The objective of the study was to construct the eukaryotic expression vector of bovine GDF-9 gene,and investigate its effect on the expression of expansion-related genes in transfected bovine cumulus cells.Eukaryotic expression vector plasmid pcDNA4 myc-his-GDF-9 was constructed by insert GDF-9 in the multiple cloning site.pcDNA4 myc-his-GDF-9 was transfected into bovine cumulus cells by liposome.The expression of GDF-9 gene was detected by Western blotting and the expression of the expansion-related genes in bovine cumulus cells was detected by qRT-PCR.The results showed that GDF-9 could express in transfected bovine cumulus cells and the expression of expansion-related genes PTGS2,PTX3 and HAS2 in transfected bovine cumulus cells were significantly increased (P< 0.05).In a conclusion,the bovine pcDNA4 myc-his-GDF-9 could effectively transfected into bovine cumulus cells,which could help to study farther functions of the GDF-9 gene.

This study was aimed to observe effect of the prostaglandin receptor agonists butaprost and estrogen on TGFβ₃ mRNA expression and illustrate whether butaprost and estrogen had coordinated regulation on bovine oviduct epithelial cells (BOECs) transforming growth factor β₃ (TGFβ₃) or not.BOECs were isolated and cultured by mechanical and pancreatic enzyme digestion method;butaprost and estrogen acted on BOECs,respectively;mRNA expression of TGFβ₃ was detected by Real-time PCR in BOECs.The results showed that after the action of estrogen,the expression of TGFβ₃ at 16,24 and 48 h compared with 0 h were extremely significantly increased (P< 0.01),TGFβ₃ mRNA expression reached minimal at 4 h (P< 0.01);The receptor agonist butaprost and estrogen had coordinated regulation on TGFβ₃.Adding indomethacin could inhibit the expression of endogenous prostaglandin for TGFβ₃.The results showed that butaprost and estrogen could regulate the expression of TGFβ₃ mRNA in BOECs.

To clone the Yemule aries inhibin α (INHα) subunit gene and express its recombinant protein,total RNA was extracted from Yemule aries ovary tissue by Trizol,and a pair of primer was designed according to the published aries INHα subunit gene sequence (GenBank No.XM_004004955.1). INHα subunit gene coding sequence was amplified by RT-PCR.The mature peptide cDNA was further amplified and cloned into the Scal Ⅰ and EcoR Ⅰ sites of the pEGFP-N1 expressing vector to generate the recombinant plasmid pEGFP-INHα.The plasmid was transformed into DH5α,after appraisal,sequencing,proves that successful build INHα subunit gene eukaryotic expression vector.The sequence result was compared with homologous sequences from animals including human,gorilla,cow and other animals alpha subunit of INHα subunit gene sequence homology comparison analysis.Sequence alignment showed that ranks of their similarities were above 50%,proved that INHα subunit gene was highly conservative.By RT-PCR and Western blotting method verified the eukaryotic expression vector of statin INHα subunit gene expression and protein expression of success in BHK cells.

The objective of this study was to solve the problem of construction a transgenic safety and site-specific integrated and marker-free LacS gene expression vector.Therefore,this experiment using plasmid pEGFP-N1 as the original framework,using PCR and restriction sites to add two with LoxP sequence at both ends of the marker genes in the pEGFP-N1 plasmid,attB sequence was added at the upstream of the multiple cloning sites for site-specific integration,then add the gene BC promoter-LacS-PolyA into the multiple cloning site.By restricted endonucleases digestion,these 3 fragments,Loxp,attB and LacS,were detected in the vector pEGFP-N1-LacS,PCR products and fragment size consistent with the expected results,the results of sequencing and oligonucleotide sequences also corresponding favorable,proved that each gene fragment was correctly connected to the corresponding position of the plasmid.In conclusion,the site-specific integrated and marker-free LacS gene vector was constructed successfully.

The study was aimed to understand the relationship between the gene sequence of SRV₉ strain of oral rabies vaccine and biological characteristics,compare its major antigen site with other domestic and overseas strains of rabies vaccine,and clear the molecular basis as oral vaccine strain.The SRV₉ N,P,M,G and L genes were cloned by RT-PCR,and connected into the pMD19-T vector,respectively,identified by enzyme digestion,sequenced by DNAStar software,and compared its sequences and the main antigen sites with other 11 strains.The results showed that the sequence homologies of SRV₉ N,P,M,G and L genes with others were 81.8% to 100.0%,the whole genome phylogenetic tree showed that,all in the same big branch,they were belonged to genotypeⅠ,the evolutionary distance among SRV₉ and ERA,SAD B19,SAG-2 were near,and were far among SRV₉,aG and RC-HL.The amino acid homology analysis showed that the homology was high among SRV₉ and SAD B19,SAG-2,ERA in extramembrance,transmembrane and intramembrane regions.The results would provide experimental basis for the study of gene recombinant attenuated vaccine strain by molecular biology techniques and development of oral rabies vaccine.

Bone marrow mesenchymal stem cells (BMSC) had multiple differentiation potentiality,but their life cycle was limited,in order to make further study on the effect of telomerase reverse transcriptase (TERT) gene on activity of telomerase,our research adopted the method of transfecting exogenous TERT into mesenchymal stem cell technology,constructed recombinant expressing vector pcDNA3.1-EGFP-TERT,and then transfecting into ovine BMSCs,and through selection and identification,recombinant expressing vector pcDNA3.1-EGFP-TERT was successfully expressed in BMSCs.Then the TERT-BMSCs were cultured in vitro and the determination of growth curve was conducted,finally,the expression of the original stem cell factor were identified by PCR technique.The results showed that TERT-BMSC had the ability of rapid amplification,cell shape was good and still had the characteristics of stem cells.We got the conclusion that the expression of exogenous TERT gene could activate the activity of BMSCs'telomerase.

The assay was aimed to explore the biological characteristics of bone morphogenetic protein 4 (BMP4) of sheep,NCBI,DNAMAN DNAStar,TMHMM Server v.2.0,PsortⅡ,SignalP various bioinformatical softwares were used to speculate the physical and chemical properties,hydrophobic property,phosphorylation site,conservative structure domain,protein secondary structure of BMP4 protein.Also,the three-dimensional structure was forecasted with the SWISS-MODEL Workspace software.The results indicated that the BMP4 of sheep had high homologies with the BMP4 of various species.The encoded protein was a hydrophilic protein which was unstable.There was no transmembrane regions and it was likely to be located in the nucleus.What was more,there was signal peptide and eighteen phosphorylation sites.Through the forecast of functional domains,the protein had two functional domains,including the transforming growth factor-beta (TGF-beta) superfamily and TGF-beta propeptide superfamily.The result was consistent with the function of BMP4 gene family,it also demonstrated that BMP4 was a growth factor and it had the function of signal transduction.The amino acid homology between the predicted 3D structure of protein and template 3bmp.1.A was 88.29%.The bioinformatics analysis of BMP4 gene could provide reference for the further study in practice.

Two pairs of primers used to respectively amplify transmissible gastroenteritis virus (TGEV) S gene and porcine epidemic diarrhea virus (PEDV) M gene were designed to develop a method of differential diagnosis of TGEV and PEDV.The established double PCR could detect S gene of TGEV with the length of 299 bp and M gene of PEDV with the length of 437 bp.Negative results using CSFV,PCV2,PRRSV and PRV as control were obtained.The detection limit of this method was 10⁴ copies/μL.68 clinical samples collected from swine farm were submitted to detect TGEV and PEDV,and the result showed that the established double PCR method with the characteristics of high sensitivity and high specificity could be widely used in clinical diagnosis and epidemiological investigation.

The purpose of this research was to establish a rapid detection serological method against avain infectious bronchitis virus (IBV).In this study,an indirect ELISA method was established using IBV as the detected antigen and a variety of testing conditions were optimized.The results showed that the optimal antigen coating concentration was 19.2 μg/mL and the optimal condition for coating was incubated at 37 ℃ for 1 h and then 4 ℃ overnight.The optimal dilutions of serum and enzyme labeled antibody were 1:800 and 1:7 000 incubated at 37 ℃ for 60 min,respectively.The optimal condition of chromogenic substrate was incubated at 37 ℃ for 10 min in the dark.The specificity,repeatability and sensitivity tests proved that the indirect ELISA did not cross-react with positive antiviral sera of other chicken diseases,had good repeatability and could detect IBV antibody when serum was diluted 1:12 800.We concluded that the established indirect ELISA was specific,repeatable and sensitive.

The aim of this study was to establish a simultaneous triple PCR detection method for Staphylococcus aureus (S.aureus),Pseudomonas aeruginosa (P.aeruginosa) and Klebsiella pneumoniae (K.pneumoniae).Three pairs of specific primers had been designed according to nuc gene of S.aureus,toxR gene of P.aeruginosa and PhoE gene of K.pneumoniae.The triple PCR reaction conditions were optimized on the basis of single PCR methods.At the same time,specificity,sensitivity and repeatability tests of the triple PCR method were studied,and the results of bacteria isolation and culture and the triple PCR method were compared.The results showed that the amplification product sizes were 484,278 and 368 bp,respectively.The optimal annealing temperature was 56 to 59 ℃,the concentrations of primers were all 0.2 μmol/L,dNTP concentration was 200 μmol/L,Mg²⁺ concentration was 2.5 mmol/L.The specificity test showed that there was no cross reaction between these three bacteria templates and other eight kinds of common bacteria templates,such as Bordetella bronchiseptica.The minimum of simultaneous detection of three bacteria genomic DNA was 10^-5 ng/μL.The results of three repeatability tests of triple PCR were the same which indicated the repeatability was good.30 samples from mice were detected by bacteria isolation and culture and the triple PCR method,the detection rate of triple PCR was slightly higher than the bacteria isolation and culture.The positive samples detected by bacteria isolation and culture were also positive detected by triple PCR.The results showed that a specific,sensitive and efficient triple PCR system had been established and could provide the technical support for bacteria detection and epidemiological investigation of laboratory animals.

The assay was aimed to isolate,culture and identify Orf virus (ORFV) in Chongqing area,and investigate the expression of the F1L gene sequence in E.coli,the virus in the scab samples collected from suspected ORFV infected lamb was isolated in MDBK cell.Moreover,the F1L gene was amplified by PCR and ligated into pET-32a for expression in E.coli.The recombinant plasmid pET-32a-F1L was constructed with the introduction of restriction enzymes NotⅠand EcoRⅠ.The isolated virus was ORFV.SDS-PAGE analysis and Western blotting showed that the target protein was highly expressed with 57.4 ku in lenght at 5 h in E.coli as inclusion bodies and had good reactivity.

In order to comparatively analyze the differences and similarities of intestinal flora from all ages of giant pandas,we built 16S rDNA clone library of three captive elderly giant pandas'fecal bacteria and made restriction fragment length polymorphism (RFLP) and sequencing analysis by using restriction enzymes HinfⅠ and MspⅠ.The results showed that the intestinal flora of elderly giant panda were mainly attributable to two bacteria:Proteobacteria and Firmicutes.Between them,the main in Proteobacteria of intestinal flora of elderly giant panda were Escherichia coli,followed by Pseudomonas sp.,Shigella and Aeromonas;The main in Firmicutes were Streptococcus,followed by Weissella,Clostridium;Of which there were a certain proportion of bacteria were uncultured bacteria.The article firstly established a rich clone library of intestinal flora from elderly giant pandas,which provided a reference to comparatively analyze the similarities and differences in structure of intestinal flora from all ages of giant pandas,as well as provided the important information about reasonable feeding elderly giant pandas and protecting health of elderly giant pandas.

In order to provide more information for further studies of miRNA's biological function,the identification of microRNA of bovine was studied.A Solexa sequencing of multiple tissues from a Simmental bull and a Holstein cow was conducted,and then bioinformatics analysis was performed.Meanwhile,we randomly selected and validated 3 miRNAs (bta-miR-2346-5p,novel_miR-48,novel_miR-86) by stem-loop RT-PCR.From this data set,we identified 604 miRNAs,including 429 known miRNAs and 175 novel miRNAs.And the stem-loop RT-PCR result showed that the 3 miRNAs were ubiquitously expressed in longissimus dorsi muscle,cardiac muscle,liver,spleen,lung,kidney,large intestine and small intestine.The study enriched the amount and kinds of bovine miRNAs which provided more credible information for the miRNAs biological functions study in various species.

This study was aimed to obtain polyclonal antibody against swine pseudorabies virus (PRV) Min A strain,and provide a theoretical basis for the study of the treatment and detection of PRV.This study was performed on PK-15 cell and proliferation of PRV was measured as TCID₅₀ 10^-7.372,the protein concentration of PRV was measured as 3.6 mg/mL.Choosing five healthy male rabbits (2.5 kg±0.2 kg) as experimental animals and using PRV obtained as the antigen,we got polyclonal antibody against PRV.Antiserum titer was 1:32 000,antigen coating dilution was 1:40,the best coating conditions was 4 ℃ 12 h,the best blocking time was 1 h,the best working dilution of enzyme labled antibody was 1:8 000,the result of cell lesions neutralization test showed that PRV antiserum prepared in this assay at 1:16 dilution could protect 50% of PK-15 cells from being infected by PRV,and negative serum couldn't protect PK-15 cells from being infected by PRV.The study successfully prepared polyclonal antibodies against PRV.

Long noncoding RNA (LncRNA) constituted a novel class of non-protein coding transcripts longer than 200 nt,and were localized in nuclear and cytoplasmic.LncRNA can regulate gene expression in cis and trans,were recognized as a kind of key regulatory molecules.LncRNA were involved in various aspects of cell biology and disease,such as cell differentiation,X chromosome inactivation,cancer,immune,neurological and cardiovascular system disorders.In this paper,we mainly summarized the origions,research status and action mechanism of LncRNA,as well as overseas and domestic research status of LncRNA in animal husbandry and veterinary.Finally,the potential research and application prospects of LncRNA in animal science were put forward.

This experiment was conducted to study the effects of different levels of compound Chinese medicine on growth performance,nutrients apparent digestibility,immune function and slaughter performance of weaning Rex rabbits.70 healthy Rex rabbits at age of 60 days were divided into 7 groups with 5 replicates per group and 2 rabbits per replicate.Group Ⅰ was fed with basic diet as control group,group Ⅱ was fed the basal diet supplemented with antibiotic (contains 50 mg/kg milligrams bacitracin zinc,10 mg/kg colistin sulfate).The other five groups (Ⅲ,Ⅳ,Ⅴ,Ⅵ and Ⅶ) were fed the basal diet supplemented with 1%,2%,3%,4%,5% compound Chinese medicine,respectively.The pre-test period lasted for 7 days and the trial period lasted for 45 days.The results showed as follows:①The ADG in group Ⅳ was significantly higher than that in groups Ⅰ,Ⅱ and Ⅲ (P< 0.05),the F/G in group Ⅳ was significantly lower than that in other groups (P< 0.05).②The apparent digestibility of DM in group Ⅳ was significantly higher than that in groups Ⅰand Ⅱ (P< 0.05),the apparent digestibility of DM in group Ⅶ was significantly higher than that in group Ⅰ (P< 0.05).The apparent digestibility of CF in group Ⅳ was extremely significantly higher than that in group Ⅰ (P< 0.01),the apparent digestibility of CF in group Ⅶ was significantly higher than that in group Ⅰ (P< 0.05).The apparent digestibility of NDF in group Ⅳ was extremely significantly higher than that in group Ⅰ (P< 0.01),and significantly higher than that in groups Ⅱ,Ⅲ,Ⅴ and Ⅶ (P< 0.05).The apparent digestibility of ADF in group Ⅳ was significantly higher than that in groups Ⅰ,Ⅱ,Ⅴ and Ⅶ (P< 0.05).The apparent digestibility of CP in group Ⅳ was significantly higher than that in groups Ⅰ and Ⅱ (P< 0.05).③ There were no significant differences in the contents of IgG,IgA and IgM of serum among all groups (P> 0.05),the spleen index in group Ⅳ was significantly higher than that in groups Ⅰ,Ⅱ,Ⅲ and Ⅴ (P< 0.05).④There were no significant differences in the slaughter rate,half eviscerated slaughter rate and all eviscerated slaughter rate among all groups (P> 0.05).In summary,adding compound Chinese medicine and antibiotics in the feed both could improve the growth performance,nutrients apparent digestibility,immune function and slaughter performance of weaning Rex rabbits,and the appropriate compound Chinese medicine level in the diet of weaning Rex rabbits was 2%.

In this paper through digestive and metabolic experiments studied the voluntary intake and apparent digestibility of cottonseed hulls (kernel-contained CH,kernel-free CH) and cornstalk on sheep to evaluate the nutrition value and digestion characteristics of cottonseed hulls.12 of adult Xinjiang merino rams were selected and divided into two groups (n=6 each group),and on the basis of 300 g/(head·day) of mixed concentrate,fed with kernel-free CH by 400 (group 1) or 800 (group 2) g/(head·day) respectively,to estimate the digestibility of CH by difference method;As well,another 18 of adult Xinjiang merino rams were selected and divided into three groups (n=6 each group),and on the basis of 300 g/(head·day) of mixed concentrate,cafeteria feeding with kernel-contained CH (group 3),kernel-free CH (group 4) or ground cornstalks (group 5) respectively,to study the effects of three diets on the voluntary intake,digestion and metabolism of sheep.The results showed that the apparent digestibility of the DM,OM,CP,cellulose,hemicellulose and energy of kernel-free CH by sheep were 46.71%,47.37%,-53.40%,66.60%,62.48% and 42.59%,respectively.There were no significant differences in the intake of DM,OM,cellulose,hemicellulose,energy and Ca between kernel-contained CH and kernel-free CH groups (P> 0.05);Intake of CP and P in kernel-contained CH group were extremely significantly higher than that in kernel-free CH group (P< 0.01);Intake of lignin in kernel-contained CH group was extremely significantly lower than that in kernel-free CH group (P< 0.01).There were no significant differences in the apparent digestibility of DM,OM and energy between kernel-contained CH and kernel-free CH groups (P> 0.05).The apparent digestibility of cellulose and hemicellulose in kernel-free CH group were significantly and extremely significantly higher than that in kernel-contained CH group (P< 0.05;P< 0.01),respectively.The apparent digestibility of CP in kernel-contained CH group was extremely significantly higher than that in kernel-free CH group (P< 0.01).The apparent digestibility of DM,OM,CP,cellulose and energy in ground cornstalks group were extremely significantly higher than that in kernel-contained CH and kernel-free CH groups (P< 0.01),however,the apparent digestibility of Ca and P were lower than that in kernel-contained CH and kernel-free CH groups (P> 0.05).Intake and retention of nitrogen in kernel-contained CH group were extremely significantly higher than that in kernel-free CH group (P< 0.01);The was no significant difference in the retention of nitrogen between kernel-free diet group and cornstalk diet group (P> 0.05).It was concluded that CH was a kind of special forage with larger intake,higher digestibility on the fiber substances and energy,but with a serious obstruction on protein digestion;The nutritional value between kernel-free diet and cornstalk diet was almost equal,and with lower nitrogen digestibility on kernel-free diet.

The aim of this study was to investigate the effect of silage sugarcane leaf replacing different ratio of King grass on growth performance,nutrient apparent digestibility and serum biochemical indices of Hainan Black goats.A total of 20 3-month-old Hainan Black goats with an average weight of (6.96±0.49) kg were selected (half male and hale female) and randomly divided into 5 groups with 4 replicates per group and 1 goat per replicate.The goats in the control group were fed the basal diet including concentrate and roughage,roughage was King grass.The goats in the groups 1 to 4 were fed with the equal concentrate,roughage (fresh King grass) was replaced with 25%,50%,75% and 100% silage sugarcane leaf,respectively.The pre-test period lasted for 5 days and the trial period lasted for 30 days.The results showed that compared with control group,the average feed intake of crude feed and average feed intake of total dry matter in group 3 were significantly increased (P< 0.05),there were no significant differences in the average daily gains,average feed intake of concentrate and F/G among all groups (P> 0.05).Compared with control group,the apparent digestibility of Ash and P in group 3 were significantly increased (P< 0.05),the apparent digestibility of Ash,NDF,ADF and Ca in group 4 were significantly increased (P< 0.05).No significant difference was showed between groups 3 and 4 (P> 0.05).There were no significant differences in serum biochemical indices among all groups (P> 0.05).In conclusion,silage sugarcane leaf replaced certain percentage of King grass in roughage could improve the growth performance and nutrient apparent digestibility to some extent,and had no bad effect on the serum biochemical indices of goats,the optimal proportion of silage sugarcane leaf in roughage was 75%.

In order to study the tolerance dose of Kunming mice to montmorillonite as a mycotoxin adsorbent in diets,the experiment selected 60 healthy male Kunming mice with initial average body weight of (17±0.12)g,randomly divided them into 5 treatments with two replicates per group and 6 mice per replicate.Five experimental diets were added to 0 (control group),0.5%,1%,2% and 5% montmorillonite,respectively.After 30 days,the growth performance,nutrients apparent digestibility,serum biochemical indices and internal organization were tested to determine the tolerance dose of montmorillonite for Kunming mice.The results showed that the ADFI was significantly increased in 0.5% group (P< 0.05),while there were no significant differences in ADFI and ADG between 1% group and control group (P> 0.05),then the ADFI was significantly decreased with the increasing of montmorillonite supplemental level (P< 0.05).The ADG in 2% group had no significant difference when compared with the control group (P> 0.05),while significantly lower than that in 0.5% and 1% groups (P< 0.05).The apparent digestibility of DM,CP and EE showed no significant effect at any level of montmorillonite (P> 0.05).Serum ALT,AST activities and MDA content were gradually increased with the montmorillonite supplemental level increasing,and that in 1%,2% and 5% groups were significantly higher than that in control group (P< 0.05),but there were no significant differences between 0.5% group and control group (P> 0.05).Tissue sections showed that mice livers were damaged in varying degrees when the montmorillonite supplemental level was 5%.In conclusion,based on a comprehensive consideration of all indexes,it could be seen that the tolerance dose of the tested montmorillonite for Kunming mice was between 0.5% and 2% when mycotoxin contents in diets were in security range.

The experiment was conducted to study the effects of compound probiotics supplementation on blood biochemical indexes and antioxidant capacity of Yili mare during lactation.A total of 24 Yili mare during lactation were divided into four groups with similar age,parity,lactation months,i.e.groups Ⅰ,Ⅱ,Ⅲ and control group.The control group and trial groups fed with the same based diet,groups Ⅰ,Ⅱ,Ⅲ were supplemented with 0.05 g of Bacillus subtilis (2.0×10¹¹ CFU/g) and 0.133 g of plant Lactobacillus (3.0× 10¹⁰ CFU/g),0.10 g of Bacillus subtilis and 0.266 g of plant Lactobacillus,0.15 g of Bacillus subtilis and 0.399 g of plant Lactobacillus, respectively.The results showed as follows:Compared with control group,total protein,albumin,blood urea nitrogen contents and GSH-Px,SOD activities of blood in trail groups were no significant differences (P >0.05);But supplementary feeding compound probiotics total protein,albumin contents and SOD activity of blood in trail groups had a upward trend.GLB content of blood in group Ⅱ was significantly higher than the control group and groupⅠ(P <0.05);CAT activity of blood in group Ⅱ was significantly higher than control group and group Ⅲ (P <0.05);T-AOC activity of blood in group Ⅱ was significantly higher than groupsⅠand Ⅲ (P <0.05);No significant difference was observed among other groups (P >0.05).Therefore,supplementary feeding plant Lactobacillus and Bacillus subtilis complexes had enhanced immune function,improved the nitrogen deposition and the body's antioxidant capacity of Yili mare during lactation,when supplementary feeding 0.10 g of Bacillus subtilis and 0.266 g of plant Lactobacillus complexs was more obvious effect.

Rice is one of the major food crops for humans.It is also an important feed source of animals.In recent years,several transgenic Bacillus thuringiensis (Bt) rice were bred by domestic and international research institutes.These varieties of transgenic Bt rice can kill insect pests such as rice stem borer,yellow rice borer and rice leaf roller.They can also reduce the use of pesticides and boost rice yields.People pay much attention to safety of transgenic Bt rice as it is one of the major food crops.As yet there is no definite evidence that the transgenic Bt rice has safety issues.But we still need to monitor the results of long-term consumption of the rice continuously.This article mainly reviews the development,potential risk and safety assessment method,nutritional value and toxicity of transgenic Bt rice,the impact of growth and immune of animals,the foreign gene and the foreign protein in vivo residual.

Carnitine,an indispensable metabolite,was found in muscle in 1905.Carnitine plays an important role in the transport of long-chain fatty acids from cytosol to the mitochondrial matrix,where fatty acid β-oxidation takes place.In recent years,carnitine,an essential nutrient in improving livestock production and reproductive performance,has more and more widely application in animal production.In this review,we summarize the biosynthesis and transport of carnitine,as well as the application of animal production,which provides theoretical basis and insight into further research on carnitine.

For observing and analyzing the motpha,distribution and quantity of mast cells (MC),the differentiation time and degree of immune organs and digestive organs in different ages of chicken were fixed by Carnoy's,stained by AB/SO.The results showed that MC concentrated in the thymus medulla;In spleen,MC distributed in the junction of red pulp and white pulp;MC were seen in the tissue around the lymphoid nodules in bursa of fabricicus tissue. MC distributed with the lamina propria and compound tubular gland in stomach;MC distributed in the lamina propria,sub mucosa of intestine;More MC in liver concentrated around sinusoids and the central vein.The number of MC in the main immune organs and digestive organs of 1 to 7 day old chicks showed an increasing trend.

In this experiment,dry chemical method and wet chemical method were used to detect electrolyte levels of Cchl1a3-R528H gene knock-in mice,we compared the two different detection methods and analyzed their correlation.Firstly,we tested the precision of the electrolyte analyzer,and then selected the same mouse,collected the whole blood from tail with dry chemical method to detect electrolyte levels and collected the whole blood from the retro-orbital venous plexus with wet chemical method to detect electrolyte levels,and compared the two different detection methods.The results showed that the CV of Na⁺,Cl^- and K⁺ detected by dry electrolyte analyzer were(2.06±0.18)%,(3.61±0.22)% and (4.40±0.19)%,respectively,all < 4.5%.Compared with the results detected by two different detection methods above-mentioned,the difference was not statistically significant (P> 0.05),and there were linear positive correlations between the two detection methods in Na⁺,Cl^- and K⁺(r of Na⁺ and Cl^- were both > 0.975).The result indicated that the dry electrolyte analyzer had high precision and good stability.The detected electrolytes results by two detection methods were with high correlation and good comparability,subsequent experiments could use dry chemical method to replace wet chemical method to detect electrolytes.

This study was designed to compare the blood physiological and biochemical parameters between plateau Juema minipigs and plain Bama minipigs,and provide a theoretical basis for Juema minipigs served as experimental animals in the future.The blood samples of Juema minipigs were taken in Gannan,and the blood samples of the Bama minipigs were taken in Xi'an,24 blood physiological and biochemical parameters were tested using an automatic analyzer of blood cells and biochemistry.Between the plateau Juema minipigs and Bama minipigs,there were significant differences in PLT,RDW-CV and RDW-SD (P< 0.05) and extremely significant differences in RBC,HGB,HCT,MCH,MCMH,GGT,GLU and CRE (P< 0.01).The plateau Juema minipig had an altitude acclimatization,so it was better than Bama minipig in plateau medical science.

The single nucleotide polymorphisms (SNPs) of ovine lymphocyte antigen DQB1 (OLA-DQB1) gene exon 2 was amplified by PCR-SSCP method from 148 healthy and 60 infected with Brucella Chinese Merino sheep and then PCR products of different alleles were sequenced to determine the polymorphism loci of the gene.The differences in gene frequency and genotype frequency of each SNP loci were analyzed statistically to analyze its correlation with brucellosis susceptibility.The sequencing result showed that 43 SNPs were detected in 270 bp DNA sequence,the gene frequencies of G196A allele had extremely significant difference in case and control samples (P< 0.01),and its genotype frequencies presented significant difference (P< 0.05).Similarly,C211T allele was significantly different in case and control samples (P< 0.05).The results showed that the polymorphism of OLA-DQB1 gene exon 2 might be a significant association gene with brucellosis susceptibility.

The aim of this study was to investigate the expression profile of Pax7 (paired box 7) gene in different tissues and its developmental expression in longissimus dorsi of pigs.The expression profiles of Pax7 gene at the levels of mRNA and protein in 12 different tissues including heart,liver,spleen,lung,kidney,stomach,small intestine,hypothalamus,cerebellum,longissimus dorsi,biceeps femoris,psoas major of Mashen pigs at birth and the developmental expression patterns in longissimus dorsi at seven stages (1,30,60,90,120,150,and 180-day old) of Mashen and Large White pigs were studied by quantitative Real-time PCR and Western blotting.The results showed that the Pax7 mRNA was expressed in longissimus dorsi,biceeps femoris,psoas major,hypothalamus,and cerebellum,wherease the Pax7 protein was only expressed in skeletal muscles of longissimus dorsi,biceeps femoris,and psoas major.The developmental expression patterns of Pax7 mRNA and protein in Mashen pig was basically in accordance with those in Large White pig.The mRNA expression amount of Pax7 gene in longissimus dorsi was the greatest at 30-day old (P <0.01),followed by the expression at 1-day old,and kept lower stable levels at other stages both in Mashen and Large White pigs.At the stages of 30-day,90-day,and 180-day old,Pax7 mRNA expression amounts in Mashen pig were significantly higher than those in Large White pig (P <0.01).The Pax7 protein expression at 1-day old was the greatest (P <0.01),followed by it at 30-day old,which were significantly greater than those at other stages,at which Pax7 expression was in lower stable levels.The Pax7 protein expression from one-day old to 180-day old in Mashen pig was significantly greater than those in Large White pig (P <0.05;P <0.01).The expression of Pax7 gene could associated with tissue,age,and background of pig breed.

The article through the analysis and research to the effects of parity of dam and breeding date on the litter size of Inner Mongolia White cashmere goats,determine the optimal mating time of Inner Mongolia White cashmere goats,in order to improve the efficiency of reproductive.Data used were 1998 to 2013 reproductive performance records collected from the Arbas stock farm in Inner Mongolia,China.The GLM procedure of SAS 9.0 software was used to determine the significant effect of the parity of dam and breeding date,the results showed that the parity of dam and breeding date had significant effect on litter size (P< 0.01).Litter size was the lowest in 1st parity does (1.37),the significantly increase in litter size with advance in parity and decline after six parity;Mating cycles for September 29th to October 16th had the highest litter size (1.76),and the estrous months and estrus peak time mainly concentrated in October,so this stage in production could be used as breeding period of Inner Mongolia White cashmere goats.

Tumor necrosis factor α (TNF-α) gene plays an important role in innate immunity and inflammatory responses,this study aimed at analyzing the relationship between the polymorphism of TNF-α gene promoter and gene expression in Large White pigs,and further providing a scientific basis for analyzing the genetic effect of TNF-α gene promoter C-791T mutation.This study detected the polymorphism at C-791T site of TNF-α gene promoter in 201 heads of Large White pigs by PCR-SSCP method.Meanwhile,the expression of TNF-α gene in 11 tissues was assayed by Real-time PCR and analyzed the differential expression among individuals with different genotypes at C-791T site.The results showed that there existed one mutation (C-791T) in TNF-α gene promoter and three genotypes were detected,which were CC, CT and TT.The mRNA expressions of TNF-α were higher in spleen,lung,thymus and lymph of weaned piglets.TNF-α gene expression levels of TT and CT genotypes individual were generally higher than CC genotype individual in 11 tissues,and the mRNA expression in TT and CT individuals were significant higher than that in CC individuals in the duodenum and jejunum tissue (P <0.05).Therefore,the C-791T mutation of TNF-α gene promoter indeed had a significant influence on the mRNA expression level of TNF-α gene,which indicated that it was necessary for us to carry out an in depth study of TNF-α gene promoter C-791T polymorphism as genetic markers of enhancing the resistance to intestinal pathogenic bacteria.

This study was aimed to investigate the distribution of genetic polymorphisms of Myf5 and MyoD genes in Hainan Wuzhishan pig.PCR-RFLP method was used to detect the Myf5 and MyoD of 80 Wushishan pigs.Some characters of Myf5 and MyoD genes were analyzed by bioinformatics tools.The main results were as follows at the HhaⅠ-RFLP locus of exon 1 in Myf5 gene,CC and CG genetypes were major in Wuzhishan pig.At the Hsp92Ⅱ-RFLP locus of exon 1 in Myf5 gene,AA genetype was major in Wuzhishan pig.At the DdeⅠ-RFLP locus of intron 1 in MyoD gene,allele A was predominant in Wuzhishan pig.In addition,bioinformatics analysis revealed that Myf5 and MyoD genes promoter and exon 1 region contained CpG islands.In this study,we obtained the sequences of Myf5 and MyoD genes which laid a foundation for further studying their functions in Hainan Wuzhishan pig.

In this study,7 945 litter records from 2 904 Large White sows were used to determine the effect of parity,mating season and the classified gestation length on total number born (TNB),number born alive (NBA),number healthy birth (NHB),number weak birth (NWB),deform fetus,number born dead (NBD),mummy fetus and stillbirth. Genetic parameters and genetic correlation of the above reproduction traits were estimated by restricted maximum likelihood (REML) procedure.The results indicated that the parity had non-significant effect on deform fetus (P=0.2619) and mummy fetus (P=0.4639),but had significant effect on other 7 reproduction traits (P <0.01).The effect of mating season on gestation length,NBA,NWB,mummy fetus,NBD and NHB were highly significant (P <0.01),while mating season effect on deform fetus was not significant (P=0.3993).Moreover,the effect of mating season on TNB and stillbirth were significant (P <0.05).Effect of different gestation length classification was significant on mummy fetus (P <0.05),and was highly significant on the other 7 traits (P <0.01).The gestation length got the highest heritability,at 0.34,while others below 0.2 as low heritability traits.There was a negative genetic correlation (r_g=-0.10) between gestation length and TNB,while a strong positive genetic correlation exists among TNB,NBA and NHB (r_g >0.80).The genetic correlation between gestation length and other reproductive traits was low,the highest one appears between gestation length and deform fetus reached -0.17.We concluded that the gestation length was medium heritability trait,it was influenced not only by environment factors (such as parity,mating season) but also reproductive traits like TNB,and gestation length could be used as an indirect indicator in pig production.

In order to understand the regularities of A-FABP gene expression in Guizhou native goats,and to establish the theoretical basis of molecular marker assisted selection for meat quality traits,β-actin gene was used as control,expression levels of A-FABP gene in livers,kidneys,hearts,lungs,longissimus muscle,semimembranosus muscle and subcutaneous fat 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.The results showed that A-FABP gene was expressed in all 7 tissues of 5 goat breeds,and the expression levels of A-FABP gene mRNA were all high in subcutaneous fat of 5 goat breeds;In subcutaneous fat,which was the main tissue of A-FABP gene expression in Guizhou native goats,and in longissimus muscle and semimembranosus muscle,which were directly associated with meat production property,A-FABP gene mRNA levels were all high in Qiandongnan small Xiang goat.The results suggested that the tissue distribution patterns of A-FABP gene mRNA in Guizhou native goat breeds were similar to each other.The excellent meat quality of Qiandongnan small Xiang goat might be related with the high expression levels of A-FABP gene mRNA in it's subcutaneous fat,longissimus muscle and semimembranosus muscle.

This study was conducted on 20 949 reproductive records from 1998 to 2012 of 5 257 Sanhe cattle in Xiertala cattle farm,Inner Mongolia,which was a synthetic breed formed in China in 1986.Age at first pregnancy in heifer (AFPH),age at first calving in heifer (AFCH),gestation length in cow (GLC),days open in cow (DOC),and calving interval in cow (CIC) were considered for genetic evaluation.SAS 9.13 and DMU software were used for data processing,and AI-REML combined EM algorithm based on multiple traits animal model was employed for estimating variance components.The heritability for each trait were then calculated,and breeding value was used to analyze the genetic trends.The results showed that the estimated heritabilities of age at first pregnancy in heifer,age at first calving in heifer,gestation length in cow,days open in cow,calving interval in cow were 0.0552,0.0638,0.0527,0.1096 and 0.0844,respectively.The heritabilities were all less than 0.1 except days open (0.1096),indicating these were low inheritable traits.In general,trends of EBVs for each trait didn't show any defined progresses and indicating good reproductive performance maintained in Sanhe cattle.These results lay a theoretical foundation for optimizing breeding programs and improving the accuracy of selection in Sanhe cattle.

The aim of this study was to investigate the association between CARD15 gene StyⅠpolymorphism and susceptibility to tuberculosis (TB) of dairy cows from Yunnan plateau.A total of 201 dairy cows (103 cases and 98 controls) from Kunming,Yuxi and Dali were used in this study.We designed a specific primer pair for PCR-RFLP analysis.By using PCR-RFLP genotyping and PCR sequencing,We analysized allele frequency and correlation between polymorphism of CARD15 gene and susceptibility to bovine tuberculosis in Yunnan province.The results showed that there were three genotypes in Sty Ⅰ enzyme site,AA,AG and GG.In cases,the genotype frequencies of AA,AG and GG were 0.03,0.42 and 0.55,respectively,while in controls were 0.01,0.05 and 0.94,and the allele frequencies of A and G were 0.24 and 0.76 in cases,while were 0.04 and 0.96 in controls.Then analysizing the genetic pattern of polymorphic loci,we found that the best genetic model was dominant and the frequency of AG-AA in cases (44.7%) was apparently higher than that in controls (6.1%) (OR(95% CI)=0.08 (OR< 1)),so extremely significant difference existed between cases and controls (P< 0.0001).The polymorphism of StyⅠenzyme cut site in CARD15 gene was significantly associated with the susceptibility to tuberculosis of Yunnan plateau dairy cows,and AG-AA might be the susceptible genotype to tuberculosis.

Oocytes and early embryo in vitro culture (IVC) was a key technology of mammalian embryo engineering and was the basis of biotechnology such as genetically modified (GM) and cloning research.The factors,which affect the efficiency of in vitro culture for oocytes and early embryo,include the composition,ion concentration and osmotic pressure of the culture medium,and the gas composition and temperature of the culture environment. Recent studies have shown that the oxygen tension in the culture systems can significantly affect the development of embryos,and this effect is mainly regulated by the hypoxia-inducible factor (HIF). This paper reviewed the effects of the oxygen tension on oocyte maturation and early embryo development,and the structure and regulation mechanism of HIF were discussed,in order to provide a theoretical basis for further explore the application of hypoxia method in the in vitro culture of oocytes and early embryo.

The study was aimed to investigate the biological characsteristics and drug sensitive tests of Haemophilus parasuis isolated in Sichuan province.This paper reported the biological characteristics,16S rRNA gene PCR and drug sensitive test of the isolates by technique of microbiological and molecular biology.The results showed that the isolates had micro anaerobic and could produce satellite phenomenon,the isolates were just same in biochemical characteristics with field isolates in other area.All the isolates amplified 821 bp 16S rRNA gene.And drug sensitive tests showed that the isolates were highly sensitive to cefradine,ampicillin,cefradine,amoxicillin,cefaclor,ofloxacin,cefotaxime and furadantin,the sensitive rate was 75.61% to 87.80%,but the isolates were high resistance to lincomycin,carbenicillin,enrofloxacin,amikacin and sulfamethoxazole,and multiple-drug resistance was found among the isolates,the number of bacterial strains resistant to antibiotics were more between five and eight,the resisitance strains were 21.95% to 30.49%,7 isolates were even resistant to 13 antibiotics.The results showed the phenomenon of drug resistance of the isolates was very serious.

In a poultry farm of Chengdu city,Sichuan province,isolation,identification and drug sensitivity test of Salmonella were conducted from 156 dead embryos.In this experiment,we used Salmonella chromogenic medium,biochemical identification of enterobacteriaceae,trisaccharide iron experiment,Salmonella polyvalent serum and 16S rRNA PCR to identify suspected strains,PCR identification of isolated Salmonella were conducted with 6 kinds of virulence genes.The results concluded that Salmonella separation rate was 15.4%(24/156),including Salmonella typhi accounted for 58.3%(14/24); Detection rates of fimY,invA and mgtC virulence genes were all 100%;The isolated Salmonella were resistant to most clinical drugs to Salmonella.

This study was designed to explore the effect of nitrogen and phosphorous on the resistance of E.coli from environment and the mechanism.Microcosms were established to study the effect of nitrogen and phosphorous on the resistant phenotype of E.coli to chloramphenicol (CHL).cat gene of isolated drug-resistant strains and susceptible strains were detected.The results showed that,different concentration of nitrogen and phosphorous could induce the formation of antibiotics resistance of E.coli to CHL.The rate of cat gene of 46 strains of chloramphenicol resistant E.coli was 89.13%,which was 0 in the 16 strains of chloramphenicol sensitive E.coli.The results indicated that,nitrogen and phosphorous in the microcosms could induce the formation and maintenance of resistance to chloramphenicol in E.coli,which had correlation with cat gene.

Infectious bronchitis (IB) is an acute and susceptible infectious disease,which has been classified as B loemia and causes a grave threat to the poultry industry.Now,the primary prevention measures of IB are vaccine inoculation.With the diversity of infectious bronchitis virus (IBV) serotype and easy variation,and the weakness of cross protection,the prevention and control of IB were a major problem in poultry industry.In this ariticle,it summarized the molecular biological characteristics, and the development and immunization strategy of the attenuated vaccine of IB to provide scientific references for the research and application of IB attenuated vaccine.

A chronic and infective disease occurred on the farmed Chinook salmon (Oncorhynchus tshaivytscha) in a farm in Huairou district of Beijing and it caused high accumulative mortality.The typical clinical symptoms indicated that it might be bacterial kidney disease (BKD).The purpose of this study was to confirm its pathogen.The typical clinical symptoms of sick fish,morphology observation of bacteria in kidney tissue smears and sequence analysis of bacteria DNA were conducted.A number of gram positive brevi bacteria were observed in kidney tissue print.16S rDNA sequence of K1 strain was more than 99% homology with that of Renibacterium salmoninarum and they were in the same cluster.383 and 320 bp fragment were amplified by PCR and nested PCR.The fragments were sequenced and analyzed.The results showed the fragment was shared 100% nucleotide identity with P57 surface protein gene of Renibacterium salmoninarum. It was indicated that these diseased fish were suffering from BKD caused by Renibacterium salmoninarum.This was the first report about Renibacterium salmoninarum detected in farmed fish in China.

The aim of this study was to investigate the resistance to fluoroquinolones and compound Chinese medicine, and control colibacillosis in fur-bearing animals.14 E.coli strains isolated from diseased fur-bearing animals were detected by PCR for the fluoroquinolone-resistant gyrA gene,and then the PCR fragment of gyrA gene was cloned,sequenced and analyzed for homology.Afterwards,the fragment was labeled with digoxigenin as DNA probe for dot-blot hybridization detection.The resistance to compound Chinese medicine was detected by Oxford-cup tests.The results demonstrated that the positive rates of PCR and dot-blot hybridization were 57.1% and 50.0%,respectively.Only 28.6% of the E.coli strains were resistant to compound Chinese medicine.The results indicated that the resistance to fluoroquinolones in E.coli strains from fur-bearing animals was severe,however,the isolates were much more sensitive to compound Chinese medicine.The results would provide basis for control of colibacillosis of fur-bearing animals.

In order to detect the main bacterial pathogens and analyze the drug resistance for providing therapeutical guidance for drug administration,4 stains were isolated from death chickens of a breeding chicken farm in Qinhuangdao area.All 4 isolates were identified by morphological characteristics,biochemical test,immunofluorescent staining,amplification of 16S rRNA genes by PCR,sequence analysis and drug susceptibility test.The results showed that all 4 isolates were gram-negative,bacillus brevis with green fluorescent,and the sequence of 16S rRNA gene of isolates were analyzed and test results showed that specific amplified band was consistent with the expected size.The isolated bacteria were identified to be Pseudomonas aeruginosa.Results of drug susceptibility test indicated that the bacterial isolates were highly sensitive to gentamycin,amikacin,spectinomycin,ceftazidine,ceftriaxone,levofloxacin,enrofloxacin,florfenicol,polymyxin B and fosfomycin,intermidiate sensitive to tetracycline and doxycyclin,resistant to neomycin,cefotaxime,cephalothin,amoxicillin,ampicillin,erythromycin,trimethoprim-sulfamethoxazole and oxygen sulfamethoxazole isoxazole.The results suggested that the resistances of Pseudomonas aeruginosa isolates to commonly used antibiotics were serious.So antibiotics should be reasonably selected based on the result of drug susceptibility test in this farm.

Avian influenza (AI) is an acute respiratory disease caused by influenza A virus.Avian influenza virus (AIV) can infect poultry,wild birds and some mammals including human.AI is a big threat to both poultry and human health because the virus can cross the species barrier to get the capacity of transmitting from poultry to human.Vaccination is the most efficient measure against AI outbreaking.Traditional vaccines include inactivated vaccine based on chick embryo and attenuated vaccine.Although the traditional vaccines play important roles in the past AI epidemics,many disadvantages have been proved to exist in traditional vaccines.Forced by major drawbacks of traditional vaccines,several studies focused on the development of novel vaccines.In this review,we reviewed recombinant live vector vaccine,subunit vaccine,DNA vaccine and virus-like particle vaccine of AI in order to provide some references for prevention and control of AI.

Bovine papilloma (BP) is a infectious papilloma induced by bovine papillomavirus (BPV).This disease is easily to recur and evolve into cancer,leading to severe loss of breeding industry.The author mainly summarize the gene types,genetic structure,function of genes and pathogenic mechanism of BPV,providing important references to further study the carcinogenic potential of BPV and to develop antiviral vaccines.