Torovirus, an important member of the Coronaviridae family in Nidovirales, contains four official species: equine torovirus, bovine torovirus, swine torovirus, and human torovirus, they are the agents cause gastroenteritis and respiratory diseases in different animal species and human. It has been 30 years since the virus firstly separated and reported in Lowa America, the progress in understanding the molecular epidemiology about torovirus and its hosts have been updating, this review introduced the most recently research progress of torovirus include finding and taxonomy, characteristic, genetic structure, mechanism of cell apoptosis, also contain variation, epidemiology, pathology, detection methods and a summary about possible research prospect.

This experiment was conducted to study the relationship among gene expression of malate dehydrogenase and lipoprotein lipase in muscle and contents of intramuscular fat and composition of fatty acids in F₁ hybrids with boar × Laiwu pigs, and to investigate the sedimentary mechanism of intramuscular fat. F₁ hybrids with boar × Laiwu pigs(called YL F₁) and pigs with Landrace × Yorkshire (40 pigs for each, 20 for male, 20 for female) were used. The dorsal-waist longissimus muscle were collected when the body weight reached 20, 35, 50, 70 and 90 kg. The developmental changes of MDH and LPL genes expression was detected by fluorescent quantitative RT-PCR method, and the relationship between the change and the content of intramuscular fat and composition of fatty acids was analyzed. The results indicated that with the increase of body weight, the MDH gene expression in muscle in F₁ hybrids with boar × Laiwu pigs and pigs with Landrace × Yorkshire pigs increased from 20 to 70 kg, and decreased from 70 to 90 kg. The LPL gene expression in muscle in F₁ hybrids with boar × Laiwu pigs and pigs with Landrace × Yorkshire pigs increased with the increase of body weight. Correlation analysis showed that in F₁ hybrids with boar × Laiwu pigs, the expression of MDH and LPL mRNA had significant positive correlationship with content of intramuscular fat(P<0.05), but had no correlation with rate of polyunsaturated fatty acid to saturated fatty acid (P>0.05). In Landrace × Yorkshire pigs, the expression of MDH and LPL mRNA had no correlationship and had significant positive correlationship with content of intramuscular fat, respectively(P>0.05 and P<0.05), and had significant negative correlationship with rate of polyunsaturated fatty acid to saturated fatty acid (P<0.01 and P<0.05). The rate of polyunsaturated fatty acid to saturated fatty acid had significant negative correlationship with the content of intramuscular fat in two pig populations(P<0.05). The results suggest that with the increase of body weight, the expression of both MDH and LPL genes in muscle of pigs have apparent developmental characteristic. We can adjust the content of PUFA in muscle by controlling the content of PUFA in diet of pigs, which can affect the expression of MDH and LPL genes, and adjust the content of intramuscular fat.

The objective of this study was to explore the expression pattern of POU2F3 gene in sheep, and to clone the full-length CDS of POU2F3 and its alternatively spliced isoforms in Chinese Merino skin (Xinjiang Junken Type). The expression of POU2F3 gene was detected by Semi-quantitative reverse transcription PCR (SqRT-PCR) in tissues. The full-length CDS regions of POU2F3 gene and its isoforms were amplified by RT-PCR, cloned into T vector, and sequenced. The sequence was analyzed using online bioinformatics softwares. SqRT-PCR analysis showed that the POU2F3 gene was only expressed in some sheep internal organs and tissues. Comparatively, it was highly expressed in body side skin. No significant differences at the expression levels of POU2F3 gene between the skins from various anatomic locations of super fine wool strain were observed (P>0.05). Similarly, no significant differences at the expression levels of POU2F3 gene in body side skin between wool and hair sheep were observed (P>0.05). Sequence analysis indicated that sheep POU2F3 gene in skin generated at least four spliced mRNA isoforms encoding four POU2F3 protein isoforms of 429, 396, 221 and 221aa, respectively. POU2F3 gene is highly expressed in the skin of Chinese Merino (Xinjiang Junken Type). Besides the full-length POU2F3, sheep POU2F3 has at least two protein isoforms in skin. This study not only lays the foundation for clarifying the function of sheep POU2F3 gene, but also provides theoretical basis for breeding high quality Merino sheep.

The study was conducted to clone sheep YAP1 CDS and construct a eukaryotic expression plasmid and a mutant that could not be phosphorylated at Ser42. The CDS of sheep YAP1 was amplified by RT-PCR from healthy sheep longissimus dorsi muscle, and was cloned into pMD19-T simple vector by T/A Ligation to sequence the DNA fragment. After double digestion, YAP1 coding region was subcloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant. PCR，restriction endonucleases analysis and sequencing were used to conform both of the recombinant plasmids. The sheep full-length YAP1 cDNA sequence was 1 712 bp in length encoding 403 amino acids. It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine at 42^ed amino acid by PCR，restriction digestion and sequencing. The results showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly, which paved the foundation for further studies on the YAP1 protein expression and its biological activities.

The study aimed to screen the SNPs in the promoter region of STAT3 gene in cattle and analyze the effect of SNPs on function elements of promoter and growth traits. Two cattle breeds (Wuchuan Black cattle and Guizhou Holstein cow) that possessed significant difference in breeds property were selected to construct DNA pools, SNP sites were screened by direct sequencing subsequently. PCR-RFLP was used to be genotyping and a total of 114 Wuchuan Black cattle were used to study the associations of SNPs with 8 growth traits. Results showed that 7 new single nucleotide polymorphism (SNP) sites including G-449A, C-341A, A-322G, C-246A, G-240A, C-225T and A-103T, respectively were found in the 5' flanking region of STAT3 gene and then submitted the information of SNPs to NCBI to obtain accession numbers. Furthermore, bioinformatics tools were used to predict the core region of the promoter and CpG island. SNPs found in this study led to the disappearance of a large amount of transcription factors bingding sites and the appearance of new sites, it also showed that SNP mutation didn’t affect the range of CpG island dramaticlly by using various softwares. Association analysis demonstrated that A-322G had highly significant effects on body weight (P<0.01), A allele was the favorable alleles. Moreover, animals with the genotype AG had the highest mean body weight than those with GG and AA genotypes, which suggested that A-322G was an important SNP marker affecting the cattle growth performance and STAT3 gene could be applied in marker-assisted selection for improving growth performance in cattle. This work could lay the experimental foundation for the identification of function of STAT3 gene in cattle.

Factor XI deficiency caused by an insertion of 15 nucleotides in bovine Factor XI gene is a newly identified genetic defect that results in blood coagulation disorder in Japanese Black cattle. To analyze the distribution frequency of the defect gene in cattle population in China in current study, two new methods were developed(capillary electrophoresis-based genotyping and allele-specific amplification) to sensitively discriminate the alleles. Using these methods, 452 individuals were genotyped from 14 cattle breeds or populations, including Japanese Black bulls, a breeding herd and a commercial herd with Japanese Black cattle blood, four Chinese local breeds (Luxi, Yanbian, Menggu, Fuzhou), two composite breeds (Chinese Holstein, Xinjiang Brown), and six foreign breeds (Simmental, Charolais, Limousine, Angus, Montbeliard, Norwegian Red). The results showed that the defect allele frequency was 0.182, 0.389 and 0.242 in Japanese Black bulls, the breeding herd and commercial herd with Japanese Black cattle blood, respectively, 1 carrier was found in Yanbian cattle, and the mutation was not found in other cattle populations. The result suggest that the mutation only exist in cattle populations in Northeast Asia and Northeast region in China. The mutation will be detected and eliminated during crossing, improving and breeding the local cattle by Japanese Black cattle and then the unfavorable allele frequency will be reduced.

This study aimed to clone the complete length sequence of Cocaine- and Amphetamine-Regulated Transcript gene (CART), analyze its structure, and reveal the distribution of SNPs (single nucleotide polymorphisms) in different yak breeds. Possible impact of the SNPs on CART gene expression was also predicted by bioinformatics analysis. This study could provide some basic information for studying the function of yak CART gene in future. The blood samples of 243 Datong yak, 208 Tianzhu yak, 208 Gannan yak, and 53 Pali yak were used to isolate the genomic DNA. Then the full length of CART gene was cloned and sequenced. Genetic variations and the distribution of 4 SNPs in the 4 yak breeds were detected by the high-resolution melting system followed by genetic statistical analysis. In this study, 4 novel SNPs located in the yak CART gene were identified, and these SNPs were located in upstream, intron, and 3′-UTR of the CART gene. Χ² tests showed that besides the V-221 in Datong population and V1816 in Gannan population, all the other loci were in Hardy-Weinberg equilibrium (P＞0.05) in the 4 yak breeds. Through population genetics analysis, the results showed that V168 and V181 loci were at intermediate polymorphic status, but V-221 and V1791 loci were at low polymorphic status. The haplotype C was predominant. Bioinformatics analysis showed that different alleles of V1791 and V1816 might change the RNA secondary structure, which could influence CART gene’s expression. The nucleotide variation of V1791 and V1816 loci reduced RNA stability that might produce negative influence on CART gene expression.

The aim of this study was to investigate the relationship between the expression level,tyrosine phosphorylation of sp32 and the activation of the boar proacrosin/acrosin system, the acrosomal membrane proteins of boar sperm for different treatments (fresh sperm, freezing-thawing, capacitation, acrosome reaction) were separated, and stained by CBB, assayed using SDS-PAGE and Western blot analysis. The results showed that the expression level of sp32 was different in conversion of proacrosin/acrosin system after treatment of boar sperm capacitation, freezing-thawing and acrosome reaction, the expression of sp32 in experiment groups of capacitation and acrosome reaction were slightly higher than the freezing-thawing experiment group, and significantly higher than that of fresh semen group. The level of sp32 tyrosine phosphorylation had a significant difference between the freezing and thawing experiment group and other experiment groups. But bands with molecular mass of 38-170 ku in the fresh semen group were more obvious; it showed that the acrosomal membrane proteins were accompanied by modification and degradation of big molecular proteins when sperm underwent capacitation and the acrosome reaction. sp32 as a proacrosin binding protein, its expression and tyrosine phosphorylation level were up-regulated in the activation of the boar proacrosin/acrosin system.

To explore the possible effects of Ghrelin and certain relationship between Ghrelin and FSH (Follicle-stimulating hormone) in regulating the proliferation of cumulus cell freed from bovine cumulus-oocyte complex (COCs) in vitro maturation process, the Ghrelin mRNA levels within bovine cumulus cells varied depending on the oocyte maturation stage, the effect of FSH and FSHBI (FSH binding inhibitor) on Ghrelin mRNA expression and the effect of FSH and Ghrelin receptor antagonist (［D-Lys³］-GHRP-6) on regulating the expression of PCNA, BCL-2 and BAX mRNA at 8 h in vitro were detected by relative real-time RT-PCR experiments. And cumulus cell proliferative activity affected by FSH and ［D-Lys³］-GHRP-6 was determined by cell proliferation activity assay technology during bovine COCs maturation. The results showed that the expression of Ghrelin mRNA within cumulus cell was the highest at 8 h and the expressed difference among 8, 0 and 24 h was significant (P<0.05). The expression of Ghrelin mRNA in cumulus cell was prominently elevate when addition of 0.01 IU·mL^-1 FSH (P<0.05), while the Ghrelin mRNA levels was significantly decrease affected by 400 ng·mL^-1 FSHBI. The expression of PCNA and BCL-2 genes and the proliferative activity of cumulus cell were observably promoted by 0.01 IU·mL^-1 FSH (P<0.05), but were markedly reduced after adding ［D-Lys³］-GHRP-6 (P<0.05). The results suggested that Ghrelin involves in regulating cumulus cell proliferation triggered by FSH and that this research could help in understanding the mechanisms behind follicular development regulated by FSH.

This study was undertaken to evaluate protein expression differences between mature and immature follicular fluids of swamp buffalo in Guangxi. The research used two-dimensional gel electrophoresis (2DE) coupled to Matrix Assisted Laser Desorption/Ionization Time-of-flight Tandem Mass Spectrometry (MALDI-TOF/TOF) to analyze the different proteins, and the establishment of a suitable 2DE system for separating buffalo follicular fluid proteins was described. The results indicated that acetone precipitation in a loading volume of 350 μg in 24 cm IPG strips (pH 4-7) improved resolution and the quality of the 2DE maps, and 11 different expression protein spots were found with Image Master 2D platinum software. Compared with the mature follicular fluid 2DE map, 5 of these proteins were up-regulated in immature buffalo follicular fluid, 3 were down-regulated, 1 deleted and 2 proteins specifically expressed. Finally, 4 proteins were successfully identified by mass spectrometry; these are peroxiredoxin-2, aldose reductase, fibrinogen gamma-B chain precursor, and transthyretin precursor. In conclusion, an appropriate 2DE system for separating buffalo follicular fluid proteins was established and optimized. Different expression proteins from mature and immature buffalo follicular fluids were successfully analyzed and identified to provide new research insight into the developmental microenviroment and maturation mechanism for buffalo oocytes.

 The objectives of this study were to evaluate the expression of Androgen receptors (AR) in various tissues and the cellular localization of gonad of the male sheep lambs. In this experiment, mRNA expression was detected by real-time fluorescent quantitative PCR. Cellular localization of AR in testis and epididymidis were examined by immunohistochemistry. Qualitative investigation of AR protein was expressed by Western blotting. The result showed that: (1) Sheep AR mRNA was expressed in all the tissues, and abundantly expressed in testis and heart. (2) Weak positive signals were detected in pseudo stratified ciliated columnar epithelium and interstitial tissue of EpH, EpB and EpT. Strong positive signals were detected in interstitial tissue, spermatogenous cells and myoid cells of sheep testis. Sheep AR was widely expressed in all the tissues. AR plays an important role in gonadal developing and sperm maturation processing and heart protecting, the mechanisms need further research.

This experiment was conducted to evaluate the effects of purified Glycinin on the permeability and the expression of tight junction protein Occludin in piglet intestinal epithelial cells (IPEC). IPEC were cultured with mediums added with Glycinin by different concentrations (0-5.0 mg·mL^-1) for 24 h. Measuring the mRNA expression of Occludin by real-time quantitative FQ-PCR and the transendothelial electrical resistant (TEER). The results showed that, compared with the control group, Glycinin could decrease the TEER and the relative expression of Occludin (P<0.001) mRNA in piglet, but it had no significant effect at the concentration of 0.5 mg·mL^-1. These results indicate that Glycinin can increase the permeability of the epithelial cells, and decrease the Occludin mRNA expression. Furthermore, this study clarify that the Glycinin directly induces intestinal damage by depressing intestinal epithelial cells growth and damaging the integrity of intestinal cells.

This study was conducted to investigate effects of maternal undernutrition during late pregnancy on growth, development and anti-oxidation capability of Mongolia ovine fetal liver. 42 Mongolia ewes treated by estrus synchronization fecundability were selected, 6 ewes were slaughtered at the beginning of the experiment and the remaining 36 animals were allocated to three different groups: restricted group1 (RG1, 0.175 MJ ME·kgw^-0.75·d^-1, n=14), restricted group2 (RG2, 0.33 MJ ME·kgw^-0.75·d^-1, n=12) and control group (CG, ad libitum, 0.67 MJ ME·kgw^-0.75·d^-1, n=10). At 140 d of gestation, 6 representative ewes in each group were slaughtered. The results showed as follows: fetal weight(P<0.01), fetal liver weight(P<0.01), DNA concentration(P<0.01), protein concentration(P<0.01), total DNA contents(P<0.01), protein/DNA(P<0.01), T-AOC(P<0.01), SOD activity(P<0.05) in fetal liver were significantly decreased and GSH-PX activity (P<0.05), MDA concentration(P<0.01) were significantly enhanced in RG1 group compared with CG group. For RG2 group, the decreases of fetal weight(P<0.01), liver weight(P<0.05), DNA concentration(P<0.01), protein concentration(P<0.01), total DNA contents(P<0.01), protein/DNA(P<0.01), T-AOC (P<0.01) were founded and MDA concentration was significantly increased inRG2 group than that in CG group(P<0.01). In conclusion, the growth, development and anti-oxidation capability of fetal livel were affected seriously by maternal undernutrition during late pregnancy. With the decreasing of nutrition levels, the negative reactions to fetal liver became more severe and the sensitivity of anti-oxidation defense was increased.

The study aimed to improve the fermentation characteristics and nutritive quality of mixed silage of hullessbarley straw and perennial ryegrass in Tibet, and enhance the utilization efficiency of straw resource in Tibet. Mixture of hullessbarley straw and perennial ryegrass (6︰4) were ensiled with Cornzyme at four levels (0，1.5，2.0 or 2.5 mL·kg^-1 of fresh weight). Triplicate silos per treatment were opened on 7, 14, 30 and 60 days after ensiling, respectively, the fermentation characteristics and in vitro degradability of silages was analyzed. Cornzyme addition significantly improved the fermentation quality of mixed silages, indicated by higher (P<0.05) lactic/acetic acid, lactic acid and water soluble carbohydrate content, and lower (P<0.05) pH value and ammonia/total N (AN/TN) than that of the control silage. The pH value of Cornzyme addition silages maintained to about 4.0, which inhibited the production of acetic acid, propionic acid and butyric acid. After 60 days of ensiling, AN/TN in Cornzyme addition silages were half of that in control, but WSC content were double of that in control. The contents of crude protein were higher and neutral detergent fiber were lower in Cornzyme addition silages. These results suggest that adding Cornzyme to mixture of hullessbarley straw and perennial ryegrass at ensiling appears to be a feasible strategy to improve the fermentation and nutritive quality of straw-grass silages.

The specific primer pairs for amplification of long terminal repeat sequences (LTR) of Reticuloendotheliosis virus (REV) was used in the PCR reaction to amplify the specific nucleic acid sequence of healthy wild birds collected in Jilin province. Positive samples were inoculated on the DF1 cells for virus reproduction.With identification of indirect immunofluorescence assay (IFA), PCR assay, two REV strains were isolated, one from the pintail (Coturnix cotu), another from mallard (Anas platyrhynchos). The two isolates were named DBYR1101 and DBYR1102, respectively. The gp90 gene encoding the most important protective protein was cloned and sequenced. Sequence analysis showed that the gp90 gene based amino acid homology between the two isolates is 99.5%. And they are more identical to the northeast China isolates (the amino acid homology of 99.2% to 99.9%) than to the early Chinese REV isolates HA9901 (94.7% and 94.2%). The two isolates in this study and the northeast China isolates tend to group together in their own distinct phylogenetic clade. The gp90 amino acid homology between DBYR1101 and 3 subtype of REV170A, SNV and CSV was 95.7%, 94% and 98.2% respectively, while DBYR1102 was 95.2%，93.7% and 97.7%. And the gp90 gene between the two isolates and the early southern isolate HA9901 were 94.7% and 94.2%. In addition, the 2 REV strains have a high identity with some REV strains in US and Taiwan, which classified as subtype Ⅲ. This is the first study to investigate the status of wild birds infected with REV, and the results of this paper will not only expands the epidemiological data of REV, but also remind us pay more attention to the role of wild bird migration in the spread of diseases. Meanwhile, it also makes a basis for further researching the pathogenic and immunosuppressive mechanisms.

The objective of this study was to investigate the inhibition of lipid associated membrane proteins (LAMPs) from Mycoplasma hyopneumoniae of different virulence on porcine alveolar epithelial cell lines. LAMPs were extracted from the culture of M. hyopneumoniae by Trion-X114, the suppression ratio was detected through MTT assay, and moreover the concentration of nitric oxide was measured by Griess reagent. Our results indicate that all of four kinds LAMPs can do toxic action on porcine alveolar epithelial cell lines, and the containment procedure was agreed with the virulence of M. hyopneumoniae. The inhibited effect caused by strain NJ and 168 was stronger than strain attenuate 168 and J, which the minimum IC₅₀ (0.13 mg·mL^－1) belonged to strain NJ and the maximum IC₅₀ (0.56 mg·mL^－1) belonged to strain J. Moreover, LAMPs also could induce NO. The inhibition on porcine alveolar epithelial cell lines induced by M. hyopneumoniae LAMPs is related with the virulence of Mycoplasma, while the inhibited effect of virulent strain is more powerful than attenuate strain.

The signaling pathway of Nuclear factor-kappaB (NF-κB) plays an important role in the viral infection, immune injury diseases and tumor diseases. Its activation level is often used as a detection indicator for immune response level and disease development. In order to obtain porcine NF-κB p65/p50 sequence characteristics and establish a real-time fluorescence quantitative PCR (real-time FQ-PCR) method to detect its mRNA expression in vitro, porcine NF-κB p65 ORF and mature p50 coding region were amplified, cloned and analyzed. Specific primers were designed to develop the real-time FQ-PCR assay to detect p65, p50 mRNA expression. Sequence analysis showed that porcine p65 ORF contained 1 662 nucleotides in full length encoding a protein of 533 amino acid residues, the p50 mature protein coding region contained 1 653 nucleotides encoding 551 aa. The nucleotide sequence of p65 and p50 genes shared a high similarity with other animals. 56.5% p65 located in the nucleus, 78.3% p50 existed in the cytoplasm, no signal peptide and transmembrane region had found in both p65 and p50. The real-time FQ-PCR results showed a good liner relationship between template copy number and circulation number, the correlation coefficient (R²) of the standard curves were all 0.999, the amplification efficiency at about 95%. These assays were highly specific and there are single specific melting peak. The assays were highly sensitive and have a detection limit of 10¹ copies·μL^－1, and it was highly repeatable and had a coefficient of variation less than 5‰. This study laid a foundation for researching biological function of NF-κB p65/p50 and its differential expression in pig diseases.

 The aim of this study was to develop a rapid detection kit for olaquindox (OLA-Kit) in animal feed. The OLA monoclonal antibody (OLA mAb) was applied to assemble the indirect competitive ELISA kit and its characterization was determined. The results showed that the calibration curve of OLA-Kit was a typical sigmoid curve with the linear detection of 1-243 μg· L^－1, and R² was 0.976 7. The kit has a half inhibition concentration (IC₅₀) of (14.29±3.75) μg·L^－1, the detection limit of 0.79 μg·L^-1, the quantitative limit of 4.0 μg·L^－1, the cut-off value of 83.37 μg·g－1 for pannage, and the matrix has no effect on the results of the OLA-Kit; The recoveries of OLA in pig feed, chicken feed and fish feed were all above 74.40%, and the inter-assay and intra-assay coefficient variation were all below 15%; The OLA-Kit had 17.93% cross-reactivity towards Kuianchun and little or no cross-reactivity towards MQCA, QCA and other quinoxaline drugs. The validity of kit in 2-8 ℃ was above six months. There was no significant difference of the recovery of OLA in pig feed between OLA-Kit and HPLC (P≥0.05). In conclusion, the OLA-Kit was sensitive, specific and accurate, could be used for the rapid determination and extensive screening of OLA in animal feed.

This experiment was conducted to study thymuses physiological and immunological functions. The age-associated changes in histology of the healthy yak's thymus at three different age groups (one-day-old, five-month-old and adult) were observed by histology, immunochemistry and statistics methods. The results showed that as the age increased, identifiable corticomedullary delineation became unclear; thymocytes in both the cortical and the medulla became sparse. These changes were accompanied by a marked increase in the content of interlobular connective and adipose tissue and thickening of the thymic capsule. S100 positive thymic dendritic cells (TDCs) were localized in the corticomedullary junction and medullary zones in each group. The average number of S100 positive thymic dendritic cells decreased per area while the age increased. And differences between each age group were significant (P<0.01). Moreover, the average number of Caspase-3 positive thymocytes per area increased while the age increased. Particularly, the average number of this cell in the cortex increased and there were significant differences between each age group (P<0.01). No difference (P>0.05) were observed between 5-month-old and adult group, but these two groups were significantly higher (P<0.01) than the 1-day-old group, respectively. The results indicated that the yak’s thymus showed obvious degeneration trend in structure and function which is possibly associated with the decrease of TDCs and increase of cortical apoptotic thymocytes. The underlying mechanism need to be investigated further.

 This experiment was conducted to study tissue residues of ceftiofur in swine, preparing for determination of the withdrawal time. A confirmative method has been improved to determine ceftiofur residues in edible tissues of pigs by high-performance liquid chromatography (HPLC). Ceftiofur was administered to 30 healthy pigs intramuscularly at a dosage of 5 mg·kg^－1 for 3 consecutive days. Animals were sacrificed at 12 hours and the 3rd, 5th, 7th, 9th days after administration. The tissues were extracted with dithioerythritol, derivatized with iodoacetamide, and cleaned up on MCX solid-phase extraction cartridges. The analyte was detected by UV absorptive spectroscopy after separation by C₁₈ column. The results showed that kidney had the highest drug concentration with 2.589 μg·g^－1 at 12 hours, while the concentration of ceftiofur in all of tissues at 3 days was lower than the Maximum Residue Limit (MRL). The pharmacokinetic parameters were estimated using Winnonlin software package. The results indicated that the elimination rate was skin/fat﹥muscle﹥liver﹥kidney﹥injection site, with the elimination half-life of 28.99, 35.80, 36.76, 55.72, 160.8 h respectively. Compared with other tissues, the area under concentration-time curve (AUC) in kidney was highest with 123.4 h·μg·g^－1. Kidney was considered as the target organ. According to the MRL set by EU, the longest withdrawal time was suggested in injection site with 98.64 h. The results demonstrated that absorption and elimination was quickly and the distribution was wide. It is proposed that the withdrawal time was 5 days.

In order to study the developmental regularity of mink oocytes during dioestrus in November and to evaluate the in vitro maturing possibility of them, ultrastructure of ovaries preantral follicles of American short-hair black minks during dioestrus were observed by transmission electron microscope. Results showed that there were a less number of cristae at the primordial follicle stage. Golgi apparatus were visible. The size of lipid droplets was different, and cortical granules dispersed around the nucleus. At the primary follicular stage, the number of mitochondrion was increasing. Golgi apparatus tended to be typical. Lipid droplets distributed evenly. Cortical granules monolayer dispersed near the plasma membrane. At secondary follicle stage, the number of cristae was increasing. Flocks of Golgi apparatus distributed under the plasma membrane. The diameter of lipid droplets was increasing and the color of those was shallower. The results suggest that the distribution of cortical granules and Golgi apparatus in mink oocytes during dioestrus in November was different from that during estrus; while the developmental trend of mitochondria, endoplasmic reticulum, lipid droplets and other organelles during these two periods was the same. Therefore it is possible that mink oocytes obtained in November develop to mature in vitro.

The objective of this research was to construct recombinant adenovirus vector specific to SREBP1 gene of Qinchuan cattle and further package and amplify recombinant adenovirus carrying SREBP1 gene, aimed at studying SREBP1 gene function at cellular level. A pair of exclusive primers was designed according to the GenBank sequence information of SREBP1 gene to amplify the complete CDS area of SREBP1 gene via polymerase chain reaction (PCR). The obtained PCR products were then sub-cloned into pMD19-T simple vector. The confirmed fragments containing CDS area of SREBP1 gene were first digested from clone vector and then?insert into the shuttle vector to construct the pAd-Track-CMV-SREBP1 plasmid. The resultant plasmid was linearized by digesting with restriction endonuclease PmeⅠ and subsequently transformed into BJ5183 containing pAdEasy-1 to obtain the expression vector pAd-SREBP1. Overall, the confirmed recombinant adenovirus plasmid pAd-SREBP1 was digested with PacⅠ and transfected into 293A cell line to package and amplify the recombinant adenovirus. The viral titer was determined by GFP labeled method. SREBP1 gene was cloned from Qinchuan cattle with two mutations in the DNA fragments. The two mutations were proved to be caused probably by the varietal difference, not by external factors. Recombinant plasmid pAdTrack-CMV-SREBP1 and pAd-SREBP1 were successfully constructed to the package and amplify the recombinant adenovirus with 1.5×10⁹ GFU·mL^－1 as its titer.

The aim of this study was to construct 4 Lenti-virus Interference Vectors for chicken zyxin gene, determine their titers, and filter the effective interference target, and lay the foundation for studying of anti-coccidial mechanism of chicken zyxin gene. The experiment was performed by extracting RNA from chicken thymus, then synthesizing the cDNA with reverse transcription, amplifying coding regions of zyxin, and cloning it into eukaryotic expression vector Pex-6 at last. The shRNA sequences for zyxin were designed, and were inserted into Lv3 lenti-virus vectors. Lv3-shRNA vectors and packaging plasmids which include pGag/Pol, pRev and Pvsv-G were cotransfected into 293T cells to package virus. Dilute virus liquid 10 times the 6 gradients, and transfect them into 293T cells respectively to determine the titer. Transfect 4 recombinant lenti-virus interference vectors and eukaryotic expression vectors Pex-6 into 293T cells, and filter the effective interference target by RT-RCR and Western-blot. The sequence identification results showed that the 4 constructed zyxin recombinant lenti-virus interference vectors including CO1, CO2, CO3, CO4 and a negative control plasmid were correctly constructed. By way of transfecting them into 293T cells and concentration, the titer reached 1×10⁸ TU·mL^－1 which showed that they were suit for being transfected into the target cells. The filter result showed that CO3 had the most significant interference effect on zyxin expression in 293T cells. Analysis of the data suggests that the zyxin recombinant lenti-virus interference vectors were constructed and filtered the effective interference target successfully, which laid the foundation for further research on zyxin gene function.