A Beijing fatty chicken embryo fibroblast cell line (NBLCHE2/2) was successfully established using the primary explant technique. Observations on morphology, dynamic growth and analysis of karyotype, isoenzymes of lactate dehydrogenase, malate dehydrogenase were carried out. The results showed that population doubling time of cells was 24 h; diploid cells were dominant of 76%～88%. The banding patterns of the isoenzymes of the two kinds of emzymes had significant difference between the Beijing fatty chicken embryo fibroblast cell line and other cell lines in our laboratory; tests for microbial conta mination of bacteria, fungi and yeasts, virus and mycoplasma were negative. The newly established cell line make the Beijing Fatty Chicken, a national important genetic resource preserved in cell level, as well as will provide an ideal experimental material for the genetic studies on the Beijing local chicken.

Bovine preantral follicles (about 100μm in diameter) were cultured in vitro in a serum -free system for 15 day. The transmission electron microscope analysis showed that the preantral follicles isolated mechanically were 1 to 3 layers of granulose cells tightly enclosing the oocyte and intact basement membrane without theca cell. There are obvious and intensive gap junctions between the adjacent granulose cells. The ultrastructural characteristics of the preantral follicles isolated freshly or cultured in vitro were mostly similar to those presented in situ. The proliferatating of granulose cells was observed on the sixth day of culture. The theca interna cell emerged in a few preantral follicles on the 15th day of culture. These results showed that the culture system is adapted for smaller preantral follicles’ growing.

Experiments were conducted to study the effect of different vitrifications, straw and open pulled straw(OPS ), on survival, in vitro maturation, in vitro fertilization, and post-fertilization development of vitrified-thawed immature bovine COCs(cumulus oocyte complex).The results as follows: The recovery of post-thaw morphologically normal COCs in straw and OPS were 59.4%±4.3% and 77.9%±4.1% respectively(P<0.01)；The maturation rates were 48.2%±5.3% and 66.0%±5.8% respectively（P<0.01）；The cleavage rates were18.5%±2.0% and 32.8%±1.4% respectively（P<0.01）；The percentage of 8-cell embryo were 148%±2.5% and 24.8%±1.5% respectively（P<0.01）；The percentages of morula in straw , OPS and fresh oocytes were 0, 5.3%±1.1%and 21.0%±3.8% respectively（P<0.01）；The percentage of blastocyst in straw , OPS and fresh oocytes were 0, 4.0%±1.0%and 17.1%±2.7% respectively （P<0.01）.In conclusion,the vitrified-thawed immature bovine COCs by OPS could develope to morula and blastocyst stages after IVF,although it was low.

Four castrated Small Tail Han ram fitted with permanent ruminal, duodenal, and ileal fistula were used to examine the feasibility of a non-isotopic dual-marker method to measure duodenal and ileal digesta flow. The sheep were fed pellet diet by automatic feeder. For measuring digesta flow, chromic oxide (Cr₂O₃) and polyethylene glycol-4000 (PEG-4000) were added in the pellet diet. Markers were directly put into the rumen through rumen fistula when measuring their recovery rate in feces. The recovery rate of chromic oxide and PEG measured in the present study was 95.75% and 91.48% respectively. The concentrations of chromic oxide and PEG in ruminal, duodenal and ileal whole digesta and fluid phases reached plateau after about 4 d successive feeding of marker-containing pellets. The averages of duodenal and ileal digesta flow rates measured in present study were 10.054 3±1.726 5 L/d and 5.629 5±0.901 9 L/d respectively. The mean retention time of chromic oxide in the rumen was 88 hours, and the mean retention time of PEG was 40 hours.

The experiment was conducted based on the method of the deletion to investigate the ideal pattern of dietary Lys,Met,Trp,Thr,Ile in 0~3 week-age Beijing ducks. When 20% of a single amino acid was removed from the amino acid pattern was regulated based upon the standards of nutrition requirement and the results of the recent research, others were not modified .Ducks were randomly allotted to six treatments and fed a basal cornstarchpeanut meal diet containing 15.93%CP,12.74MJ/kg ME, 0.46% Lys,0.19% Met,0.12% Trp,0.42% Thr and 0.42% Ile. The results indicated that the ideal amino acid pattern in 0~3 weekage Beijing ducks was Lys100, Met 42,Trp 22,Thr 38,Ile 48.

The influence of selective Chinese medical herbs (CMHs) on production and immunity of laying hens in normal and high temperature environment were investigated in the present study. The experiment was concluded two periods. In period one, one hundred and forty-four 51-weeks old laying hens were used. Experiment birds were divided into 6 groups. Group 1, 2, 3 were fed a basal diet supplemented with 1% Ligustrum Lucidum (LL), Schisandra Chinensis (SC) or Si Jun Zi Tang (SJZT), respectively. Group 4 were fed the basal diet supplemented with 10 mg/kg daidzein (DA). Group 5 were fed the basal diet supplemented with 5mg/kg flavomycin. Group 6 were fed the basal diet. The experiment lasted 6 weeks. The environment temperature was 18~26℃. In period two, ninety-six 58-weeks old laying hens were used. Experiment birds were exposed in high temperature environment (32℃), treatments of experiment birds in experiment two were same with experiment one.The results showed that little influences were observed on production, serum corticosterone (Cor) and estradiol (E₂) content by supplemental LL, SC, SJZT and DA (P>0.05). But there were significantly increasing of serum antibody formation and T lymphocyte proliferation by supplemental LL, SC, SJZT and DA (P<0.05) in normal environment temperature. Supplemental LL, SC, SJZT and DA significantly increased production, serum antibody levels and T lymphocyte proliferation (P<0.05), and significantly decreased serum Cor content (P<0.05) in high environmental temperature.

The experiment was conducted to study the effects of high zinc on immune function in chicken. 200 one-day-old Avian broilers were randomly divided into four groups and fed on diets as follow: basic diet (Zn 100 mg/kg, control ［Ⅰ］) and high zinc diets (Zn 1 500 mg/kg ［Ⅱ］, 2 000 mg/kg ［Ⅲ］, 2 500 mg/kg ［Ⅳ］) for seven weeks. The chicken in groups Ⅱ, Ⅲ and IV grew very slowly. At the same time， lymphocytes of the thymus, bursa of Fabricius and spleen were significantly depleted, degenerated and necrotic histopathologically. The weight and growth index of lymphoid organs，proliferating index of lymphocytes， the ANAE⁺ ratio of peripheral blood lymphocytes and erythrocyte C₃bRR ratio markedly decreased and ICR ratio increased markedly in high zinc groups when compared with control group. The above mentioned results indicated that proliferations of lymphocytes were impaired by high zinc.

The changes of the types of the submucous neurons in the small intestine of the piglets aged 0 days, 5 days and 28 days were studied qualitatively and quantitatively by histochemical and immunohistochemical methods. The results were as follows: The submucous plexus includes external submucosal plexus (ESP) and internal submucosal plexus (ISP). The properties of the nerve fibers in ESP and ISP are different. Most of the nerve fibers in ESP are myelinated nerve fibers, while most of the nerve fibers in ISP are nonmyelinated nerve fibers. As the piglets grow, the submucous plexuses develop fast. And the difference between ESP and ISP becomes more obvious. The study showed that the piglets altered the shapes and the ratio of the different types of submucosal neurons to make the function of the submucous plexuses more perfect.

The histogenesis of choroid plexuses in goat brain ventricle were observed by using histological technology,and immunohistochemical ultra sensitive SP method has been used to examine the expression of nerve growth factor (NGF) and its high-affinity receptor (TrKA) in developing choroid plexuses simultaneously.The results suggested that during embryonic development,the choroidal epithelium lining the brain ventricle transited from psedostratified columnar to simple columnar, and became simple cuboidal layer at last.The epithelial cell changed from column to cube in shape,from big to small in diameter.Well-developed acidophilic brush border could be observed at the top of epithelium during development.It was established that during the period studied choroidal epithelium of NGF and TrKA positive reactivity were present.There were little differences between cellular localizaton of two markers,NGF positive products were mainly occurred on nucleus,especially on nucleus membrane,but TrKA positive products were mainly on nucleus membrane.Endogenous NGF in choroidal epithelium suggested major roles in development and maturation of itself,and also in development of brain by circulation of blood-cerebrospinal fluid.

Caprine hemolymph nodes were studied by observing afferent vessels and efferent vessels. It was discovered that these caprine hemolymph nodes are lymphonodus locating in lymphatic circulation pathway and filtering lymph contained numerous erythrocytes.

19 representative type strains and some isolates of Riemerella anatipestifer were detected by slide and tube agglutination and agar gel precipitin tests. Homologous and heterologous reactions of R. anatipestifer strains showed that three serotyping methods correlated well, but gave differences on detecting cross reactions between heterologous stains.Slide agglutination test is a convenient method to screen large number of isolates quickly, but it is not suited to correctly serotype isolates. Isolates could be serotyped by tube agglutination test using type strains as control, but it is difficult to serotyped isolates which produced titers with minor dilution difference with antisera to more than one representative strains. Agar gel precipitin test is also suitable for further detection of isolates, but antiserum with higher-level antibodies should be prepared. Serum absorption removed all cross-reactions in three serotyping tests, and mono-specific antiserum could be prepared. However, all monospecific antiserum showed serotype-specificity only against the known serotypes. Further investigations indicated that some isolates which agglutinated in mono-specific antiserum against one serotype could belong to a new serotype or sub-serotypes of a known serotype.

Chicken interleukin-18 (ChIL-18) mature protein gene was amplified from LPSstimulated MDCC-MSB1 cells by RT-PCR.PCR product was cloned into the pMD18-T vector. Sequencing analysis showed that the nucleotide sequence of this ChIL-18 mature protein gene was 5l0bp including the stop coden and the same as the published ChIL-18 cDNA sequence by Schneider K. A prokaryotic expression plasmid of ChIL-18, pGEX-ChIL18, was obtained by subcloning the encoding region of the ChIL-18 mature peptide into pGEX-6P-1. The recombinant ChIL-18 (rChIL-18) was expressed efficiently in pGEX-ChIL18transformed BL21(DE3)LysS induced by IPTG, the yield was accounted for 32% of the total bacterial protein.

Immunohistochemical method was used to detect avian leukosis virus subgroup J from egg-type chicken, which was preliminarily diagnosed to appear subgroup J avian leukosis. The organs from these chickens were examined by ALV-J gp85 monoclonal antibody, these organs included marrow, liver, spleen, kidney, lung, heart, pancreas, oviduct, ovary, ventriculus glanduaris, skeletal muscle, cerebrum, ischiacticus nerve. The results from immunohistochemistry using monoclonal antibody against gp85 envelope protein of avian leukosis virus subgroup J（ALV-J） revealed that all examined organs contained detectable antigen of ALV-J. The result of immunohistrochemistry agrees with that of pathology diagnosis. It is the first study at home and abroad that discovered and reported natural disease of ALV-J in egg-type chickens.

The SPF pigs were infected with porcine reproductive and respiratory syndrome virus(PRRSV) experimentally. The results indicated that there were not only antibodies(Ab1) to PRRSV, but also the anti-idiotype antibodies(Ab2) to the Ab1 in the serum samples collected from the pigs infected with PRRSV. According to the differences of proteins with different isoelectric point, various kinds of IgG in the serum samples were separated and purified by using isoelectrofocusing(IEF). Ten SPF pigs were immunized with purified Ab2 to anti-PRRSV-GP5 and anti-PRRSV-M respectively, and then they were infected with PRRSV through nasal cavity 7 days after immunization. The serum samples were collected from the pigs infected with PRRSV, and the strains of PRRSV in the serum samples were isolated by using MARC-145 cell or identified with competitive IFA. Five samples collected from pigs immunized with the Ab2 to anti-PRRSV-GP5 were positive from 3 to 7 days post infection; 3 samples were negative from 14 to 63 days post infection; 2 samples were positive from 14 to 35 days post infection and became negative between 42 and 56 days post infection, one of them became positive 63 days post infection. Five samples collected from pigs immunized with the Ab2 to anti-PRRSV-M were positive from 3 to 7 days post infection; 2 samples were negative from 14 to 63 days post infection; 3 samples were positive from 14 to 35 days post infection and became negative between 42 and 56 days post infection, one of them became positive 63 days post infection. The results suggested that the immune response against PRRSV of Ab2 to anti-PRRSV-GP5 and antiPRRSV-M is obviously,and these Ab2 could be used as substitute antigens of PRRSV-GP5 and PRRSV-M to stimulate swine to produce neutralization antibody providing protection against infection of PRRSV.

The cell culture character, safety, efficacy and antibody duration of gene-deleted Pseudorabies virus vaccine strain (SA215), which lacked gE, gI and thymidine kinase (TK) genes, but can express LacZ gene were tested in this study. SA215 could adapt to Vero cells and induce plaque formation. It was safe to 1-day-old piglets, pregnant sows, cattle, sheep, goats and rabbits, moreover, the animals vaccinated with SA215 strain were less clinically affected and the SA215 strain virus couldn’t be recovered from vaccinated animals. Inoculation of SA215 could protected pigs from challenge of Fa strain,the virulent Pseudorabies virus, at a dose of 10⁷ PFU. The periods of fever and growth arrest and titer of excreting virus of the pigs vaccinated with SA215 were lower than those of the pigs vaccinated with Bartha strain, but were much lower than that of the unvaccinated pigs. The vaccinated pigs with SA215 strain produced high-level anti-PRV neutralizating antibodies that could provide protection from infection till 16 weeks after vaccination. The results showed that SA215 strain was a safe vaccine that had good immunogenicity.

In order to clarify the pathogenesis of endotoxin-induced acute renal failure, the blood coagulation changes and the pathomorphologic changes of kidney were investigated while the renal function was being measured in the rabbits given intravenous injections of E.coli O₁₁₁B4 endotoxin. The platelet count and content of plasma fibrinogen were significantly reduced (P<0.01), and bleeding, clotting, prothrombin time and kaolin partial thromboplastin time were markedly prolonged (P<0.05, P<0.01) as compared with the saline-injected control rabbits. On the other hand, the intravenous injections of endotoxin triggered disseminated intravascular coagulation (DIC) in all the rabbits given endotoxin, microthrombi were found in 71.4 percent of the glomeruli, and the mean index of fibrin concentration per glomerulus was 60.6 percent. In addition, the degeneration and necrosis of epithelial cells and the formation of tube cast were found in 96.2 and 84.1 percent of renal tubules, respectively. However, none of the control rabbits showed similar pathologic changes. These results suggested that the coagulation changes induced by endotoxin triggered glomerular capillary thrombosis, the blood flow in the capillaries was blocked , thus the glomerular filtration rate was reduced. This may be the chief mechanism of endotoxin-induced acute renal failure. Additionally, the degeneration and necrosis of the epithelial cells of the renal tubules may impair their reabsorption and secretion capability, and the formation of tube cast block the lumen of the renal tubule and increase the pressure in Bowman’s capsule. These may also be responsible for the pathogenesis of endotoxemic acute renal failure.

A method was described for the determination of the residual sulfamethazine and its metabolite, N⁴-acetylsulfamethazine in sturgeon muscle by high performance liquid chromatography. The sample was extracted with acetonitrile, followed by alumina column clean-up procedure, and analysized using a reversed-phase C¹⁸ column and a mobile phase of water-acetonitrile-acetic acid(76∶24∶0.05, V/V/V). The average recoveries of sulfamethazine and N⁴-acetylsulfamethazine from fish muscle fortified with 20,100 and 2 000 μg/kg were ranged from 80.4%～85.3%, 88.4%～96.0% respectively. The detection limits were 10 μg/kg for each drug.

A RP-HPLC method was used for the determination of eprinomectin concentrations in sheep plasma following i.v. and s.c. administration at a single dose of 0.2 mg/kg. The extract of plasma samples were loaded onto a C₁₈ catridge. After solvent exchange,the methanol eluate was derivatized via the addition of 1-methylimidazole and trifluoroacetic anhydride in acetonitrile.Then the fluorescent derivative was analyzed by using fluorescene detector. Eprinomectin in plasma within 2.5~200 ng/mL ranges had a good linear relationship (R=0.996 8) .The average recovery of the method was 99.65%±3.84%.The RSDs of within-day and between-day assays were less than 10%,12% respectively. Drug concentration-time data in plasma were founded to be fitted to a two-compartment open model and one-compartment open model after i.v.and s.c. administration respectively.The main pharmacokinetic parameters demonstrated that eprinomectin was distributed widely and eliminated slowly after i.v. adminstration, After s.c. adminstration it was absorbed completely and had longer residue time in sheep ,eliminated slower than that of i.v. adminstration.

Tilmicosin was administrated intravenously (i.v.) and subcutaneously (s.c.) to adult healthy crossed sheep to determine its serum concentrations by a high performance liquid chromatography (HPLC) method. Pharmacokinetic analysis of serum concentration-time data after i.v. and s.c. administration was carried out using a computer program (3p97,China Pharmacology Association). Serum concentration-time data after i.v. and s.c.administration were best described by a two-compartment open model.After i.v. injection of 5 mg/kg bw，the parameters were as follows, t _1/2a 0.611±0.017 h，t _1/2β 23.215±0.459 h，AUC 11.815±0.396(μg/mL)·h, CL(s) 0.424±0.014 L/(kg·h).After s.c. injections of 10 mg/kg bw, the parameters were: t _1/2a 1.751±0.557 h，t _1/2β 22.896±2.747 h，t _1/2Ka 0.100±0.025 h，AUC 25.828±1.479(μg/mL)·h，CL(s)0.393±0.017 L/(kg·h)，T _max 0.500±0.065 h，C _max 1.424±0.156 μg/mL, F was 109.28%±6.25%. After s.c. injections of 30 mg/kg bw, the parameters were: t _1/2a 1.342±0.244 h，t _1/2β 20.052±1.236 h，t _1/2Ka 0.086±0.015 h，AUC 57.575±6.760(μg/mL)·h, CL(s)0.527±0.068 L/(kg·h),T _max 0.437±0.039 h, Cmax3.343±0.512 μg/mL, F was 81.22%±9.54%.The results showed that tilmicosin was absorbed quickly, distributed widely and eliminated slowly in sheep after i.v. and s.c. administrations; The bioavailability of tilmicosin after s.c. administrations of 10 and 30 mg/kg b w were 109.28%±6.25% and 81.22%±9.54%, respectively, indicating that tilmicosin was almost absorbed completely.

Virgin female mice weighting 18～25g were superovulated, and then mated with male mice. The acquired pregnant mice were divided into 6 groups for embryotoxicity research of toosendanin on embryo of mice in 2-cell, morula, blastocysts stages. Control group was set up in each of these three stages. Every pregnant mouse of treated and control groups in the three stages was given the dose of 1/30 LD₅₀ toosendanin and same amount of PBS 2 times by intraperitoneal injection respectively. Superovulation need not be carried out for embryotoxicity study of toosendanin in implantation stage, but each pregnant mice in treated and control group was given the dose of 1/30 LD₅₀ toosendanin and PBS 3 times by intraperitoneal injection respectively. Finally, the number and configuration of embryo in uteri of all groups were counted and examined. Results indicated that total embryo of treated group in 2-cell, morula and blastocysts stages had no significance difference comparing with corresponding control group (P> 0.05); Number of normal and abnormal embryo of treated group in 2-cell stage had no significance difference comparing with control group either (P> 0.05); Number of normal and abnormal embryo of treated group in morula and blastocysts stages had great significance difference comparing with corresponding control group (P< 0.01). Due to injection to the treated group 3 times in implantation stage, almost all the fetuses in uteri had been melting, and counting of them could not be performed, while healthy fetuses could be seen and counted clearly in control group. There were only mild histological changes founded in maternal heart, liver, lung, kidney and uteri in all treated groups. All above means that the intraperitoneal injection of a small amount of toosendanin to pregnant Kunming mice may has special embryotoxicity.

Ninety-three cows were separated into 3 groups.31 cows in group 1 were treated with idophor by teat dipping.32 cows in group 2 were treated with Chinese medicine teat disinfectant which were made up of cordate houttuynia,safflower and alum by teat dipping too.30 cows without dipping were raised as control.The practice continued for 35 daysThe result showed as follows:Both Chinese medicine teat disinfectant and idophor could reduce the incidence of clinical mastitis (P＞0.05);The recessive mastitis incidence of the group 1 decreased from 58.1% to 48.4%,and the divergence against control was remarkable (P＜0.05)The recessive mastitis incidence of the group 2 dropped from 81.3% to 75%,and the divergence against control was remarkable (P＞0.05).The udder region incidence of the group 1 was not remarkable against that of the group 2 (P＞0.05),and the incidence against control was significantly remarkable (P＜0.01).The Chinese medicine teat disinfectant could decrease remarkably both SCC and activity of LDH (P＜0.05).The activity of NAGase was not changed significantly (P＞0.05).The Chinese medicine teat disinfectant did not affect the milk yields

Porcine primordial germ cells(PGCs) were isolated from day 25 to 28 genital ridges of fetuses from inbred Wuzhishan miniature pig (WZSP).To support undifferentiated growth, cell lines were derived and stable maintained on STO,which was used for in vitro culture,clone,isolation and passage of embryonic germ cell(PEG).The culture basic medium (CBM) was DMEM with 15% FCS+0.1mmol/L 1-mer-captoethanol+2 mmol/L glutamine and so on.The result was shown that if CBM with growth-stimulating factors such as SCF,bFGF and LIF,the EG’s clone was coming out more early than CBM alone and also with fast growth rate and undifferentiate characteristics than CBM clone.The PGCs of fetuses between day 27~28 of pregnancy (estrus=day 0) was easy and more suitable for collection.PEG’s cell could be survival in vitro culture after frozen and thawing and passaged over F11.
The experiment of making chimeric pig by PGCs was carried out from Sept.to Dec.2002 at experimental farm on Institute of Animal Science,CAAS.Total 252 embryos at blastocyst stages was collected from 22 donors of Large White pig at day 5.5 to 7 of breeding(estrus=day 0).123 of 226 embryos were used for PGCs injection,each embryo was injected 5 to 15 cells of PGCs on average.226 embryos were transferred to 9 recipients,7 of them were return to estrus after transfer at day 15 to 22,one of them remained pregnancy,and the other needs to be future tested.8 piglets were delieved at January 8,from the recipient which was transferred 30 embryos (18 embryo injected and 12 embryo no-injection).2 of 8 piglets were showing coat colour skin,that is called a piglet’s chimeras.The future evaluation of molecular genetic testing is needed to do.