Porcine Circovirus Type2 (PCV2), a single-stranded DNA virus associated with Postweaning multisystemic wasting syndrome(PMWS) of swine, has two major open reading frames, ORF1 and ORF2. The genomic size and molecular weight of ORF2 is respectively 699bp, 28kDa. ORF2 encodes the capsid protein (structural protein) that has type-specific epitopes and is very immunogenic and associated with the induction of neutralizing antibodies, suggesting its potential use in diagnostic assays as well as vaccine development. For efficient production of the capsid proteins, we expressed the PCV2 ORF2 gene with baculovirus in the insect cells. In this study, PCV2 ORF2 was appropriately ligated into the baculovirus transfer vector, pBacPAK9 and pB9-Acpol19-110-EK. Sf21 cells were transfected with a mixture of the purified recombinant transfer vector and bAcGOZA. We generated and purified recombinant viruses containing PCV2 ORF2, and named rAc-B9-PCV2ORF2 and rAc-B9-19-110-EK-PCV2ORF2, respectively. Expression levels of capsid fusion proteins with a partial polyhedrin region of AcNPV more increased than recombinant proteins from non-fusion expressed. Also, expression efficiency increased over time and differed at MOI. As a results, fusion expression of porcine circovirus type2 ORF2 using baculovirus could be utilized as an alternative expression method to produce recombinant antigen against PCV2 infection and is worthy of further investigation.

Aujeszky's disease (AD), also called pseudorabies, is an infectious viral disease caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. Aujeszky's disease virus (ADV) virions contain several envelope glycoproteins. Among them, glycoproteins gB, gC and gD are regarded as the major immunogenicity proteins and the antibodies induced by them can neutralize virus in vitro or in vivo. In this study, we investigated expression of these glycoproteins using the bacterial and baculovirus expressionn system. Successful expression of ADV glycoproteins in E. coli was confirmed by SDS-PAGE and Western blot analysis and their optimal expression condition was determined. However, the recombinant proteins generated in the bacterial expression system which lacks glycosylation process frequently lose their biological activity. We tried to express the ADV glycoproteins using the baculovirus expression vector system. The recombinant gB, gC and gD were detected at approximately 100, 60 and 50 kDa on SDS-PAGE and Western blotting, respectively. The optimal expression conditions were determined for MOI(multiplicity of infection) and post-infection days. One MOI and 4 or 5 days post-infection were the best conditions for the expression of the ADV glycoproteins in Sf21 cells. We are currently investigating the antigenicity of recombinant proteins using experimental animals.

The pig has been considered to serve as an appropriate model of human disease. Therefore, establishment of porcine embryonic stem cell lines is important. The purpose of the present study was to further work in this direction. We produced porcine parthenogenetic embryos, and separately aggregated two of each of two-cell (2×2), four-cell (2×4), and eight-cell (2×8) embryos derived by parthenogenesis. After culture for 4 days, the developmental ability of the aggregates and total blastocyst cell numbers were evaluated. The percentage of blastocysts was significantly higher in both 2×4- and 2×8-aggregated embryos (58.3±1.9% and 37.2±2.8%, respectively) than in the control or 2×2-aggregated embryos (23.6±1.1% and 12.5±2.4%, respectively). Total blastocyst cell numbers were increased in the 2×4- and 2×8-aggregated embryos (by 44±3.0% and 45±3.3%, respectively) compared with those of control or 2×2-aggregated embryos (30.5±2.1% and 30.7±2.6%, respectively; p<0.05). The levels of mRNA encoding Oct-4 were higher in both the 2×4- and 2×8-aggregated embryos than in the control. When blastocysts derived from 2×4- aggregated embryos or intact normal embryos were cultured on mouse embryonic fibroblast feeder cells to obtain porcine stem cells, blastocysts from aggregated embryos formed colonies that were better in shape compared with those derived from intact blastocysts. Together, the data show that aggregation of porcine embryos not only improves blastocyst quality but also serves as an efficient procedure by which porcine embryonic stem cells can become established.

Transducin-like enhancer protein 1(TLE-1) is protein associated with cell proliferation. This study analyzed change of TLE-1 mRNA expression during in vivo and in vitro maturation in porcine oocytes. Oocytes and granulose cells were collected from follicles of <2 mm, 2～6 mm and >6 mm in diameter in slaughtered pig’s ovaries. Oocytes collected from follicles of 2～6 mm in diameter were used after in vitro maturation for 0, 10, 20 and 44 h. Cumulus cells and granulose cells were collected after treatment with hyaluronidase. In results, TLE-1 mRNA expression in oocytes collected from follicle >6 mm in diameter is increased, TLE-1 RNA expression in cumulus cells and granulosa cells from follicles <2 mm, 2 mm 6 mm and >6 mm in diameter. However, there is no significant difference. On the other hand, TLE-1 mRNA expression from oocytes cultured for 10 h and 44 h is increased, TLE-1 mRNA in cumulus cells cultured for 10 h is significant increased(p<0.05) than other culture periods. In conclusion, these results show that TLE-1 is expressed in all cell types of oocytes, cumulus cells and granulose cells, and associated with oocyte maturation.

This study was performed to estimate the seroprevalence of PRRSV in breeding farms in Jeju 2008 using a commercial enzyme-linked immunosorbent assay (ELISA) kit. The tested sera were randomly collected from a total of 1,947 sera from 9 breeding farms unvaccinated in Jeju. As a result, all breeding farms were seropositive for PRRSV. Seven hundred-eighty six of 1,947 sera (40.4%) were positive for PRRSV. Seropositve rate of PRRSV infection in 9 farms showed various levels: 1%, 8.9%, 9.1%, 43%, 46.9%, 48.2%, 51.6%, 60.9%, 85.5%, respectively. The results confirmed that PRRSV infection has been prevailing in breeding farms in Jeju. Also, these results must be taken into a consideration in strategy establishment for the control and eradication of PRRS.

The lung and lymph node samples were collected from 786 pig farms associated with wasting and respiratory syndrome during 2005～2009. All samples were tested for the detection of porcine reproductive and respiratory syndrome virus (PRRSV) and the differentiation of its genotype using reverse transcriptase-polymerase chain reaction (RT-PCR). 643 farms (81.8%) of the pig farms examined were positive for PRRSV, of which 57.2% accounted for PRRSV type 1 and 70.2% accounted for PRRSV type 2. Furthermore, 37.5% of the farms positive for PRRSV, showed the coexistence of two genotypes. The results indicate that the PRRSV infections of single genotype or two genotypes are very common in Korean pig farms.

Infertility of the pig is directly affects on economic loss and failure of the embryo transfer. In the present case report, we show one rare case of porcine infertility resulted from oviductal obstruction. A gilt showing normal heat behaviors was selected as a recipient of embryo transfer. During the laparotomy surgery, abnormality of the reproductive tract was founded. Several large sized cyst-like structures were founded on infundibulum and body of uterus. Severe enlargement of oviduct represents that obstruction of the oviduct. Sign of fibrosis on the surface of uterus and other internal organs revealed that the obstruction was come arise from prior peritonitis. Mild neutropenia and elevated number of monocytes, eosinophils and platelets in blood smear represent that the peritonitis might be due to chronic parasitic infection. Ovarian function was seems to be normal due to blood progesterone concentration was higher than basal level. The pig was culled because she cannot be recovered by surgical or hormonal treatment.

During normal early embryonic development in mammals, the global pattern of genomic DNA methylation undergoes marked changes. The level of methylation is high in male and female gametes. Thus, we cloned the cDNA of the porcine DNA methyltransferase 1 (Dnmt1) gene to promote the efficiency of the generation of porcine clones. In this study, porcine Dnmt1 cDNA was sequenced, and Dnmt1 mRNA expression was detected by reverse transcription-polymerase reaction (RT-PCR) in porcine tissues during embryonic development. The porcine Dnmt1 cDNA sequence showed more homology with that of bovine than human, mouse, and rat. The complete sequence of porcine Dnmt1 cDNA was 4,774-bp long and consisted of an open reading frame encoding a protein of 1611 amino acids. The amino acid sequence of porcine DNMT1 showed significant homology with those of bovine (91%), human (88%), rat (76%), and mouse (75%) Dnmt1. The expression of porcine Dnmt1 mRNA was detected during porcine embryogenesis. The mRNA was detected at stages of porcine preimplantation development (1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stages). It was also abundantly expressed in tissues (lung, ovary, kidney and somatic cells). Further investigations are necessary to understand the complex links between methyltransferase 1 and the transcriptional activity in cloned porcine tissues.

The aim of this study was to investigate what protein(s) of porcine epididymal fluid (pEF) are able to enhance the nuclear maturation of porcine germinal vesicle (GV) oocytes in vitro. Proteins of pEF were fractionated by affinity, ion exchange, and gel filtration chromatography. Porcine cumulus-oocytes complexes (COC) from follicles were cultured in tissue culture medium (TCM 199) containing various fractions obtained by chromatography. Porcine COCs were also cultured in TCM 199 containing various meiosis inhibitors and pEF. After 24 or 48 h culture, oocytes were examined for evidence of GV breakdown, metaphase I, anaphase-telophase I, and metaphase II. When porcine COCs were cultured in the medium with meiosis inhibitor such as, dibutyryl cAMP (dbcAMP) and forskolin (Fo), more than 80% of oocytes were unable to resume meiosis. However, porcine COCs supplemented with pEF were able to overcome the inhibitory effect of dbcAMP and Fo. Maturation rate of oocytes was significantly (p<0.05) increased in the media supplemented with cationic protein(s) during in vitro maturation than in those with anionic protein(s) (44.1% vs 20.0%). When oocytes were cultured in the TCM 199 with fractions obtained by gel filtration, the maturation rate of oocytes was significantly (p<0.05) higher in fraction 11 containing 18 kDa than other fractions. The present study suggests that 1) dbcAMP and Fo prevent the spontaneous maturation of oocyte after isolation from follicles, and that pEF contain a substance(s) that improves meiosis resumption in vitro of porcine COCs, 2) cationic 18 kDa protein(s) are responsible for promotion of MⅡ stage.

Porcine reproductive and respiratory syndrome (PRRS) virus causes not only breeding disorder such as abortion, stillbirth and premature birth to pregnant sows but economic damage like high estrous return and low delivery rate. The presented study was conducted to confirm seroprevalence of PRRS in Jeju herds. PRRS positive rate was examined with pig serum from hog farms located in Jeju. Serum samples were extracted from the 11 of sow farms and 10 of hog farms, The groups were divided into 20, 40, 60, 80, 100 and older than 120 days of age and pregnant sows in hog farms. Anti-PRRSV antibody titers in sera were analyzed by ELISA. All the breeding farms and hog farms (10/10) showed PRRS positive except one breeding farm (1/11). Serological patterns determined by ELISA did not show any difference regardless of whether pigs were vaccinated or not. Nevertheless, the farms unvaccinated pigs displayed low productivity in terms of piglet loss rate and prolonged period of shipment. Therefore, vaccination against PRRSV appears to be a crucial factor in sanitary management of hog farms. Taken together, since PRRSV was spread widely in Jeju already, stabilization of sows by PRRS vaccination after adapting PRRSV free sow by all-in-all-out method in farms is recommended for the control strategy for PRRS.

The present study was to investigate the source of contamination during semen processing for in vitro uses. In the present study, frozen semen was prepared from liquid semen in our laboratory for in vitro fertilization (IVF) experiments due to lack of fresh semen. Antibiotics were added in the frozen semen extender (kanamycin and gentamicin) and in vitro culture (IVC) medium (gentamicin) for further inhibiting growth of microorganisms. Nevertheless, proliferations of microorganisms were observed in IVC culture drop during culturing of IVF embryos using frozen semen. Randomly 3 samples were taken from the liquid semen, frozen semen and egg yolk. Contaminated IVC medium, frozen-thawed semen, liquid semen and egg yolk were cultured in de Man, Rogosa and Sharpe (MRS) agar medium. Whitish colonies were detected in contaminated IVC drop, frozen-thawed semen samples and egg yolk but no colonies were formed in liquid semen samples. Gram-negative and rod-shaped identical bacteria were found in both frozen-thawed semen sample and contaminated IVC drop and egg yolk samples. Enterobacter cloacae were confirmed by API 20E kit according to manufacturer's instruction with identification value (% ID) 94.3% and T index 0.88. Antibiotic susceptibility tests were done according to Clinical and Laboratory Standards Institute (CLSI) by using ampicillin, amikacin, cephalothin, gentamicin, kanamycin, tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin test. Among them Enterobacter cloacae were resistant to ampicillin, amikacin, cephalothin, gentamicin, kanamycin but susceptible to tetracycline, oxytetracycline, sulfamethoxazole trimethoprim, norfloxacin and ciprofloxacin. From these findings it could be suggested that this contamination sources might be from egg yolk.

Optimization of the preimplantation mammalian embryo culture condition was widely focused on refining medium composition under the name of chemically defined media. However, recent research revealed that the alteration of physical environment can be a crucial factor to a successful embryo development. In this study, under the same embryo density, a novel culture device named oil-free micro tube culture (MTC) system was evaluated using porcine parthenogenetic embryos. The activated oocytes were placed into the 0.2 ml thin-wall flat cap PCR tube and cultured to the blastocyst stage. As a preliminary step, embryo density and culture medium volume were optimized under a standard drop culture system. The optimal embryo density range for in vitro culture was 0.5 embryos per in drop (20.5%) and 1.0 embryos per in drop (20.6%). Based on these results, we compared drop culture system and 'MTC' system in terms of the developmental rate to the blastocyst stage. In medium volume, the 'MTC' system showed similar blastocyst formation rate when compared with drop culture system (20.2% versus 20.5%, respectively) while the 'MTC' system showed lower blastocyst formation rate than drop culture system in one (12.7% versus 20.0%, respectively). Therefore the MTC system may be an alternative incubation system for short-distance embryo transport without carrying the incubator and this provides novel embryo culture device to clinical veterinary embryologists.

In the present study, effects of concentration and time of culture in presence of roscovitine on nuclear maturation and meiotic spindle configuration, chromosomal alignment were examined in porcine oocytes. In experiment 1, porcine cumulus oocyte complexes (COCs) were cultured at in a 5% atmosphere in North Carolina State University 23 (NCSU-23) supplemented with 25, 50, 75 or roscovitine for 22 h and then were cultured for additional 22 h after removal of roscovitine. Nuclear maturation and morphology of the meiotic spindle and chromosomal alignment were examined to determine the optimal concentration of roscovitine in oocyte maturation. In experiment 2, COCs were cultured in NCSU-23 supplemented with roscovitine for 17, 20, 27 or 42 h and then an additional 22 h without roscovitine was followed to determine the optimal time of culture. The optimal concentration of roscovitine to arrest and resume meiosis of porcine oocyte was by examining nuclear status (p<0.05) and normal spindle and chromosome configuration. The optimal time of culture in presence of roscovitine to arrest meiosis of porcine oocyte was 17 h (p<0.05), although MII rates and normal morphology of the meiotic spindle and chromosomal alignment were not significantly different among various times of culture. In conclusion, the optimal concentration and time of culture in presence of roscovitine to arrest porcine oocytes are and 17 h, respectively.