• Journal of Embryo Transfer
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• v.16 no.3
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• pp.203-211
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• 2001

This study was carried out to determine sex of porcine embryos produced by in vitro fertilization. Porcine oocyte-cumulus complexes were cultured in BSA-free North Carolina State University (NCSU) 23 medium containing porcine follicular fluid (10%), cystein (0.1 mg/ml) and hormonal supplement (10 IU eCG and 10 IU hCG per ml) for 20~22 hrs. They were then cultured in the same medium but without hormonal supplement for additional 20~22 hrs. After culture, cumulus cells were removed and oocytes were co-incubated for 6 hrs with four different concentrations (5$\times$10$^4$, 2.5$\times$ 10$^{5}$ , 5.0$\times$10$^{5}$ and l0$\times$10$^{5}$ ) of porcine sperm. After fertilization, oocytes were transferred into NCSU 23 with 0.4% BSA medium. The cleavage and blastocyst formation rates were evaluated at 48 and 144 hrs, respectively. In this study, the polymerase chain reaction (PCR) was used to determine the sex of porcine embryos in the stage of blastocyst. The PCR was performed using a set of oligonucleotide primers (5‘-TCATGGACCAGGTAGGGAAT-3', 5’-GAAAGACACGTCCTTGGA GA-3') for 491 bp fragment of porcine male-specific DNA sequence. In the flour different sperm concentration (5$\times$10$^4$, 2.5$\times$10$^{5}$ , 5.0$\times$10$^{5}$ and l0$\times$10$^{5}$ ) for fertilization condition, the cleavage rate was 55.95, 67.88, 60.18 and 47.60%, respectivety, and the development rate of blastocysts was 16.03, 20.40, 21.41 and 12.37%, respectively. At 5.0$\times$10$^4$and 2.5$\times$10$^{5}$ of sperm concentrations per ml cleavage rate and development rate of blastocyst were higher than those of 5.0$\times$10$^4$and l0$\times$10$^{5}$ of sperm concentration (P<0.01). The male of porcine embryos was detected at 491 bp by PCR, and 18 of the 31 porcine blastocysts were the male (58.1%) and the rest 13 were the female(41.9%).

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2001.10a
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• pp.47.1-47
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• 2001

One of the problems associated with in vitro culture of primordial gern cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors and antioxidants, on the ability of the porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally, \ulcorner2-macroglobulin, a protease inhibitor and cytokine carrier, and N-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (p<0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layer, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of \ulcorner2-macroglobulin and antioxidatns can increase the number of PGCs in vitro by suppressing apoptosis.

• Proceedings of the KSAR Conference
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• 2001.10a
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• pp.47-47
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• 2001

One of the problems associated with in vitro culture of primordial germ cells (PGCs) is the large loss of cells during the initial period of culture. This study characterized the initial loss and determined the effectiveness of two classes of apoptosis inhibitors, protease inhibitors and antioxidants, on the ability of porcine PGCs to survive in culture. Results from electron microscopic analysis and in situ DNA fragmentation assay indicated that porcine PGCs rapidly undergo apoptosis when placed in culture. Additionally,？ 2-macroglobulin, a protease inhibitor and cytokine carrier, and N-acetylcysteine, an antioxidant, increased the survival of PGCs in vitro. While other protease inhibitors tested did not affect survival of PGCs, all antioxidants tested improved survival of PGCs (p〈0.05). Further results indicated that the beneficial effect of the antioxidants was critical only during the initial period of culture. Finally, it was determined that in short-term culture, in the absence of feeder layers, antioxidants could partially replace the effect(s) of growth factors and reduce apoptosis. Collectively, these results indicate that the addition of ？2-macroglobulin and antioxidants can increase the number of PGCs in vitro by suppressing apoptosis.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.358-363
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• 2008

The intracytoplasmic sperm injection (ICSI) procedure has recently been utilized to produce transgenic animals and may serve as an alternative to the conventional pronuclear microinjection in species such as pigs whose ooplasm is opaque and pronuclei are often invisible. In this study, the effects of sperm membrane disruption and electrical activation of oocytes on in vitro development and expression of transgene green fluorescent protein (GFP) in ICSI embryos were tested to refine this recently developed procedure. Prior to ICSI, sperm heads were treated with Triton X-100+NaCl or Triton X-100+NaCl+NaOH, to disrupt membrane to be permeable to exogenous DNA, and incubated with linearized pEGFP-N1 vector. To induce activation of oocytes, a single DC pulse of 1.3 kV/cm was applied to oocytes for $30{\mu}sec$. After ICSI was performed with the aid of a micromanipulator, in vitro development of embryos and GFP expression were monitored. The chemical treatment to disrupt sperm membrane did not affect the developmental competence of embryos. 40 to 60% of oocytes were cleaved after injection of sperm heads with disrupted membrane, whereas 48.6% (34/70) were cleaved without chemical treatment. Regardless of electrical stimulation to induce activation, oocytes were cleaved after ICSI, reflecting that, despite sperm membrane disruption, the perinuclear soluble sperm factor known to mediate oocyte activation remained intact. After development to the 4-cell stage, 11.8 (2/17, Triton X-100+NaCl+NaOH) to 58.8% (10/17, Triton X-100+NaCl) of embryos expressed GFP. The expression of GFP beyond the stage of embryonic genome activation (4-cell stage in the pig) indicates that the exogenous DNA might have been integrated into the porcine genome. When sperm heads were co-incubated with exogenous DNA following the treatment of Triton X-100+NaCl, GFP expression was observed in high percentage (58.8%) of embryos, suggesting that transgenic pigs may efficiently be produced using ICSI.

• Proceedings of the KSAR Conference
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• 2004.06a
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• pp.196-196
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• 2004

By use of RT-PCR coupled with DHPLC technique (WAVE analysis), pattern of isoforms of porcine cytochrome P450 aromatase gene was investigated. Relatively higher expression of aromatase mRNA was observed in testis than in ovary and this result accounted for the previous findings of higher blood estrogen level in male compared with female in this species. (omitted)

• Proceedings of the Korean Society of Veterinary Pathology Conference
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• 2003.10a
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• pp.31-31
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• 2003

Porcine circovirus (PCV) is the smallest virus that replicates autonomously in mammalian cells. PCV2 is recongized as the casual agent of postweaning multisystemic wasting syndrome (PMWS).[1] Although the role of PCV2 in reproductive failure requires further clarification, there have been several reports of PCV2-associated reproductive failure. Consistent clinical signs of affected farms include elevated abortion, stillbirths and fetal mummification. The objective of this study was to determine the prevalence of PCV2 in aborted fetuses and stillborn piglets. Second objective was to determine the distribution of PCV2 DNA and antigen in aborted fetuses and stillborn piglets by in situ hybridisation and immunohistochemistry. (omitted)

• Korean Journal of Veterinary Research
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• v.39 no.2
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• pp.353-358
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• 1999

New emerging post weaning multisystemic wasting syndrome (PMWS) threatening swine industry worldwide and investigation of the etiological agent is underway. The porcine circovirus (PCV) consistently identified from PMWS pigs and research result indicate that there is strong relationship between PCV and PMWS. Fauns with PMWS submitted pigs suffered from various PMWS typical signs and necropsy finding showed lymph node anomalies. The PCV DNA was amplified from inguinal lymph node collected from pigs with PMWS. PCV specific primers were successfully amplified PCV DNAs and were able to differentiate PCV type I and II. We have identified noble PCV virus with genetic variation. The virus showed insertion of the nucleic acid at the 5' of the genome but did not have PCR product with primer set corresponding to PCV type II virus.

• Korean Journal of Veterinary Research
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• v.37 no.1
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• pp.177-183
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• 1997

One cytopathogenic virus was isolated in alveolar macrophages from pig without any apparent respiratory clinical signs. Biophysical properties and electron microscopy of the isolate showed the characteristics of adenovirus. Intracytoplasmic inclusion bodies were seen in virus-inoculated cells. The genetic analysis indicated the presence of DNA with the size of >20Kb. In a serological survey of 40 serum samples collected from two different farms in slaughter house, 9 sera were positive for neutralizing antibody against the isolate. The potential implications of the isolate as the causative agent in respiratory disorder were discussed.

• Journal of Embryo Transfer
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• v.17 no.1
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• pp.45-53
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• 2002

The main goal of this study was to produce transgenic porcine embryos by direct injection of sperm-mediated exogenous DNA. Spermatozoa (6$\times$10$^{6}$ sperms of final concentration) were mixed with pcDNA LAC Z (20 ng/$\mu$l) and subjected into electroporation (300~750 volts, 25 $\mu$F, 0.4 cm electrode). After sperm injection, the oocytes were activated electrically (1.7 KV/cm, 30$\mu$sec, single pulse) in 0.3 M mannitol solution or not. The sperm injected eggs were cultured in NCSU 23 medium (0.4% BSA) at 39$^{\circ}C$, 5% $CO_2$ in air fur 144 h. The rates of cleavage and development into blastocyst stage in activation group were significantly higher than those of non-activation group (79.6% and 24.1% vs. 46.3% and 14.4%, respectively, p<0.05). Control oocytes and shame injection were developed to blastocysts low (2.5%). Sixty five (27.1%) out of 240 embryos observed in activation and non-activation groups were showed positive by X-gal staining. However, all embryos in both groups were expressed partial or mosaic pattern. These results suggested that electrical stimulation far oocytes activation after sperm injection enhances the incidence of both fertilization and development fellowing sperm injection in the pig. Our study also suggested that sperm-mediated transfer of exogenous DNA by ICSI would be used as a valuable tool for the production of transgenic porcine embryos.

• Korean Journal of Veterinary Service
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• v.14 no.2
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• pp.110-120
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• 1991

28 porcine enteroviruses were isolated from 86 pig-feces of 9 swine farms located in south region, Chung-buk, from March to September 1990. Physico-chemical properties and pathogenicity of isolates were investigated. Results obtained throughout experiments are summarized as follows. According to the age, weanlings(40-90 days), sucklings(10-30 days) and adult pigs(6 months over) showed the isolation rate of 67%. 8% and 4%, respectively. By physico-chemical tests, YD-90/22, YD-90/43 and YD-90/64 strains were found to be ether, chloroform and PH stable. Nucleic acid test suggests the virus to have a DNA genome. Most of the Isolates were not evident of hemagglutinin using erythrocytes from various mammalian & avian. 22 strains among the isolates were shown CPE type I and the remainders were CPE type II. 3 strains among isolates of CPE type I strains were neutralized with high titers to serotype 2 antiserum. In the study on virus growth curve in PK-l5 cells, YD-90/22, YD-90/43 and YD-90/64 strains showed the maximum infectivity titers($10^{6.0}-l0^{6.5} TCID({50}ml$) at 4days post inoculation(PI). When 30 day-old commercial piglets were inoculated only intraoral route with the YD-90/22 strain at $10^{6.0} TCID_{50}ml,$ piglets not showed the symptoms. But piglets inoculated by intramuscle route, intraoral and intramuscle route after pretreat with dexamethasone(2.5mg /kg) for 5 days were shown the symptoms of anorexia, diarrhea, pyrexia and ataxia at 4th-6th days PI. The viral reisolation in the virus-inoculated piglets was examined from feces. The viruses were recovered intermittently from 2nd to 16th day PI and at 4th-6th day PI, all piglets excreted viruses.