In order to further study the problem of persistent infection and investigate the molecular mechanism of transformation between different biotypes of bovine viral diarrhea virus(BVDV),cytopathic BVDV (CP BVDV) and non cytopathic BVDV (NCP BVDV) were used to infect MDBK host cell and the cells were observed and collected per 12 h from 12 to 72 h,and at the same time the transcriptome analysis were used to analyse the 24 and 48 h MDBK cells.The results showed that at 24 h,compared to MDBK cell infected by NCP BVDV,there were 2 849 up-regulated and 3 347 down-regulated differential genes,while 2 933 up-regulated and 2 831 down-regulated differential genes at 48 h in MDBK cell infected by CP BVDV.The results of GO function classification statistics showed that the molecular function of differential gene mainly involved in catalytic activity,binding activity, enzyme regulator activity,molecular transduction activity and protein binding transcription factor activity. Pathway saliency map showed that the genes involved in autophagy, apoptosis, signal transduction related expression of immunoregulatory factor were preliminary screened. The found of differential gene laid the foundation to further reveal the molecular mechanism of viral pathogenesis,also provided molecular targets for the development of new anti BVDV preparations.

To study the diversity and composition of the bacterial community from cecum samples from Yacha pig, Qingyu pig and Wujin pig, the investigation was designed to reveal mechanism of roughage resistance. Illumina amplicon sequencing of 16S rDNA Tag was used to analyze the cecum microbial diversity of Yacha pig, Qingyu pig and Wujin pig at 90 kg liveweight. The results indicated that the core flora of the three breeds were Bacteroidetes and Firmicutes at the phylum level and Prevotella and Bacteroides at the genus level. Meanwhile bacteria associated with cellulose decomposition were found in all three breeds, in which the number of Fibrobacter and Clostridium in Yacha pig were significantly higher than that in Qingyu pig and Wujin pig (P<0.05),and the number of Prevotella in Yacha pig was significantly higher than that in Wujin pig (P<0.05). The number of Bacteroides in Qingyu pig was significantly higher than that in Yacha pig (P<0.05).The number of Spirochaetes in Qingyu pig was significantly higher than that in Wujin pig (P<0.05). It could conclud that the composition of the cecum bacteria community was similar in above pig breeds, but there were significant differences in distribution and quantity. According to analysis of microbial related to cellulose digestibility, Yacha pig was the strongest among the three breeds, followed by the Qingyu pig and Wujin pig.

This study was aimed to construct the eukaryotic expression plasmid of β₂ adrenergic receptor (β₂AR) and mutants including D130N, C285S and D130N/C285S and demonstrate the plasmids could be expressed in HEK293T successfully. The β₂AR gene was amplified by PCR from pig genome DNA, then subcloned into pcDNA3.1(+) vector by homologous recombination to construct the eukaryotic expression plasmid, added the c-myc tag in the plasmid which named myc-pcDNA3.1(+)-β₂AR. We made the eukaryotic expression vector as the template to construct the mutant, transfected the plasmids into HEK293T, extracted the protein and demonstrated the expression by Western blotting. The results of sequencing showed that the 130th amino acid had been mutate from aspartic acid to asparagine, the 285th amino acid had been mutate from cysteine to serine. Also, we confirmed that the plasmids could be expressed in HEK293T successfully. The results showed that the recombinant plasmid myc-pcDNA3.1(+)-β₂AR, myc-pcDNA3.1(+)-β₂AR-D130N, myc-pcDNA3.1(+)-β₂AR-C285S and myc-pcDNA3.1(+)-β₂AR-D130N/C285S were constructed correctly and could be expressed in HEK293T, which laid the foundation for further studies on the function and the pharmacology activity of β₂AR.

Bovine viral diarrhea virus (BVDV) is one of the most important pathogenic viruses which mainly causes bovine viral diarrhea disease (BVD). BVDV can not only cause serious clinical symptoms, but also lead to decrease of immunity of livestock and infect other pathogens, resulting in significant increase of morbidity and mortality of sick animals, and causes significant losses to the cattle industry. With the development of molecular biology theory and technology in recent years, the research on BVDV has been deepening, and some new understandings have been made to the molecular biology of the virus. In this paper, the progress of molecular biology of BVDV in recent years is described from three aspects of the composition and function of virus, the epidemic situation of BVDV gene and the genetic and mutation of BVDV gene.

To investigate the impact of porcine oocytes in vivo and in vitro maturation (IVM) on the development of porcine somatic cell cloned embryos,the somatic cell cloned embryos cultured in vitro and the sows were treated with hormones to collect mature oocytes in vivo,and the cleavage rate, blastocyst rate and embryo implantation were compared. The results showed that the average number of ovulation in PGC+PMSG+HCG group was significantly higher than that of PGC+HCG,PMSG+HCG and the natural estrus groups (P<0.05). The oocytes collected in vivo could be used for the construction, and the available oocytes rate reached more than 90%,and there was no significant difference among the four groups (P>0.05),which indicated that groups treated by hormone could obtain more available oocytes and the quality of oocytes was not significant different. In vivo and in vitro matured oocytes were used as nuclear transfer embryos of recombinant receptor,the fusion efficiency (80.31% and 79.29%) and cleavage rate (90.40% and 86.51%) were not significant different (P>0.05), but the proportion of in vivo matured oocytes cloned embryos developed into the blastocyst stage was significantly higher (P<0.05). The reconstructed embryos made from in vivo and in vitro matured oocytes were transplanted into surrogate sows (transferred 30 or 60 embryos),10 piglets were born in in vivo maturation of cloned embryo transfer group,while there was no implantation in in vitro maturation of cloned embryo transfer group. The results showed that high quality oocytes obtained by superovulation could significantly increase the blastocyst rate of embryos,reduce the number of embryos transferred and improve the pregnancy rate of surrogate sows.

In order to study the hypoglycemic effect of soluble epoxide hydrolase inhibitors (sEHI) on diabetic mice,the diabetic mice model were successfully induced by high-fat diet and streptozotocin (STZ). The diabetic mice (C57BL/6J) were randomly divided into three groups,including normal control group,diabetic control group and sEHI group,the mice in three groups were fed high-fat diet+1 mg/kg sEHI (t-AUCB),high-fat diet+same volume normal saline,normal diet+same volume normal saline,respectively,and intervention last for 4 weeks. During the experiment,the body weight,blood glucose were recorded,and performed oral glucose tolerance test was conducted. After the experiment,indicators of serum lipids,liver peroxidation and pathological analysis of pancreatic tissue were measured. The results showed that compared with diabetic control group,the fasting blood glucose of mice in sEHI group was significantly reduced (P<0.05),and their glucose tolerance was improved. And the TC (P<0.05),TG (P<0.05) and LDL (P>0.05) in sEHI group were decreased,while the LDL/TC was significantly increased (P<0.05).The SOD and CAT activity in liver of diabetic mice treated with sEHI were significantly increased (P<0.05) and MDA activity was significantly decreased (P<0.05).Based on mouse pancreatic tissue sections,we speculated that sEHI had ability to repair activity on damaged islet tissue. In conclusion,the sEHI could reduce the blood glucose level and the free radical damage,inhibit lipid peroxidation,repair of damaged islet tissue in mice.

In order to understand the blood physiological and biochemical reactions after long-distance transport, 23 Akhal-teke horses (male horse 14, mare 9, aged 2 to 6 years old), were selected and transported from Russian to Alashankou of Xinjiang (5 d road transport). 7 and 35 d after reaching the destination, the jugular vein blood were collected for the determination of physiological and biochemical indicators and stress-related hormone levels. The remote control horses without transport (15 local Akhal-teke horses,male horse 10, mare 5, aged 6 to 10 years old) were used for comparative study. The results showed that on the 7th and 35th day after transport, the number and proportion of granulocytes (Gran), hematocrit (HCT), blood platelet (PLT) and alkaline phosphatase (ALKP) were significantly or extremely significantly higher than those of the local Akhal-teke horses (P<0.05; P<0.01), while the number and percentage of intermediate cells (Mid) and the level of blood glucose (GLU) were significantly or extremely significantly decreased (P<0.05; P<0.01); On the 35th day after transport, the mean red blood cell volume (MCV), phosphorus (PHOS) and total protein (TP) were significantly or extremely signifcantly increased (P<0.05; P<0.01), and the percentage of lymphocytes (LY) was significantly decreased (P<0.05); On the 7th day after transport, the levels of antidiuretic hormone (ADH), growth hormone (GH), UREA and insulin (INS) were significantly or extremely signifcantly decreased (P<0.05; P<0.01), and the total bilirubin (TBIL) and triiodothyronine (T3) were signigicantly increased (P<0.05), but there was no significant difference between the two groups (P>0.05) on the 35th day after transport. Based on the above results, after the long-distance transport, there had a corresponding transient stress response in Akhal-teke horses, which required some adjustment after the recovery period (35 d) to let most of the indicators were consistent with those without long-distance transport. So the horses could not be used until they were in a normal state, in order to avoid long-term irreversible lesions.

The study was conducted to separate and purify the hemin from yak blood and the optimal process of preparing the hemin was confirmed using blood meal,glacial acetic acid and sodium acetate methods. The purity and the yield of the extracted hemin in different methods were compared. The results showed that the purity of hemin in yak blood was 96% and the yield was 7.38 g/L using blood meal method under the optimal process of 40 min for ultrasonic treatment, 30 min for extraction,120 mL acid acetone and 9 mL sodium acetate. The purity of hemin was 80% and the yield was 6.10 g/L when the amount of glacial acetic acid was 3 times as much as blood cells amount using the glacial acetic acid method.The purity of hemin was 90% and the yield was 3.44 g/L when the amount of chloroform was 2 times as much as blood cells amount using sodium acetate method. The purity of the hemin from high to low was blood meal method,sodium acetate method and glacial acetic acid method,and that of the hemin yield was blood meal method,glacial acetic acid method and sodium acetate method by comparing the three preparation methods. So the blood meal method was more conducive to industrial production.

The study was conducted to investigate the effects of testosterone propionate supplementation on tissues residue of broilers,and its residual effect on serum biochemical parameters,testicular growth and development of broiler chickens,to provide a basis for the protection of animal products safety. A total of 160 male 60-day-old broilers with (2.2±0.1)kg body weight were selected and assigned to control group and testosterone propionate supplementation group. The chicken in control group were fed basal diets while that in testosterone propionate supplementation group were fed diets supplemented with 20 mg/kg testosterone propionate for 7 d. The broilers was slaughtered at the day of 2,6,11 and 17 d after the testosterone propionate supplemented diets were ending. The crest,liver,testis and musle were collected to measure the testosterone propionate residues and testosterone concentration by lquid chromatograph mass spectrometer mass spectrometer (HPLC-MS/MS). The serum samples were collected from the veins to measure the serum biochemical profiles by automatic biochemical analyzer. Testis samples were collected to measure the testis cell morphology and mastocyte accounts by HE and toluidine blue staining. The results showed that:①The testosterone propionate concentration in serum was the highest,followed by the crest. However,there was no testosterone propionate residues in breast muscle,leg muscle,liver and testis. The testosterone propionate concentration in crest was not affected by the withdrawal time (P>0.05). The content of testosterone propionate at 17 d of the withdrawal time was extremely significantly lower than that of 2,6 and 11 d (P<0.01),and there was a regressive between the residues of testosterone propionate in the blood and crest and withdrawal time.②Compared with the control group,the concentration of propionate in testis at the withdrawal time of 2,6,11 and 17 d was reduced 71.45%,63.46%,62.86% and 73.61% (P<0.01),respectively.③The relative weight of testis was significantly lower at the withdrawal time of 2 d (P<0.05). But there was no significant difference in liver relative weights among the two groups in all the withdrawal time (P>0.05).④Most of serum biochemical parameters (TP,ALB,A/G,CHO,TG,LDH,ALT and AST) were not significantly affected by testosterone propionate supplementation (P>0.05).⑤Testis lose the profiles, outed of the order of spermatogenic cells in tubes by testosterone propionate supplementation. Degranulation mass cell accounts,mast cells were higher than control group. In conclusion,the testosterone propionate could result in atrophy of the rooster testis,which could be detected by blood and crest samples,and the results could provide a basis for the study of livestock products safety.

The high-yield protease was obtained by screening high-yield protease Aspergillus oryzae and optimizing its solid-state fermentation conditions, and then the effect of fermented product on nutrient digestion in broiler was studied. After the screening and re-screening of 8 different Aspergillus oryzae spores, a new Aspergillus oryzae strain 0-8 with high acid protease was obtained. The fermentation conditions of acid protease were optimized by single factor and orthogonal test experiment. The results showed that the optimum temperature was 30℃, the most suitable fermentation time was 66 h, the initial water content was 47.40%, the inoculation quantity was 1.5×10⁷ spores/mL, the content of bran was 80%, C/N was 1/3, the optimized vigor of acid protease reached 14 416.64 U/g, which was 157.02% higher than that before optimization. 500 mg/kg Aspergillus oryzae 0-8 fermented product was added in the broiler diet, and the mutrient digestibility of broiler were measured after 10 d of adaptation and 5 d of test period. The results of the nutrient digestion of broilers showed that feeding Aspergillus oryzae 0-8 fermentation increased the CP and energy digestibility of broiler in a certain degree compared to control group, but there's no significant difference (P>0.05). Dietary supplementation of Aspergillus oryzae fermentation was beneficial to the production of broilers, but its additive dose or feeding pattern should be further studied.

In order to investigate the effects of cecropin and synbiotics on the intestinal mucosal morphology and intestinal mucosal immune cells of AA broilers. 480 of 1-day-old healthy AA broilers were chosen and randomly divided into 4 groups with 4 replicates per group and 30 broilers per replicate. The broilers in group Ⅰ(control group) were fed basal diet,while that in groups Ⅱ to Ⅳ were fed the basal diet supplemented with 0.5% cecropin,0.3% synbiotics,0.5% cecropin+0.3% synbiotics,respectively. The trial lasted for 42 days. The result showed that compared with group Ⅰ,the villus height of duodenum and ileum in group Ⅳ were extremely significantly increased (P<0.01).The crypt depth of jejunum and ileum in group Ⅱ,and that of small intestine in group Ⅲ were significantly or extremely significantly decreased (P<0.05;P<0.01).The villus height/crypt depth of small intestine in groups Ⅱ and Ⅲ,and that of duodenum and ileum in group Ⅳ were significantly or extremely significantly increased (P<0.05;P<0.01).The villus width of jejunum in group Ⅱ was significantly decreased (P<0.05),while the villus width of duodenum and jejunum in group Ⅲ,that of jejunum and ileum in group Ⅳ were extremely significantly decreased (P<0.01).The mucous thickness of jejunum in groups Ⅱ and Ⅲ were significantly increased (P<0.05),while that of small intestine in group Ⅳ was extremely significantly increased (P<0.01).The number of the intraepithelial lymphocyte of ileum in group Ⅱ was significantly increased (P<0.05),and that of duodenum in group Ⅲ and small intestine in group Ⅳ were extremely significantly increased (P<0.01).The number of the goblet cell of duodenum in group Ⅲ,duodenum and ileum in group Ⅳ were extremely significantly increased (P<0.01).The mast cell of jejunum in group Ⅳ was significantly increased (P<0.05).In conclusion,both the cecropin and synbiotics could improve the small intestine mucosal structure and promote the small intestine mucosal immunocompetent cells proliferation in AA broiler, and the effect of combined utilization would be better.

In order to study the effects of Fagopyrum dibotryis superfine powder on the immune function and growth and development of broilers,160 one-day-old White feather broilers were chosen and randomly divided into 4 groups. The nasal drops were given with Newcastle disease live vaccine at 7 days old,and Newcastle disease-avian influenza inactivated vaccine was given by subcutaneous injection (0.3 mL/only).After 12 h,each group was fed with different feeds,the broilers in the low and high dose groups were fed diet supplemented 4.50‰,8.75‰ Fagopyrum dibotryis superfine powder,respectively,administration for 7 d. The broilers in the positive control group were fed diet added 400 g/L Astragalus polysaccharide, continuous administration for 7 d,and feed of blank control group did not add any drugs. The lymphocytes and serum were collected and then used for the separation of lymphocytes and serum at 7,21 and 35 d after the end of the administration. The proliferation of peripheral blood lymphocytes was measured by CCK-8 method. The titer of serum was evaluated by the hemagglutination inhibition test.The body weight of each group was weighed at the same time.After that,the rats were sacrificed,the spleen and bursa of Fabricius were used to calculate the immune organ index. The results showed that compared with the control group,the F/G of high dose group,low dose group and Astragalus polysaccharide group were decreased by different degrees at 7,21 and 35 d after the end of the administration (P>0.05). In addition,the proliferation ability of the peripheral blood lymphocytes,immune antibody level and the body weight in broilers of administration groups were increased,in which the titers of AIV and NDV antibodies were extremely significantly increased in high dose group compared with the control group (P<0.01).Meanwhile the Fagopyrum dibotryis superfine powder also could increase the spleen and bursa of Fabricius indexes. In conclusion,the Fagopyrum dibotryis superfine powder could significantly improve the cellular immunity, humoral immunity and nonspecific immune function of broilers,and significantly increased the body weight. According to the specification of the results of the instructions, we could determine that it had the effect to comprehensively improve the immunity and growth of White feather broilers. The study could lay the foundation of the future development of Fagopyrum dibotryis superfine powder into a new natural herbal medicine and feed additives.

In order to develop product and improve breeding for Guangxi partridge chickens,the slaughter performance,amino acids,inosine acid and fatty acids contents in muscle were measured and evaluated for Guangxi partridge chickens at 120 days old,which were raised at same nutrition level and management. Carcass traits and meat characteristics were evaluated. The results were showed that the dressing percentage and eviscerated yield percentage of cocks and hens were 90.15%,92.57% and 69.12%, 68.08%,respectively. The total amino acids in the breast and leg muscles of cocks and hens were 20.37,18.40 and 21.30, 18.44 g/100 g,respectively. The ratio of essential amino acids to total amino acids in breast and leg muscles of cocks and hens were 39.32%,42.33% and 42.72%, 43.06%,respectively,and essential amino acid index (EAAI) were 84.33,88.82 and 90.44, 90.77,respectively. The content of delicious,sweet,branched chain amino acids and inosinic acid in breast muscle were 0.85 g/100 g,0.42 g/100 g,0.56 g/100 g and 1.55 mg/g higher than that in leg muscle,respectively,and the content of sweet amino acids in hens was 0.40 g/100 g more than that in cocks. The first limiting amino acid was Met+Cys in muscle of Guangxi partridge chickens. Oleic acid and linoleic acid were mainly components of unsaturated fatty acids in muscle,palmitic acid and stearic acid were mainly components of saturated fatty acids in muscle. The relative content of polyunsaturated fatty acids and essential fatty acids in leg muscle were higher than that in breast muscle, and their contents in hens were more than that in cocks. All these showed that the Guangxi partridge chickens had good meat production,flavor and high nutrition value,meanwhile the nutritional values in hens were superior than that in cocks.

The study was conducted to determine the effects of different levels of selenium yeast(SeY, added as selenium content) on growth performance and some biochemical indexes in Jinding laying ducks under high temperature (29-35℃). 144 Jinding laying ducks of rearing period (10-16 weeks old) were divided into 4 groups, control group, experimental groups Ⅰ,Ⅱ and Ⅲ, supplemented with 0, 0.15, 0.45 and 0.75 mg/kg Se, respectively. The results showed that compared with the control group, the addition of SeY to the diets could significantly increase the average daily feed intakes (ADFI) of ducks at 14-16 weeks old (P<0.05); The body weights of ducks in group Ⅰ at 13-15 weeks old were significant higher than that in the control group (P<0.05),and the body weight at 16 weeks old was higher than the other three groups significantly (P<0.05),there was no significant difference between the body weight and standard body weight at 16 weeks old in group Ⅰ(P>0.05), but the body weights of other three groups were extremely significantly lower than the standard weight (P<0.01); There was no significant difference among the four groups for feed to gain ratios (F/G), while the group Ⅰ was the lowest; AST activities of group Ⅰ were significantly lower than group Ⅱ(P<0.05), LDH activities of groups Ⅰ,Ⅱ and Ⅲ were lower than the control group significantly (P<0.05), and LDH activities of groupⅠ were lower than groups Ⅱ and Ⅲ significantly (P<0.05). TCH concentrations of groups Ⅰ and Ⅱ were higher than group Ⅲ significantly (P<0.05). It is concluded that the addition of 0.15 mg/kg of Se (added as SeY) could obviously improve the growth performance of the laying ducks in reared cages, protect the function of liver, promote the TCH concentrations of duck serum.

In order to measure the nutritional characteristics of Black-White rabbit meat,the nutrient components,the conventional nutrient,amino acid and fatty acid content of leg muscle in Black-White rabbit were tested and analyzed,and the protein nutritional values were evaluated based on the National Food Security Standard and chemical analytical method of product quality. The results showed that the moisture,crude proteins,crude fats and ash in leg muscle of Black-White rabbit were 73.43%,21.67%,3.83% and 1.13%,respectively. There was no significant difference on the nutrient components between female and male rabbits (P>0.05). The total amino acids,essential amino acids and delicious amino acids were 196.53,78.64 and 70.80 mg/g,respectively. According to amino acid score (AAS),the first limiting amino acid of Black-White rabbit was Val and the second limiting amino acid was Met+Cys. However,based on the standard of chemical score (CS),the first limiting amino acid was Met+Cys and the second was Val. Additionally,the ratio of unsaturated fatty acids (UFA) to saturated fatty acids (SFA) was 1.74,and no significant difference was found on the amino acids between female and male rabbits (P>0.05). More specifically,except the fatty acid of C16:1 and C18:3, there was no significant difference in the fatty acid compositions between female and male rabbits (P>0.05). In conclusion,Black-White rabbit meat was composed of high proteins,delicious amino acids and essential amino acids suggesting that it had better balance of amino acids. The unsaturated fatty acids in Black-White rabbit meat were higher than saturated ones meaning high nutritional value and better dietary values.

In order to explore the differential expression miRNAs and their expression patterns in the control of buffalo in lactation and non-lactation, the miRNAs of 18 buffalo breast tissues differentially expressed in lactation and non-lactation were screened by Solexa high-throughput sequencing and bioinformatics analysis. Viable miRNAs primers for reverse transcription and Real-time quantitative PCR were used to validate the expression patterns of miRNAs. The Real-time quantitative PCR results were basically consistent with sequencing results. The expression levels of miR-103, miRNA-125a, miRNA-30a-5p and miRNA-148a in lactation were significantly higher than those in non-lactation (P<0.05). The expression level of miRNA-29a in non-lactation was higher than that in lactation (P<0.05). The expression levels of miRNA-141, miRNA-125b and miRNA-497 in lactation were higher than those in non-lactation,but the difference was not significant (P>0.05). The low-expression levels of miRNA-490 and miRNA-592 in lactation were significantly higher than those in non-lactation (P<0.05). There was no significant difference in the expression of Novel-123b between lactation and non-lactation (P>0.05). Novel-57 was extremely significantly in lactation (29.79 times) higher than non-lactation (P<0.01). This results provide available specific reverse transcription primers and Real-time quantitative PCR primers of 16 miRNAs for further study on buffalo,and 6 miRNAs such as miRNA-103, miRNA-125a, miRNA-30a-5p, miRNA-148a,miRNA-29a and Novel-57 deserve further research.

This study was aimed to investigate the effects of the different compatibility of medroxyprogesterone acetate (MPA) with prostaglandin(PG), N-methyl-D-aspartate (NMDA) and letrozole (LE) on plasma related hormones levels of reproductive ewes. Thirty-nine and 3-year-old multiparity Suffolk ewes with average weight (75.51+11.55)kg were selected,and assigned into 3 groups,test group Ⅰ:Basis diet+34 mg/d MPA+1 mL/ewe PG, test group Ⅱ:Basis diet+34 mg/d MPA+600 mg/d NMDA, test group Ⅲ:Basis diet+34 mg/d MPA+ 14 mg/d LE, respectively, the trial period lasted for 18 days. The results showed as follows:On the basis of supplemental feeding MPA with PG treated,the levels of FSH in plasma of ewes were extremely significantly increased (P<0.01); Treated with NMDA, the levels of E₂, MLT,FSH and LH in plasma of ewes were extremely significantly increased (P<0.01), respectively;Treated with LE, the levels of MLT,LH and T in plasma of ewes were extremely significantly increased (P<0.01), respectively. This study suggested that MPA+NMDA and MPA+LE could be used as medicines for regulate and control oestrus of ewes, and the effect of MPA+NMDA was the best.

This study was aimed to investigate the expression level of myosin light chain,phosphorylatable,fast skeletal muscle (MYLPF) gene in the tissues of Black cattle, and its expression level in the longissimus dorsi muscle of Black cattle and Luxi cattle, and to lay the foundation for the study of its relationship with muscle growth and development. The MYLPF gene expression level in 10 tissues of longissimus dorsi, heart, lung, liver and spleen and so on were detected by Real-time quantitative PCR, and then the changes of MYLPF gene expression level in longissimus dorsi of Black cattle and Luxi cattle at 2, 6, 10 and 12 months of age were studied. The results showed that MYLPF gene was highly expressed in the longissimus dorsi, lowly expressed in the heart, and the other tissues were almost not expressed. The MYLPF gene expression level in the longissimus dorsi was high in 2, 6, 10 and 12 months, with the increase of age,the MYLPF gene expression level decreased gradually, the expression level of 2 months of age was the highest, which was extremely significantly higher than that of the other three periods (P<0.01). The MYLPF gene expression level in the longissimus dorsi of Black cattle was higher than that of Luxi cattle in four period, which in 10 months of age were extremely significantly different (P<0.01), 2 and 12 months of age had significant difference (P<0.05), the difference in 6 months of age was not significant (P>0.05). The results suggested that MYLPF gene in the longissimus dorsi might play a regulatory role in the growth and development of skeletal muscle, which might provide a reference for studying the mechanism of growth and development of skeletal muscle and the molecular mechanism of muscle growth.

This study was aimed to set up a simple, economic and pure method to culture and identify buffalo mammary epithelial cell in vitro, which were collected from the fresh milk. The milk was collected in the late lactation period of the high yield milk buffalo, then it was mixed with the PBS in the ratio of 2:1 and centrifugated. A few suspernatants and the pellets were resuspended with the PBS when the milk fat were decanted, then recntrifuged. The final pellets and 1 mL of suspernatants were seeded in petri dish without fat, respectively. The isolated cells were mammary epithelial cells which were identified by cell morphology, immunofluorescence, growth curve, cell viability and RT-PCR. The buffalo mammary epithelial cells were successfully isolated from both the pellets and supernatants. The cells and impurities were less in the supernatants than that in the pellets. The cells were cobblestone-like without fibroblasts and most in cell division. In the post-confluent culture, mammary epithelial cells formed dome structures and layer-separated growth. The cells expressed cytokeratin 18 was identified by immunofluorescence which was the marker of mammary cells. The cell growth curve and cell survival rate were measured. The results showed that the buffalo mammary epithelial cells were activity and easy to attach. The mammary epithelial cells were expressed of milk protein and milk fat related genes detected by RT-PCR. The buffalo mammary epithelial cell line were successfully isolated and identified from fresh milk, which estabilished foundation for the study of the mechanism of lactation, the amelioration of the quality of milk and the improvement of milk yield of buffalo.

A method was created to reduce the sampling number in order to make the preservation monitoring simpler and accurate for the Shenxian pigs' population. A total of 179 Shenxian pigs as a population was sampled in a certain gradient,each gradient randomly selected 10 times. The genetic diversity indexes of population and sampling groups were calculated using microsatellite primers marker technique. The average and standard deviation of the genetic diversity indexs of 10 groups were compared with that of the selected population, and the minimum sampling size with less changes in genetic diversity was determined as the appropriate sampling scale. The results showed that:For the group with sampling size of 30,the average allele number was 4.6609, the average effective allele number was 3.2325, the average observed heterozygosity was 0.1569, the average expected heterozygosity was 0.6284, the average PIC was 0.5794. The results indicated that the main indexes of genetic diversity in group with sampling size of 30 were similar to those in the whole population, so the sampling size of 30 pigs could be used to represent and instead of large population of Shenxian pigs.

Dorper sheep is native to South Africa, it is one of the world famous mutton sheep breeds, it is a good terminal male parent of mutton sheep hybrid production, after be introduced into China, it has been distributed in many provinces (municipality, municipalities directly under the central government), and has played an important role in promote the commercial mutton sheep production in China. In this paper, through introducing the rapid growth and development of Dorper sheep, good meat performance of germplasm characteristics, and by introducing Dorper sheep at different regions in China,the first filial generation of hybrid with local sheep varieties respectively compared with local sheep in growth and development, meat performance, we found that the first filial generation of Dorper sheep as male parent hybrid with local sheep all showed strong heterosis, and could be used as excellent male parent varieties of cultivate the new mutton sheep varieties and hybridization and improvement in China.

This experiment was aimed to study the changes of reproductive hormone and regulation of follicle development during the reproductive cycle of Yili goose in Xinjiang. 2 years old Yili goose were chosen which were in good health and similar weight. 32 samples were randomly selected to take blood samples for hormone detection during the laying, broodiness and ceased periods, and 15 samples were randomly selected to gather ovarian tissue for the observation of the development of ovary and follicular. The results showed that during the laying period, the GnRH of the Yili goose was extremely significantly higher than the broodiness and ceased periods of 16.78% and 58.29% (P<0.01);PRL was significantly lower than the broodiness period of 13.00% (P<0.05), and was extremely significantly higher than the ceased period of 28.94% (P<0.01); FSH was higher than the broodiness period of 6.98% (P>0.05),and was extremely significantly higher than the ceased period of 21.12% (P<0.01); LH was extremely significantly lower than the broodiness period of 8.38% (P<0.01),and was extremely significantly higher than the ceased period of 16.84% (P<0.01); E₂ were extremely significantly higher than that the broodiness and ceased periods of 29.80% and 112.40% (P<0.01); P₄ levels were extremely significantly higher than the broodiness and ceased periods of 28.89% and 30.34% (P<0.01). The ovary volume and weight of Yili goose were extremely significantly higher than that of broodiness and ceased periods (P<0.01). In the laying period, the mature follicles of Yili goose were significantly larger than that of the broodiness and resting periods, the number of secondary follicles was more and the number of primary follicles was lesser. Ovarian growth and mature follicle atresia, granule layer shrinkage, inward depression, cytoplasmic cavity, follicular atrophy at all levels in the broodiness period. The follicle cytoplasmic cavity of the ovary was larger in the ceased period than broodiness period, and inward concave further deeper, a large number of primordial follicles evenly arranged. Thus, during the laying period, the responses of GnRH, E₂ and LH of Yili goose were active and played a leading role on the development of ovarian and follicular. PRL and LH maintained a high level of secretion, cooperated to maintain the broodiness behavior during the broodiness period.

African swine fever (ASF), which caused by African swine fever virus (ASFV), is an acute, highly contagious disease characterized by high fever. It leads to serious economic losses in pig industry. 3 assemblies of primers and probes targeting were designed to amplify ASFV B646L (p72) gene using recombinase polymerase amplification (RPA) technology. The Real-time fluorescent RPA method was established after screening of primers and probes, optimization of reaction conditions, tests of sensitivity, specificity and repeatability. The results showed that the method could detect 10 copies of DNA within 20 min at 39℃. No cross-reaction was found when testing swine fever virus, porcine circovirus type 2, porcine parvovirus, pseudorabies virus. According to the fluorescence intensity from 5.5×10⁶ to 5.5×10⁰ copies/μL at each time point, the coefficient of variation was 0.38% to 28.30%. In conclusion, this method could be used for the qualitative detection of ASFV pathogen, which might provide technical support for the early diagnosis of ASFV infection in China, and was also of great significance for the development of corresponding control measures.

To find out the reason of the reproductive failure in pregnant sows in a hoggery in Guangdong, the mixture with brain, lymph nodes and lungs of farm abortion stillbirth were identified by PCR, virus isolation and culture, tissue culture infective dose (TCID₅₀) assay, homology and phylogenetic analysis of the important functional genes (gB, gC, gD and gE) and animal test. The results showed that the mixture was proved to be porcine pseudorabies virus (PRV) positive samples. The typical cytopathogenic effect was induced in the third passage of Vero cell and the titer of the fifth passage was 10^-6.8/0.1 mL. The sequence analysis and phylogenetic relationship of gB, gC, gD and gE genes showed that it was a Chinese PRV variant, which was named as LC strain. The typical pseudorabies clinical and pathological symptoms were presented in 12-week-old piglets inoculated with LC strain. The results demonstrated that a local pseudorabies virus had been isolated, suggesting that the Bartha-K61 vaccine was not fully effective for controlling the current epidemic of pseudorabies in China.

African swine fever (ASF) is an infectious disease caused by the African swine fever virus (ASFV). In order to ensure the accuracy and reliability of the test results,it is necessary to develop positive standard control products used in the kit. The study was aimed to develop the virus-like particles containing African swine fever virus (ASFV) nucleic acid sequence and its application in detection method. Fristly,the full-length gene fragment of p72 gene was amplified, and the ASF DNA virus-like particles containing p72 gene was constructed using insect baculovirus system. In order to further validate the reliability of the virus-like particles in the application of the method,DNA nucleic acid was extracted simultaneously with the cultured virus and infected tissue sample and applied in the Real-time quantitative PCR method. The results showed that the virus-like particles prepared by this study could replace the ASFV as a positive control product in the Real-time quantitative PCR method, and could act as quality control during nucleic acid extraction process. In the fluorescent PCR detection kit, the lowest packaging concentration of virus-like particles was 10² TCID₅₀. Further studies had shown that the virus-like particles were also suitable for common PCR and LAMP detection methods, the minimum concentration were 10³ and 10¹ TCID₅₀, respectively. The results of this study would be important for the ASF detection method, promoting the application of the method and ensuring the accuracy and reliability of the test results.

Nanotechnology is the emerging development of the core information technology in the 1980s, and it has been widely used in life medicine, optoelectronics and other fields. Nanotechnology is effectively combined with parasitic disease reagents instrumentation, drug treatment, vaccine prevention and control, and has a comprehensive application in the nano-gold standard test strip test kit and equipment, nano-carrier targeted anthelmintic drugs, new nano-vaccine adjuvant of research and development, etc. It penetrates deeply into the medical field and is widely used in parasitology. With the starting point of the application of nanotechnology in parasitology, this paper focused on the pathogenic detection, anthelmintic drugs and vaccine adjuvants of parasitic diseases. The authors described the application of nano-technology, such as colloidal gold labeled immunoaffinity chromatography, microparticle material synthesis and biosensing, in the diagnosis and diagnosis of parasitic pathogens. The progress of new nano-insect repellent drug lipid carrier, chemical synthesis medicine and herbal medicine research and development had been discussed here, in addition to liposomes, polymer particles, and cytokines in vaccine adjuvants. This review could be expected as a reference for nanotechnology in in-depth research and better application in parasitology.

Small colony variants (SCVs) have been shown to be part of the regular growth cycle, which are highly dynamic or stable and can be selected during various harsh conditions. The features of SCVs include slow growth, colony morphology irregular and biochemical characteristics abnormal, making the separation and identification of SCVs into trouble. SCVs can cause chronic persistent infection, but not lead to strong immune response to host. Furthermore, SCVs are serious resistant to antibacterials normally. In this paper, we introduce the phenotype classification of SCVs, the relationship with the host's immune response and the effects of antibacterials on SCVs, which will provide theoretical support for further research of SCVs.

This study was aimed to establish an indirect ELISA to detect antibodies against different strains of porcine epidemic diarrhea virus (PEDV). Puried nonstructural protein 7(Nsp7) was used as coating antigen, and the indirect ELISA was established by optimizing the ELISA reaction conditions. The results showed that the optimal reaction conditions were as follow:The amount of coating antigen was 0.20 μg/well, and the coating condition was at 37℃ incubation for 1 h then 4℃ overnight; The working dilution of serum samples and HRP-labelled secondary antibody were 1:300 and 1:10 000, and the incubation time were 2 and 1.5 h, respectively; TMB substrate incubation time was 15 min. Serum sample was determined as positive when its S/P>0.1694 and negative when its S/P<0.1398. The ELISA was specific, reproducible and sensitive. Forty samples of suspected PEDV serum samples were tested by the established ELISA, and the coincidence rate between the ELISA and the commercial kit was 95%. The ELISA established in this study could be used clinically to detect the antibody level of different strains of PEDV, and it also had the potential for early diagnosis of PEDV, providing a basis for the development of effective measures to control PEDV.

This study was aimed to clone and express the BPSS0180 gene of Burkholderia pseudomallei (B. pseudomallea), and perform bioinformatics analysis of its protein. A pair of primers was designed according to the BPSS0180 gene sequence information of B. pseudomallea K96243 strain in GenBank. BPSS0180 gene fragment was obtained by PCR amplification of B. pseudomallea hn-1 strain. The BPSS0180 gene fragment was ligated into the pET-28a(+) vector to construct the pET-28a(+)-BPSS0180 recombinant plasmid. The recombinant plasmid pET-28a(+)-BPSS0180 was transformed into E.coli DH5α competent cells, and the plasmids were identified by restriction enzyme digestion. Then, the recombinant plasmid pET-28a(+)-BPSS0180 was transformed into E.coli BL21 (DE3) competent cells. The expression was induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Bioinformatics analysis of BPSS0180 gene sequence was carried out using DNAMAN, ProtParam, SOPMA and Protscale. The results showed that the length of BPSS0180 gene was 1 146 bp; The expressed His-BPSS0180 fusion protein was about 45 ku, and was predominantly in the form of inclusion bodies; The molecular weight of the BPSS0180 protein was 40.6 ku (C₁₇₇₉H₂₈₀₉N₅₄₅O₅₃₆S₇); The extinction coefficient was 40 575; The hydrophobic index was 85.43; The instability coefficient was 46.52,which belonged to unstable protein;The theoretical isoelectric point (pI) was 5.54 and was acidic protein; The total average hydrophobicity (GRAVY) was -0.261,as hydrophilic protein; The secondary structure of the protein were mainly α-helix (58.79%) and random curl (32.02%), and its half-life of reticulocytes in mammals was predicted to be 30 h. This study provided a theoretical basis for further exploring the fuction of BPSS0180 gene of B. pseudomallei.

This study was aimed to develop a simple and rapid method for the porcine parvovirus (PPV) with recombinase polymerase amplification (RPA), which could be a novel and reliable tool for the control and detect of PPV. The RPA was developed using specific primers for the conserved region of PPV VP2 gene. The reaction time was optimized,the RPA reaction could amplify the PPV, and was performed successfully at 38℃ for 30 min in a water bath. The results showed that the detection limit of RPA was 102 copies of plasmid DNA, which was the same as the Real-time quantitative PCR applied in this study, and 100 times more sensitive than conventional PCR. For the clinical samples from the suspected PPV-infected pigs, the positive detection ratio was 82.6% for RPA, which was lower than that of Real-time quantitative PCR (86.9%), but was much higher than conventional PCR (66.7%). The PPV RPA assay developed in the study was simple, rapid and reliable, and was suitable for rapid detection of PPV.

This study was aimed to explore the use of baculovirus expression system to secrete peste des petits ruminants virus (PPRV) F gene protein and use it as subunit vaccine. The gene fragment encoding F protein of PPRV was cloned into the baculovirus pFastBac Ⅰ transfer vector with a honeybee melittin signal peptide.The constructed F-pFastBac was transformed into Escherichia coli DH10Bac,resulting the recombinant baculovirus DNA (F-Bacmid) which was confirmed by blue-white plaque assay and antibiotic resistance selection.The F-Bacmid was then transfected into Sf9 insect cells by the cellfectin transfection reagent.The recombinant F protein was expressed in High Five cells in the serum-free medium.The SDS-PAGE and Western blotting analysis of recombinant protein showed that the protein could be expressed in insect cells and secreted into the culture medium. For the immunogenicity study,the recombinant protein was then inoculated into BALB/c mice, the results showed that the recombinant protein was able to stimulate B cells to produce special antibodies. In conclusions,the recombinant baculovirus expressing F protein of PPRV were successfully constructed.This study applied a basis for the development of PPRV subunit vaccine.

To realize the rapid detection of duck tembusu virus (DTMUV), a set of specific primers was designed according to the highly conserved sequence of DTMUV E protein gene published in GenBank. A sensitive and convenient RT-LAMP amplification method was established by optimizing the concentrations of each component, reaction time and temperature in the reaction system. The results showed that the influence of betaine on the reaction system was not obvious under the optimal conditions. This method was 100 times more sensitive than the conventional RT-PCR. The RT-LAMP method specifically amplified tembusu virus but not other common avian viruses. The results could be differentiated by naked eyes. 74 clinic suspicious samples were tested and 65 of them were positive. These results suggested that the RT-LAMP assay could be used as a method for the diagnosis and detection of clinical cases, and molecular epidemiological investigation of duck tembusu virus disease.

Antibacterials are used for the treatment or prevention of animal diseases by direct administration or feed additives. The inappropriate use of antibacterials in aquaculture inevitably lead to excessive residue of antibacterials in animal food. Microbiological methods are the earliest methods for the detection of antibacterials residues. Compared with other detection methods, microbiological methods are widely used for detecting residual antibacterials in livestock and poultry, milk and others animal food because it is easy to operate, low cost and high throughput. The purpose of this paper is to review the development history of microbiological methods and its application status for the detection of residual antibacterials in milk, feeding livestock and poultry, eggs, aquatic products and honey, which provides a theoretical and practical basis for the subsequent development of new microbiological methods. This review also makes a prospect of the development of microbiological methods.

In order to develop a sensitive and specific method for the detection of estradiol residues (E₂) in milk, a colloidal gold immunochromatographic method was established. Colloidal gold particles were prepared by trisodium citrate reduction method and labeled with rabbit polyclonal antibodies (PcAb) by physical adsorption method. After optimization of reaction conditions such as the amount of coating antigen and labeled antibody to assemble test strip, the sensitivity, specificity, repeatability and accelerated preservation of the test strip were determined. The estrogen analogue cross reaction and milk matrix interference reaction were also measured. The results showed that the optimized concentration of E₂-OVA antigen was 2 mg/mL and the optimized antibody concentration was 20 μg/mL, visual detection limits of E₂ was 10 μg/L in PBS within 10 min. The cross reaction rate of this method with estriol was 40%, there were no negligible cross-reactivities with other estrogen compounds including estradiol valerate, estradiol benzoate, estrone, diethylstilbestrol, quinestrol, ethinyloestradiol and nonylphenol. Milk samples only needed two times diluted before analysis, and the results could judge by naked eye after 10 min with cut-off of 20 μg/L. The results demonstrated that the developed method was suitable for rapid on-site screening of E₂ residues in milk samples.

In order to screen the Chinese herbal medicine for the treatment of Demodex canis, canine acariasis spirit was prepared by Foeniculum vulgare, Fructus cnidii and Bauhinia by water alcohol precipitation and distillation extraction methods. To observe the effect on Demodex canis, canine acariasis spirit was set to high, medium and low concentrations, the content of crude drug were 2.0, 1.0 and 0.5 g/mL, respectively. The experiment of killing Demodex canis was carried out by drop method. On this basis, 12 dogs inoculated with Demodex canis were treated. In order to verify the therapeutic effect, 45 clinically diagnosed dogs with Demodex canis were divided into three groups. The results showed that 3 doses of the traditional Chinese medicine canine acariasis spirit all had strong inhibition effect to kill mites, the high dose group could completely kill Demodex canis only 4 h after treatment, the action of 1% ivermectin control group was 8 h after treatment; 3 weeks later, a large number of new hair grew, rash and skin nodules disappeared, the skin lesion had healed in the affected area of high dose group, in 1% ivermectin treatment group, the dog skin improved, only a small amount of new hair grew, it couldn't cure standard; In the high and middle dose group, the average number of the ratio of skin scraping mites and the recurrence rate after drug withdrawal was 0 in one month later; The average number of mites proportion and recurrence rates of skin scrapings in 1% ivermectin treatment group were 10.63% and 9.17% in one month later; After 3 weeks of treatment, the total average eggs negative rate, cure rate and average cure days were 100.00%, 93.33% and 19.56 d in high dose traditional Chinese medicine canine acariasis spirit and 1% ivermectin combined treatment group, the results were better than the traditional Chinese medicine canine acariasis spirit and 1% ivermectin alone treatment group. The results showed that the effect of the high dose group self-made Chinese medicine canine acariasis spirit on killing Demodex canis in vitro, inoculation of Demodex canis and clinical therapeutic effect of natural infection cases were significantly better than 1% ivermectin control group, canine acariasis spirit as a traditional Chinese medicine preparation would have a good market development prospects.

The purpose of this study was to validate the microbial limit test method for anticoccidial drug Dichroa febrifuga oral liquid (DFOL),and provide data base for its quality control and researching the new veterinary drug. The direct inoculation method was used to validate the antibacterial activity for DFOL, and the bacterial counting,determination of recovery rate and inspection of control bacteria were measured using a validated test condition and method (conventional method).The results showed that DFOL had no or faint bacteriostasis verified by validated test method, the determination and inspection of bacteria could be determined by conventional method. The normal plate counting were used to detect the amount of Escherichia coli,Staphylococcus aureus, Bacillus subtilis, Candida albicans and Aspergillus niger in DFOL in which the recovery rate were all more than 70%,and the results were not disturbed by sample and diluents. The control bacteria including Escherichia coli,Salmonella paratyphi B,Staphylococcus aureus,and Pseudomonas aeruginosa were tested according to conventional method,whose growth were detected in the test group and positive control group, while no growth were detected in the negative control group and sample group. In conclusion,the microbial limit test method for DFOL had been validated in this study,and the conventional method could be applied to DFOL for bacteria,moulds and yeast counting as well as the inspection for control bacteria. The process of test was in accordance with the regulations of microbial limits test method,the method was convenient and simple,and the results was reliable.

The purpose of this experiment was to study the acute toxicity and long-term sub-chronic toxicity of the extract of Rehmannia glutinosa in mice, evaluate the clinical safety of medication and provide theoretical basis for clinical application. Mice were chosen to measure the median lethal dose (LD₅₀) and maximal tolerance dose (MTD). In the sub-chronic toxicity test, 80 SD rats were divided into four groups:Low dose, middle dose, high dose test groups with the extract of Rehmannia glutinosa and control group with normal saline for 30 d. On the 31th day, the rats were killed and the blood routine index, biochemistry index and the organ coefficient were measured. The MTD was 61.54 g/kg, according to the judgmental standard of the acute toxicity, the extract of Rehmannia glutinosa was safe. The sub-chronic toxicity test results showed that there were no significant difference in body weight and blood chemistry indexes and organ coefficient among the four groups (P>0.05) and there were no pathological change because of the medication. The result indicated that the extract of Rehmannia glutinosa was no acute toxicity under the condition of this test according to acute toxicity classification standard of exogenous by WTO, and it was no sub-chronic toxicity too, which suggested the extract had a good clinical safety.

In order to provide therapeutical guidance for drug admistration, the bacteria of three sick minks suffering from typical diarrhea symptoms provided by mink farms in Jilin province were isolated and identified, and the drug sensitivity was tested. The bacteria were isolated with TSA plates, and identified using biochemical methods and PCR assay. The virulence of the isolates was determined by infecting BALB/c mice. The antimicrobial susceptibility of the isolates to antimicrobial agents was investigated using the K-B method. PCR was used to detect the resistance genes and Ⅰ integrons. A total of 3 Shigella isolates were obtained from sick minks. The virulent determination showed that all isolates could cause mice diarrhea. The drug sensitivity results showed that 3 strains were sensitive to fluoroquinolone, cephalosporin, florfenicol and polymyxin, but they were resistant to aminoglycoside, tetracycline, chloramphenicol, penicillin and ampicillin. There were seven resistance genes were detected,bla_TEM-1,aadA1, aac(3')-Ⅱc, aac(6')-Ⅰb, aph(3')-Ⅶ, tet(M), cat2 and three class Ⅰ integrons carrying aadA 1 gene cassette. All of the isolates were virulent and caused the mice diarrhea. The resistance of the 3 strains were very serious and mainly for multiple drug resistance phenomenon. The resistance genes detected in the mink were various, and could bring enormous implications for clinical treatment.

This study was aimed to detect the efficacy of antiviral drugs against feline infectious rhinotracheitis virus. F81 cell was used to construct a model for drug screening against feline infectious rhinotracheitis virus in vitro. Virus inhibition rate was detected with MTT assay and calculated with SPSS software. The IC₅₀ of acyclovir, ribavirin, L-lysine, isatis root and Astragalus polysaccharides were 9.5,3.3,3.4,161.0 and 4.7 μg/mL, respectively,the IC₅₀ of polyinosinic was 6.0 mg/mL. The TI of them were 76.8, 39.3, 2 588.0, 4.5, 78.7 and 5.8, respectively. The effect of L-lysine and Astragalus polysaccharides were most significant.