T cell epitopes, identification of epitopes based on the structure and interaction of MHC and antigenic peptides, screening epitopes by means of high throughput sequencing, screening epitopes by means of newly genome editing, the up-to-date research in TAP-deficient T cell epitopes, etc. Finally,future prospect for research direction of peptides was further forecasted.

The food and mouth disease virus (FMDV) vaccination is a key method in prevention and control of FMD, and the assay of antibodies to FMDV is always used to evaluate the immunity and preventive ability to FMD for its simplicity. However, the antibody titer is not always paralleled with the immunity to FMDV, for cellular immunity is also an important factor in FMDV response. So, we set up and optimized a ELISpot technique to detect specific interferon gamma(IFN-γ) to FMDV. Both the peptide pool and whole virus particle of FMDV(O/Mya98/XJ/2010) were used as stimulators, and the phytohaemagglutinin (PHA) was used as positive control. The final concentration of PBMC, whole virus and peptide pool and incubation time were optimized. The results showed that the optimized condition for ELISpot to detect specific IFN-γ to FMDV were as followed, more than 90% viability of fresh isolated or cryopreserved PBMC were seeded at the suitable concentration of 2×10⁵ cells/well, stimulated with whole virus particle containing 100 ng/mL 146 S or T cell epitope peptide pool consisting of 10 μg/mL each peptide or 20 μg/mL PHA and incubated for 16 hours. The foundation of ELISpot technique to detect specific IFN-γ to FMDV would provide a valuable means to evaluate the cellular immunity to FMDV of swine inoculated with FMD vaccine. The relevance of specific IFN-γ level to FMDV with 50% preventive dose (PD₅₀) of challenge test needed to be further investigated in the future.

To provide basic information for researching the biological function of antimicrobial peptide P3 from bovine hemoglobin and its analogs (JH-0, JH-1, JH-2 and JH-3), tools on line were utilized to analyze some bioinformatic features of these five peptides. The results showed that the isoelectric point of these five peptides were all more than 8.00. All of these five peptides had positive charge, no transmembrane domain, no signal peptide sequence, no glycosylation sites,were hydrophobins and located at extracellular. The secondary structure of peptide P3 and JH-2 had α-helix and β-sheet, JH-3 was all α-helix, while JH-0 and JH-1 were coil. Peptide P3, JH-2 and JH-3 had a glycosylation site at the thirteenth site of amino acid (Thr, T), respectively. Based on these results, we conjectured that antimicrobial peptide P3 and its analogs could kill bacteria through the interaction with cell wall or cell membrane.

This trial was conducted to investigate the effect of dietary Picriafel-tarrae Lour extracts on serum biochemical indexes and immune function of Guangxi Partridge chickens. 500 Guangxi Partridge chickens with 1 day old were randomly divided into 5 groups with 5 replicates per group and 20 chicks per replicate.Groups A,B and C were treatment groups and fed diets with 0.70%,0.35% and 0.175% Picriafel-tarrae Lour extracts,respectively,group D was a medical control group fed a diet with 0.01% colistin sulfate premix,and group E was control group fed basal diet. Serum samples were collected from 10 chicks in each group at 21,35 and 49 days old to analyze the biochemical and immune indexes. The results showed that compared with group E, the contents of GLU and TP were not significantly affected by the Picriafel-tarrae Lour extracts (P>0.05).However, in 21 and 35 days old, the TBILI content of groups A,B and C were extremely significantly or significantly reduced (P<0.01;P<0.05);At 35 days old,the activity of serum AKP of groups A,B and C were extremely significantly higher than that of group E (P<0.01),and the activity of serum GOT of group C at 21 and 35 days old were significantly higher than that of group E (P<0.05);At 49 days old,the serum IgG content of groups A,B,C and IL-2 of groups B and C were extremely significantly or significantly lower than those of group E (P<0.01;P<0.05);At 49 days old,the T-AOC of group B was significantly higher than group E (P<0.05);The GSH content of groups A,B and C at 49 days old were significantly increased than group E (P<0.05).In conclusion,the dietary Picriafel-tarrae Lour extract could significantly decreased the TBILI content,reduce the contents of IgG,IL-2 and IL-6, improve the activities of AKP and GOT,and the contents of T-AOC and GSH in some degree. The 0.35% additive amount had the better effects.

The study was aimed to analyze the effect of transplanted bone marrow mesenchymal stem cells (BMSCs) on the mammary gland development and serum hormone level of Saanen dairy goat.16 healthy goats with 2 months old and similar body weight were randomly divided into control group and experimental group. The BMSCs which was amplified to the P6 generation was injected to the goat via jugular vein,and the control group was injected with the same amount of normal saline. Feeding and management of the two groups were the same. Blood samples were collected monthly,and mammary gland tissue samples were collected at 3,5 and 7 months old. Frozen sections of mammary gland tissue was prepared, the ducts and acinus numbers were observed and recorded. The results showed that after transplantation of BMSCs,at 5 and 7 months old,the ducts and acinus number of goat in experimental group were significantly higher than that of control group (P<0.05).At 5,7,8 and 9 months old,the serum E₂ level of goat in experimental group was significantly higher than that of control group (P<0.05);At 5 to 9 months old,the serum P level in experimental group was significantly higher than that of control group (P<0.05).At 7 to 9 months old,the serum PRL and INS levels in experimental group were significantly higher than that of the control group (P<0.05);At 8 and 9 months old,the serum levels of GnRH level in experimental group was significantly higher than that of the control group (P<0.05).The serum LEP,CRH and ACTH levels were higher than that of the control group at the whole test process,but the difference was not significant (P>0.05).In conclusion, injection of BMSCs could increase the duct and acinus number of dairy goat mammary,improve the hormone level in serum,and promote mammary gland development.

This study was to determine the effect of anionic salts on health and production performance of Holstein cows during the peripartum period. Twenty-four Holstein cows during the peripartum period were selected and divided into control group and experimental group. Twelve Holstein dairy cows in control group were fed diets with original concentrate,while twelve cows in experimental group were fed diets with concentrates adding anionic salts for the last 21 days before expected calving. All animals were ad libitum fed with the same diets after calving. The results showed that urine pH maintained 5.7 to 6.9,and blood pH was decreased significantly compared to control group (P<0.05). However,the contents of plasma Ca,glucose and alkaline phosphatase (ALP) activity in experimental group were significantly increased in 1 d after calving (P<0.05). What's more, the incidences of hypocalcemia,milk fever,retained placenta,displaced abomasum and mastitis were reduced in experimental group and the incidences of retained placenta was significantly decreased (P<0.05). No significant difference was detected in milk yield between two groups (P>0.05). In summary,supplementation of anionic salts concentrate could help to keep the health and plasma Ca homeostasis of Holstein cows.

To study the effect of Astragalus polysaccharide on short-distance transport stress in beef cattle,15 healthy and 12-month-old Simmental crossbred beef cattle were selected and randomly divided into control group,transport stress group and Astragalus polysaccharide test group with 5 cattles in each group.The cattles in Astragalus polysaccharide test group were fed with 10 g/d per head Astragalus polysaccharide during the experimental period.The cattles both in transport stress group and Astragalus polysaccharide test group were transported for 3 h at the speed of 60 km/h, and blood samples were collected and used for measuring some biochemical parameters. The daily gain and morbidity were measured simultaneously. The results showed that,in transport stress group,serum Na⁺ increased significantly in 1 d after transportation comparing to before transportation (P<0.05). serum Na⁺, K⁺, Ca²⁺ and Cl^- showed no significant difference during the experimental period in control group and Astragalus polysaccharide test group (P>0.05).After transport (1 and 7 d),blood Na⁺/K⁺-ATP activities were extremely significantly decreased (P<0.01), SOD and GSH-Px activities were significantly decreased (P<0.05), while serum MDA contentswere significantly increased (P<0.05) in transport stress group comparing with that at 1 d before transport.These indicators of the control and Astragalus polysaccharide test group showed no significantly difference during the experimental period (P>0.05); 7 d average daily gain after transportation of control group and Astragalus polysaccharide test group were extremely significantly higher than transport stress group (P<0.01), incidence rates of respiratory and gastrointestinal of Astragalus polysaccharide test group was lower than transport stress group. The results demonstrated that Astragalus polysaccharide could be applied to the transport stress for prevention and treatment in beef cattle.

This experiment was conducted to study the effects of Lactobacillus probiotics on growth performance,nutrient digestibility and serum biochemical indexes in arctic foxes during the winter fur-growing period. Forty 135-day-old female arctic foxes with a similar body weight were randomly divided into 4 groups with 10 replicates per group and 1 fox per replicate. The foxes in control group were fed basal diets,while that of groups Ⅱ,Ⅲ and Ⅳ were fed basal diet+0.02% colistin sulfate, basal diet+1 mL Lactobacillus probiotics (active Lactobacillus was 3×10⁹ CFU/mL) and basal diet+10 mL Lactobacillus probiotics,respectively. The results showed that:Compared to the control group,the addition of Lactobacillus probiotics significantly increased the average daily gain (P<0.05) and significantly decreased the F/G of arctic foxes during the winter fur-growing period (P<0.05).The apparent digestibility of EE and GE were significantly increased when arctic foxes fed diet supplemented with Lactobacillus probiotics (P<0.05).Supplementation of high level Lactobacillus probiotics could significantly decrease serum total cholesterol (P<0.05). Additionally,serum alanine aminotransferase concentration of group Ⅱ was extremely significantly higher than that of the other three treatments (P<0.01). Moreover,the serum aspartate aminotransferase concentration was significantly higher than the control group (P<0.05). In conclusion,the addition of Lactobacillus probiotics had beneficial effects on growth performance and apparent digestibility of EE and GE when arctic foxes got 3×10⁹ CFU/mL active Lactobacillus from the probiotics per day. The total cholesterol in serum was significantly decreased when arctic foxes get 3×10¹⁰ CFU/mL active Lactobacillus from the probiotics per day. It might cause the liver damage when arctic fox got colistin sulfate for a long time.

The experiment was conducted to study the effects of fermented vegetable soybean straw (FSBS) on reproductive performances, colostrum quality and digestion in adult female goats.A total of 16 Chongming White pregnant goats with similar parity were randomly divided into 4 groups and were fed granular TMR with 0 (control group),17.5% (group A),35.0%(group B) and 52.5%(group C) FSBS, respectively. The results showed that:Compared with the control group and group A, the apparent digestibility of DM, CP, CF and OM of 30 days after delivery in group C significantly increased (P<0.05).The contents of urea nitrogen among 4 groups had significant differences (P<0.05)and group C was the lowest;Lambing birth weight,weaning weight,the content of milk fat, protein, lactose, TS and SNF in group C were significantly higher than that in control group (P<0.05).In conclusion, FSBS could improve the reproductive performance and nutrient digestibility of adult female goats,its appropriate level in TMR should be 52.5%.

In vitro culture method is an important way to research in ruminant dietetics, including batch and continuous culture methods. Because the continuous culture method can better simulate the conditions of fermentation in rumen, it was applied more widely. There are many operation parameters of rumen simulation technique involved in continuous culture, such as temperature, pH, dilution rate, the proportion of buffer to rumen fluid, stirring rate and way, meanwhile, different operation parameters and their combinations will have a great influence on the fermentation results. On the basis of summarizing the results of previous studies, the authors review the effects of different parameters on volatile fatty acid, dry matter disappearance rate, pH, enzyme activity, number of protozoa and microbial protein, in dual-flow continuous culture system in this paper.

To study the genetic variations of Ascaridia galli (A. galli) and the phylogentic relationships with other Ascaridata, fragment of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene and complete sequence of NADH dehydrogenase subunit 4 (nad4) gene of 15 adult A. galli isolated from Xichang city in Sichuan province were amplified by PCR, then analyzed the sequences. NJ trees and Bayes trees based on pcox1 and pnad4 genes were reconstructed. The partial sequences of cox1 gene and complete sequence of nad4 gene were 1 152 and 1 236 bp, respectively. There were 33 and 40 variable sites in the pcox1 and nad4 gene sequences, the intraspecific sequence variations within A. galli were 0 to 2.1% for pcox1 gene, 0 to 2.3% for nad4 gene. 8 and 11 haplotypes were detected in pcox1 and nad4 genes, the global haplotype diversity were 0.733±0.124 and 0.933±0.054, the nucleotide diversities were 0.00433±0.00153 and 0.00541±0.00157, and the average genetic distances were 0.004 and 0.005, respectively. Phylogenetic trees based on pcox1 and pnad4 genes with Neighbour-Joining and Bayesian analysis method revealed that all A. galli were clustered in one clade, and they were more closely related to A. columbae than any other members of Ascaridata. These findings indicated that 15 isolates of A. galli from Xichang city kept low genetic variation, nad4 gene was more suitable and effective than cox1 gene as molecular marker for detecting genetic variation of A. galli.

To study the influence of estrogen receptor (ESR), osteopontin (OPN) and retinol binding proteins 4 (RBP4) genotypes on reproductive performance of Yorkshire sows, PCR-RLFP technique was used for genotyping, and the effects of genotypes on reproductive traits were analyzed. The results showed that A allele of ESR and OPN genes were predominant allele, and B allele of RBP4 gene was predominant allele. TNB,NBA of ESR gene AB and BB genotypes were higher than AA genotype;TNB of OPN gene AA genotype was the highest, but the NBA, NHB and WB for OPN gene genotypes were BB > AA > AB;The TNB,NBA,NHB and WB for RBP4 gene genotypes were AA > BB > AB. Then combing genotype analysis showed that ABAA of ESR-RBP4 and BBAA of OPN-RBP4 were the most prolific alleles, respectively. The present data revealed that ESR, OPN and RBP4 genes polymorphisms were associated with reproductive performance of Yorkshire sows.

Through dairy herd improvement (DHI) data analysis to predict somatic cell count (SCC) of Ningxia area in time, which providing a reference for the prevention and treatment of mastitis in dairy cows, making the DHI data more effective and timely in guiding the dairy industry production. Using the difference method to make the average cow somatic cell data form September 2011 to February 2016 stabled, then used the seasonal ARIMA model to analysis, fitting and forecasting data. The auto.arima function of R software had been used to calculate the optimal time series that finally confirmed the model was ARIMA (1,1,0)(1,1,0)_[12], AIC was -3.67. The Acf test showed that the residual had no significant autocorrelation; Ljung-Box test showed that all P-value>0.5, indicating that the residual was white noise, and this model could be used to make predication for the next 24 months. The forecast function of the R software was used to predict the dairy cows SCC from March 2016 to February 2017, and the forecast map was drawn. The predicted results showed that the SCC of the entire Ningxia area were showing a downward trend. The SCC would be the least in January 2017,and the predictive value was about 253 100 per mL. It would be the largest in March 2016, was about 439 600 per mL. The results also showed that the dairy cows SCC in Ningxia was higher than the critical value of 20 million of subclinical mastitis. It suggested that the prevention and treatment of dairy cow mastitis need to be strengthened in Ningxia area. At the same time, if the data of the dairy cows SCC was added in timely, the data model should be updated to make it more close to the true value, which would be more meaningful to the actual instruction.

In order to search the molecular genetic markers for breeding of Baicheng fatty chicken, the relationship between the polymorphism of adipocyte fatty acid binding protein (A-FABP) gene and the productive performance of the male chickens at different ages was studied. Ninety two chickens with age of 6 weeks were randomly selected. The polymorphism of A-FABP gene was detected by PCR-RFLP method. In each genotype group, fifteen chickens with similar body weight were categorized, each group was randomly divided into three sub-groups with equal numbers. The productive performance of the chickens was measured at their age of 12, 18 and 24 weeks. The results showed that there were three genotypes of A-FABP gene:AA,AB and BB, BB genotype was the dominant genotype,allele B was the advantageous allele,and it was not in Hardy-Weinberg equilibrium (P<0.01). At the age of 12 weeks,no significant difference was found among the measured productive performance (P>0.05). At the age of 18 weeks, it was found that the intramuscular fat width of BB genotype was significantly higher than AA genotype (P<0.05), the abdominal fat weight of BB and AB genotypes were extremely significantly higher than AA genotype (P<0.01),and the abdominal fat weight percentage of BB and AB genotypes were significantly higher than AA genotype (P<0.05). At the age of 24 weeks, it was found that the body weight, intramuscular fat width and abdominal fat weight of BB genotype were significantly higher than that of AA genotype (P<0.05). Therefore, this results suggested that A-FABP gene could be used as a potential candidate marker for the association of fatty traits in male Baicheng fatty chicken.

The study was aimed to investigate the role of porcine oocyte nuclear factors during reprogramming. Somatic cell nuclei was introduced into intact MⅡ oocytes to establish tetraploid somatic cell nuclear transfer (SCNT) embryos containing both somatic nuclei and oocyte nuclei. And then the influence of the oocyte nucleus on tetraploid SCNT embryo development was examined by assessing characteristics including cleavage rate and blastocyst rate. The results showed that the cleavage rate of tetraploid SCNT embryos,diploid parthenogenetic embryos and haploid parthenogenetic embryos was extremely significantly higher than that of standard diploid SCNT embryos (P<0.01). The blastocyst rate and the total number of cells in tetraploid SCNT embryos were extremely significantly higher than that of standard diploid SCNT embryos (P<0.01).Overall,tetraploid SCNT embryos had a higher developmental competence than standard diploid SCNT embryos. In conclusion, the embryonic model was established in which a fetal fibroblast nucleus and an oocyte M Ⅱ plate coexist. Tetraploid SCNT represented a new research platform that was potentially useful for examining interactions between donor nuclei and oocyte nuclei. This platform should facilitate further understanding of the roles played by nuclear factors during reprogramming.

This study was designed to investigate the effect of different types and different concentrations of sugar on in vitro maturation(IVM) and developmental competence of yak oocytes, for being further research and optimization culture system of yak oocytes for efficient maturity yak oocytes and productivity of embryos. Immature yak oocytes were matured in vitro on culture medium with different concentrations (0,5 and 10 mmol/L) of glucose and sucrose in incubator for 24 h or 2 h pretreament with sugar and 22 h without sugar. Subsequently, then the maturation of oocytes,the cleavage rates and blastocyst formation rates after in vitro fertilization(IVF) were evaluated. The results showed that a medium with 5 and 10 mmol/L glucose IVM could significantly increase the yak oocytes maturation and cleavage (P<0.05), and the highest blastocyst formation rates in 10 mmol/L glucose group was significantly higher than 0 mmol/L glucose (P<0.05).10 mmol/L sucrose could increase significantly the nucleus maturation rates (P<0.05),and there was no significant difference of the blastocyst formation rates after IVF between 0 and 10 mmol/L sucrose (P>0.05). Furthermore, the nucleus maturation rates,IVF cleavage rates and blastocyst formation rates of yak oocytes which pretreated with 10 mmol/L glucose were the highest in these groups, and were higher than 0 mmol/L glucose (P<0.05). It manifested that the appropriate concentration of sugar could improve the quality of yak oocytes and embryos in vitro developmental competence, so it influenced in vitro development of yak oocytes indirectly.

Placenta is the link between the fetus and the maternal body during pregnancy.Therefore, the pregnancy outcome of the key was related by the normal development of the placenta.Wnt signaling pathway plays an important role in the development of embryo and placenta formation, several Wnt pathways which have been clear study and the formation process of early embryo and placenta in cattle are reviewed in this article,the early expression of the Wnt pathway components in the somatic cell nuclear transfer (SCNT) cattle and normal insemination of bovine embryos is introduced. The expression of DKK-1 and Fzd4 have a special role in the formation and development of early placenta, but its molecular mechanism is not clear. In addition, the Wnt signaling pathway is activated by inhibiting the MAP2K and GSK3, led to the inner cell mass and trophoblast cell number increased and blastocyst development speed. But it is similar to the extent of phosphorylation of E-cadherin and beta-catenin protein in the early placenta of cows with SCNT and normal insemination, therefore, the role of Wnt signaling pathway in the early formation and development of bovine placenta should be further understood and studied.

In this study, different concentrations of glycerol were added to semen cryoprotective extenders of Banna Mini-pig inbred line (BMI), different thawing solutions and thawing methods were studied. After thawing, the motility, the rates of plasma integrity and acrosome integrity were compared to optimize the cryopreservation method of BMI semen. The results showed that the motility, the rates of plasma integrity and acrosome integrity were significantly higher in 2% and 3% glycerol groups than those in 1%, 4% and 5% glycerol groups (P<0.05);The motility, the rates of plasma integrity and acrosome integrity were significantly higher in thawing solution Ⅰ than those in thawing solution Ⅱ, Ⅲ and Ⅳ (P<0.05); Through comparing the motility, the rates of plasma integrity and acrosome integrity, the thawing effect in 40℃ 6 s and 50℃ 6 s groups were better than that in 38℃ 30 s (P<0.05). In conclusion, the optimal semen cryoprotective method for BMI was 2% or 3% glycerol as cryoprotectant, using thawing solution Ⅰ to thaw at 40℃ 6 s or 50℃ 6 s.

This study was aimed to assess mitochondrial DNA D-loop sequence diversity and origin of Lueyang Black-bone chicken.The mtDNA D-loop sequence of 30 individuals from Lueyang Black-bone chicken were amplified by PCR and subsequently sequenced.The mtDNA D-loop sequence of other chicken were collected from GenBank and used as reference sequences to analyze the diversity and origin of Lueyang Black-bone chicken.The results revealed that the average values of base composition of A,C,G and T in mtDNA D-loop the sequence of Lueyang Black-bone chicken were 26.6%,26.6%,13.4% and 33.4%,respectively.26 nucleotide polymorphic sites were transition.The average nucleotide diversity (Pi) of the sites and haplotype diversity (Hd) were 0.00705 and 1.000,and the value of Tajima's D was -0.47272.Phylogenetic tree showed that samples were clusted in 4 clades. In the research, it could be concluded that the genetic diversity was relatively rich and wealthy and there were 4 maternal origins to Lueyang Black-bone chicken population.

In this study, the single nucleotide polymorphism of prolactin receptor (PRLR) gene and its correlation with the lactation traits in Jiangyue donkeys was tested, and the molecular genetic marker of breeding Jiangyue donkeys was explored.120 individuals were analyzed association of PRLR gene polymorphism with lactation traits in Jiangyue donkeys. Genetic polymorphism of PRLR gene flanking region was detected by PCR-SSCP method. The results showed that there was 1 mutation in PRLR gene and 2 genotypes of AA and AB, and allele A was the dominant allele. The SNP g.29764584 C>G was identified of PRLR gene by sequencing, and it was a synonymous mutation, did not cause amino acid changes. Statistical results indicated that the SNP was significantly associated with average daily milk yield and protein percentage in Jiangyue donkey (P<0.05),there was no significant in the lactin percentage(P>0.05). The mutation might be as crucial DNA genetic marker for lactation performance selection in Jiangyue donkeys.

The glycoproteins B (gB) gene of porcine lymphotropic herpesviruses 2 (PLHV-2)was amplified by PCR and sequenced, then bioinformatics analysis were conducted. The gB protein was composed of 876 amino acids, the molecular formula was C₄₅₀₀H₆₉₇₅N₁₁₉₉O₁₃₅₁S₄₀, the molecular mass was 100.77 ku, the value of theoretical isoelectric point was 6.45, and the instability index was 38.58. The prediction of secondary structure revealed that some alpha helixes and beta sheets exist in protein while random coil was major pattern. It had a signal peptide in the position 1-39 amino acids and a transmembrane helice in the position 761-780 amino acids. It contained several prosites and several intrinsically disordered proteins. Tertiary structure model of gB protein showed a fusion loop. Multiple antigenic epitope located in gB through comprehensive analysis prediction method. It was suggested that gB gene could be as a candidate antigen for vaccination of PLHV-2.

To evaluate the feasibility of synthetic polypeptides O157:H7 Ivy146-157 as a vaccine antigen, a bioinformatics method was employed to analyze the secondary structures, hydrophilicity plot, flexibility plot, surface probability plot and antigenic index of the Ivy. A B-cell epitope peptide sequence was identified and synthesized. BALB/c mice were immunized with Ivy146-157, antibody titers and splenic lymphocytes proliferations in mice were tested. The results showed that the antibody titers were 1:6 400 after immunized mice three times. And the native Ivy protein of O157:H7 R14 strain was identified by polyclone antibody Ivy146-157. The MTT assay results indicated the splenic lyphocytes were increased after immunized polypeptides Ivy146-157, and had significant difference with the control group (P<0.05). These results indicated that polypeptides Ivy146-157 could induce a strong humoral and celluar immune respond, and it would provide a theory as a vaccine antigen for further researches.

To explore the molecular characteristics of protein L2R from sheeppox virus(SPPV), genomic DNA was extracted from SPPV GY strain. The specific primers were designed and used to amplify the L2R gene from the genomic DNA by PCR. Then the PCR product was ligated into pGEM-T Easy vector. After transformation into E. coli Trans 5α, the positive clones were sequenced and the sequences were analyzed by the bioinformatic softwares. The result showed that L2R gene sequence contained an open reading frame (ORF) of 279 nucleotides and deduced protein consisted of 92 amino acids with the theoretical molecular weight of 10.92 ku and isoelectric point was 6.56. Analysis of secondary structure of protein L2R revealed that α-helix, β-strand, random coil and extended strand were 69.57%,9.78%,8.70% and 11.96%,respectively. Analysis of multiple sequence alignment showed that L2R gene from different capripox virus isolates were highly conserved, phylogenetic analysis showed that GY and NK, TU and SA was in a branch, indicating with a close genetic relationship among them. The present results laid a foundation for further studies of biological functions of protein L2R and interaction among the early proteins of capripox virus.

CRISPR/Cas system is an immune system present in bacteria and archaea that includes the RNA which has the ability to target viruses invading the host or exogenous DNA fragment of the nucleic acid and the Cas9 nuclease cutting the fragment. In view that the system is easier to operate, more efficient, lower cost, has been widely used in various fields of genetic engineering. Here, we demonstrated the mechanism of CRISPR/Cas systems and summarized the application in respect of the parasite.

In order to obtain the P66 protein from Borrelia garinii (B. garinii) SZ, the first strand cDNA was synthesized based on the total RNA extracted from B. garinii SZ, and then the targeted P66 gene was amplified by PCR. The fragment was linked into the pET-30a(+) vector, and transformed into Escherichia coli BL21(DE3). After identified by PCR, double restriction enzyme digestion, and nucleotide sequencing, the recombinant protein was expressed and purified. Polyclonal antibody was then prepared from New Zealand rabbit immunized with purified recombinant protein. The recombinant protein was about 70 ku in size confirmed by SDS-PAGE, and Western blotting analysis indicated that the recombinant P66 protein could recognize the mouse monoclonal anti-His-tag, positive sera of spirochete from mouse, and anti-P66 polyclonal antibody. Additionally, the anti-P66 polyclonal antibody could recognize native P66 protein. In this study, we successfully expressed the recombinant P66 protein and obtained the anti-P66 polyclonal antibody, which provided the foundation for further functional studies of P66 protein from B. garinii SZ.

In this study, we isolated a strain from diseased Clariasfuscus. Through morphological observation, physiological and biochemical tests, 16S rDNA and phylogenetic tree analysis method, it was concluded that the pathogen was Morganella morganii. Artificial infection experiment results showed that the pathogen had strong pathogenicity to Clariasfuscus, and also had strong pathogenicity to totilapia, loach and carp.The drug sensitivity test results showed that the KD01 strains had high resistance to 6 medicines, including penicillin G, ampicillin and novobiocin, and so on. And it had the sensitivity to the 15 medicines, including chemitrim, enrofloxacin and ciprofloxacin, and so on.The test results of hemorrhage of intestinal necrosis disease study provided a strong basis for the study of Clariasfuscus, also could guide farmers to the rational use of drugs.

In order to comprehend the status of Erysipelothrix rhusiopathiae carried in swinery in Shanghai area, samples of pigs were collected from some standard abattoirs in Shanghai area, including nasal swab, lung, tonsil and mandibular lymph nodes. Strains were isolated from these samples for detecting the Erysipelothrix rhusiopathiae. Several kinds of methods were used, such as VITEK 2 Compact system and VITEK MS system. A pair of primers was designed to amplify the 16S rRNA gene. 16 PCR products selected randomly were sequenced and 16S rRNA homology analysis was conducted. As a result, strains were isolated from 49.07% (105/214) samples and were identified as Erysipelothrix rhusiopathiae.16S rRNA homology in strains of isolation and reference were 99.6% to 100.0%. The result indicated that the situation was very severe for so many carriers of Erysipelothrix rhusiopathiae in health group in nursery.

In this study,in order to map the key amino epitope of VP7 protein of blue tongue virus serotype 1 (BTV1),we used monoclonal antibody against VP7 protein of BTV1 to screen 7 mer phage display random peptide library. The selected phages including VP7 protein consensus sequence were amplified and purified,immunoreactivity between epitope of selected phages and monoclonal antibody against VP7 of BTV1 was analyzed by ELISA and competitive ELISA (c-ELISA).The result showed that the epitope of phages including LNWPMVR sequence could specially combine monoclonal antibody against VP7 of BTV1,and the combination could be inhibited or blocked by BTV1,it demonstrated that 7 mer phage simulated the antigenic determinant of BTV protein which could combine monoclonal antibody against VP7 of BTV1.The results suggested that the amino acides 163 to 189(LNAGARGDVQQIFQGRNDPMMLYLVWR) were the specific epitope of VP7 protein of BTV.

In order to understand the prevalence of the duck infectious serositis in different regions,and further to determine the sensitivity of the commonly used drugs for the Riemerella anatipestifer in these area.Bacteria were isolated from suspected infected ducks in Yunfu, and Kaiping in Guangdong province and Changshan in Zhejiang province.The isolated bacteria were identified by differential culture, microscopic examination and biochemical test. Micro dilution method was carried out in drug sensitivity test, and the MIC values of the isolated strains to the commonly used drugs were determined.As a result, a total of 16 strains of Riemerella anatipestifer were isolated and identified from three areas. Data of the MIC showed that all isolates were highly sensitive to cephalosporins and florfenicol;Doxycycline andspectinomycin were sensitive too,but enrofloxacin, neomycin and other tested drugs showed different degrees of resistance.The present study provided a systematic method for the isolation and identification of the Riemerella anatipestifer and for the drug sensitivity test.At the same time, it also provided theoretical guidance for the rational drug control of the duck infectious serositis.

Newborn calves in a dairy farms suffered from severe diarrhea disease with high mortality in Heilongjiang province.In order to make a definite diagnosis,the pathological samples were collected aseptically and the viral antigens and microorganism were detected by virus detection,bacteria separation,biochemical test,PCR for the specific gene of virus and bacteria,animal pathogenicity test and antimicrobial susceptibility test. The results showed that the virus detection of bovine rotavirus was positive and specific gene fragment of bovine rotavirus was amplified. One E. coli strain with K99 and F41 adhesin antigens which belonged to highly pathogenic enterotoxigenic E. coli was isolated. This strain was sensitive to enrofloxacin,cefamedin and chloramphenicol,however,it showed resistance for other antibiotics. A polyinfection of bovine rotavirus and E. coli were eventually confirmed through epidemic investigation and pathological diagnosis. It had significant preventive effect by taking effective measures according to the diagnosis results.

To establish a rapid, simple, sensitive and adapting field application assay for water buffalo Fasciola gigantica, the monoclonal antibodies of hybridoma cell strains 7D1 and 7D4 against excretory-secretary antigen(ES Ag) of Fasciola gigantica were prepared and purified. Meanwhile, the colloidal gold particle was prepared by the sodium cirrate reduction method, then the affinity purified 7D1 monoclone antibodies was labeled with the 30 nm colloidal gold particles. The 7D4 monoclonal antibodies and the goat anti-mouse IgG antibody using as secondary antibodies were coated on the surface of nitrocellulose filter(NC) membrane as the test line (T line) and control line (C line), respectively. The immune colloidal gold test strip was developed by optimizing the experiment conditions. The test results showed the prepared strip had a detecting prescribed minimum of 0.94 μg/mL of Fasciola gigantica excretory-secretary antigen, and it had no any cross reaction with the antigens of Eurytrema pancreaticum, Paramphistome, Chlamydia, Toxoplasma gondii and Trypanosoma evansi. Using the prepared strip to investigate the 334 fresh feces from buffaloes in Guangxi and the positive rate was 38.62%. These results indicated that this strip method was simple, rapid and easy determination, good specificity, high sensitivity, and adapted field application assay for Fasciola gigantica.

To investigate the molecular mechanism of the inflammatory response in the piglets infected with enterotoxigenic E. coli (ETEC) K88, piglets were infected with ETEC K88,the IL-8 content in serum of piglets were assayed by ELISA,and the mRNA relative expression levels of TLR2/4 and its signal transduction pathway related genes (MyD88,Tollip and Bcl3) in mesenteric lymph nodes were detected by quantitative Real-time PCR. The results showed that compared with control group,the content of IL-8 in serum and the expressions of TLR2/4 in lymph nodes were all extremely significantly or significantly increased at 6 and 24 h after infection (P<0.01;P<0.05),and the IL-8 content and TLR2/4 mRNA expression at 24 h after infection were all significantly lower than those at 6 h after infection (P<0.05).In addition,the expressions of MyD88,Tollip and Bcl3 in lymph nodes were all extremely significantly increased at 24 h after infection compared with control group (P<0.01), but there was no significant difference between experimental group and control group at 6 h after infection (P>0.05). In conclusion,ETEC K88 infected piglets might produce inflammatory cytokines IL-8 through the TLR2/4-MyD88 signaling pathway,which could promote the inflammatory reaction in piglets. This inflammatory response might be regulated by Tollip and Bcl3,which could weak the inflammatory intensity in piglets.

To investigate the distribution and characteristic of E. coli in the microbial-fermentation bed of piggery, litters with different levels of fermentation were used for the isolation, identification and drug sensitivity test of E. coli, to analyze the pathogenicity and drug susceptibility of E. coli in the microbial-fermentation bed of piggery. The results showed that, E. coli could be isolated only from litters with low degree of fermentation (0<△E≤7.63), and totally 41 strains were isolated from litters with 10^-5 dilution, but none from litters with higher degree of fermentation (△E>7.63). The number of E. coli reduced along with the fermentation of litters. Among the 41 strains of E. coli, 15 were intestinal pathogenic strains, 9 of which had astA gene (22%), and 2 strains had sepA gene (4.8%). 11 strains were resistant to doxycycline, accounted for 27% of the total.The two strains containing sepA gene had strong resistance to doxycycline. In conclusion, the microbial-fermentation bed fermentation bed had a inhibitory effect on E. coli which promoted the application of fermentation bed and reusing of litters.

This experiment was aimed to study the effects of mixed Caulis Lonicerae japonicae extracts on fracture healing and inflammation. Rabbit right radius fracture model was induced by dental drill in order to study the medicine's effect on fracture healing. Moreover,the contents of Ca,P and alkaline phosphatase in serum were measured to explore the influences of mixed Caulis Lonicerae japonicae extracts on serum biochemical indexes of fractured rabbits.RAW264.7 cell was induced by LPS in order to research the effect of medicine on inflammation. The concentrations of TNF-α,IL-1β,IL-6 and NO were measured by ELISA. The results showed that the rabbit fracture model was successfully prepared by dental drill, and the extracts of Caulis Lonicerae japonicae could increase the levels of Ca and alkaline phosphatase and reduce the P content in the serum of fractured rabbits. The TNF-α,IL-1β and NO levels in RAW264.7 cell induced by LPS were significantly inhibited by the extracts with concentrations ranging from 50 to 200 μg/mL (P<0.05). However, the extracts had no significant effect on the level of IL-6 (P>0.05). These results demonstrated that the extracts of mixed Caulis Lonicerae japonicae could promote the fracture healing and possessed good resistance inflammation effect.

In order to verify the effect of Baihu Dingchuan oral liquid of new veterinary drugs,the effects of infectious bronchitis virus (IBV) on the proliferation inhibition test, tracheal cilia movement test and SPF chickens infected with IBV were tested.The test solution was mixed with an equal amount of IBV solution,inoculation of 11-day-old SPF chicken embryo,incubation at 37℃ for 168 h.RT-PCR was performed to detect IBV in kidney of live chick embryo.The results showed that the survival rate of 168 h chicken embryo was 80% in the drug group,but the positive control group was 20%, and there was significant difference between two groups (P<0.05). In the test drug group, no band was detected by RT-PCR, but the target band appeared in the positive control group.After 40 days old SPF chickens were administered continuously for 3 days, 0.02 mL of ink was injected into the trachea. The distance of ink movement in 1 min was measured. The results showed that the migration distance of ink was extremely significantly higher than that of saline control group (P<0.01).In the 17-day-old SPF chickens, IBV-M41 was infused into the nasal mucosa with 0.3 mL. After 24 h, 1 mL/kg was administered for 5 days. The results showed that there were significant differences in clinical symptom score and cure rate between the test drug group and the control group (P<0.05). There was no significant pathological changes in the tracheal mucosa of the experimental group, but the pathological changes in the positive control group were significant. The above results showed that Baihu Dingchuan oral liquid had good inhibitory effect on IBV in vitro, which could promote the movement of tracheal cilia, and had good clinical control effect on infectious bronchitis.

To study the preparation technology of inclusion compound carrying monensin, monensin was encapsulated with β-cyclodextrin using saturated water solution. The phase identification of inclusion compound was examed by infra-red spectrum (IR).Based on a comprehensive evaluation of inclusion efficiency, the optimal formulation and process conditions of inclusion compound carrying monensin were determined by orthogonal design methods.IR result showed that inclusion compound carrying monensin was formed;The best optimum of the preparation technology were as follows:The proportion of the quality of β-cyclodextrin and monensin was 3:1,the temperature of inclusion was 40℃,and the time of inclusion was 1.5 h. The inclusion rate was 57.54%.This preparation technology was feasible,and enlarged the use of monensin. The study laid a foundation for improvement about dosage form of monensin.

As an adjuvant, cytokine plays a key role in regulating the innate and adaptive immunity and enhancing the vaccine specific immune responses. The researches on the interleukin, interferon, granulocyte macrophage colony stimulating factors, tumor necrosis factors, and chemotactic factor have made considerable progress. Now we reviewed the cytokine adjuvant application in order to provide reference for the future application.