• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.66-66
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• 2001

Since the first birth of pig derived from embryonic cells by nuclear transfer, many researches to produce cloned pig have been carried out. Recently, two reports about the birth of somatic cell cloned pigs using in vivo oocytes and also Betthauser et al. (2000) reported the birth of somatic cell cloned pigs using in vitro oocytes. So here we investigated the effect of activation method and culture medium on in vitro development of porcine nuclear transfer embryo using fetal fibroblast. Oocytes derived from slaughter house obtained ovaries were matured for 42 to 44 h in TCM 199. Matured oocytes were denuded using 0.1% hyaluronidase and then Oocytes with the first polar body were used for enucleation by aspirating the first polar body and adjacent cytoplasm in TCM 199 supplemented with 7.5 $\mu\textrm{g}$ cytochalasin B. Petal fibroblast cells were prepared from 35 days old fetus. To be used as donor cells, fetal fibroblast cells were serum starved for 3 to 5 days and then isolated into single co:1 by trypsinization. Nuclear transfer embryos were fused using 2 times 1.25㎸ for 30$mutextrm{s}$. Fused NT embryos were activated with calcium ionophore (CI) and 6-dimethyl-aminopurine (6-DMAP). Activated oocytes were cultured in NCSU 23 or BECM 3 for 6 days. There was no significant difference between chemical activation and no chemical activation for blastocyst development rate(11.6 vs. 14.8%). However, cell number was significantly higher when NT embryos were activated with CI and 6-DMAP (31.2 vs. 22.6). When NT embryos were cultured in NCSU 23 or BECM 3, blastocyst development rate was 16.4 and 13.2%, respectively, and cell number was 31.5 and 24.1, respectively. These results suggest that chemical activation after fusion and culture in NCSU 23 could increase cell number of porcine NT embryos.

• Journal of Animal Science and Technology
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• v.63 no.5
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• pp.984-997
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• 2021

This study sought to evaluate DNA damage and repair in porcine postovulatory aged oocytes. The DNA damage response, which was assessed by H2A.X expression, increased in porcine aged oocytes over time. However, the aged oocytes exhibited a significant decrease in the expression of RAD51, which reflects the DNA damage repair capacity. Further experiments suggested that the DNA repair ability was suppressed by the downregulation of genes involved in the homologous recombination (HR) and nonhomologous end-joining (NHEJ) pathways. The expression levels of the cell cycle checkpoint genes, CHEK1 and CHEK2, were upregulated in porcine aged oocytes in response to induced DNA damage. Immunofluorescence results revealed that the expression level of H3K79me2 was significantly lower in porcine aged oocytes than in control oocytes. In addition, embryo quality was significantly reduced in aged oocytes, as assessed by measuring the cell proliferation capacity. Our results provide evidence that DNA damage is increased and the DNA repair ability is suppressed in porcine aged oocytes. These findings increase our understanding of the events that occur during postovulatory oocyte aging.

• Korean Journal of Animal Reproduction
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• v.19 no.3
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• pp.217-226
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• 1995

The objective of this experiment was to test the effect of EG F on nuclear maturation of pig immature oocytes in vitro. Basic medium used TCM-199 supplemented with 0.2 mM pyruvate, 1 ${\mu}\textrm{g}$/ml estradiol-I7$\beta$ and 25 ${\mu}\textrm{g}$/ml gentamycin, this medium treated with EGF, FSH and FBS. Experiment 1 examined to the effect according to the addition of FSH or EGF (0, 1. 10 and 100 ng EGF/ml) in oocytes maturation. Nuclear maturation rates (M ll %) of 1, 10 and 100 ng EGF/ml (83.0. 8fi.7 and H7.5%) treatments were significantly higher than those of non- and FSH-treated groups (27.3 and 60.3%, p < 0. 001). Experiment 2 examined to the interactive effects of EGF. FSH or FBS during oocytes maturation. Nuclear maturation rates (M ll %) of EGF alone, EGF plus FSH, EGF plus FBS, FSH plus FBS, and EGF plus FSH added FBS treatments (86.7, 90.2, 87.1. 89.6% and 92.6%) were significantly higher than those of non, FSH, and FBS alone treatments (22.3, 52.2 and 42.3%, p < 0.001). Also, cumulus cells expansion of oocytes maturation was examined to total treatments. Normal cumulus cells expansion was shown by FSH plus FBS, EGF or EGF with FBS combination treatments, but cumulus cells of oocyte complexes were still clumped together in EGF-treated groups although they had separated from oocytes. However, EGF showed a positive on nuclear maturation. These results conclude that EGF alone can stimulate nuclear maturation in pig immature oocytes.

• Clinical and Experimental Reproductive Medicine
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• v.23 no.1
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• pp.33-39
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• 1996

This objective of this experiment was to test the effect of EGF on GVBD and MII of nuclear maturation of pig immature oocytes in vitro. Experiment 1 examined to the effect of EGF on nuclear maturation of pig immature oocytes according to different maturational times. The percentage of GVBD of EGF 10mg/ml treated groups were significantly higher than untreated groups after 24hr (p < 0.001). Experiment 2 examined to the effect of duration of exposure of oocytes to EGF supplement in maturation medium. Nuclear maturation rates (M II) of EGF treated groups (during 0-24: 72.8% and 0-42hr: 84.8%) were significantly higher than 53.5 and 26.1% of EGF treated group (during 24-42hr) and untreated group (p < 0.001). Also, experiment 3 examined to the effect of EGF on nuclear maturation of CEOs or CFOs. Nuclear maturation rate (M II) 84.6% of EGF treated group of CEOs was significantly higher than 53.0, 27.6, and 44.2% of EGF treated group of CFOs and untreated groups of CEOs and CFOs (p < O.001). These results conclude that EGF alone can stimulate GVBD and M II of nuclear maturation in pig immature oocytes.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.13-13
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• 2001

This study was undertaken to evaluate the effects of catalase using xanthine (X) - xanthine oxidase (XO) system on in vitro maturation and fertilization in pig. When follicular oocytes were cultured in maturation medium with X and/or XO, the maturation rates were not significantly different between in medium with and without catalase despite of different culture periods. However, significantly (P<0.05) higher maturation rates were obrained in culture with X-XO system. The rates of degenerated oocytes were increased with culture periods prolonged, and were significantly (P<0.05) higher in medium without than with catalase at 120 h of culture. On the other hand, the parthenogenetic oocytes were observed with high proportions at 72 h of culture, hut were not different in medium with and without catalase at various times of culture. In another experiment, the frozen-thawed boar spermatozoa treated with X-XO system for in vitro fertilization. The penetration rates were higher in medium with that than without catalase during the in vitro fertilization with, none (P<0.05), XO and X＋XO. On the other hand, when sperm were treated with none, X, XO and X＋XO, lipid peroxidation were higher in medium without that than with catalase. However, the changes in sperm penetration and lipid peroxidation showed opposite patterns. The sperm suspensions were also treated with X and/or XO for assay of sulfhydryl (-SH) group content. Under the above all conditions, sperm-SH group were higher detected In medium with that than without catalase. The activity of sperm binding to zona pellucida was also evaluated through binding to salt-stored porcine oocytes. In control group, sperm binding to zona pellucida were higher than in medium with X, XO and X＋XO groups. No significant differences, however, were observed between medium with and without catalase. In conclusion, the exposure of follicular oocytes and spermatozoa to X-XO system may be caused stimulating in vitro maturation and fertilization in pig. This work was supported by grant No. 2000-1-22200-001-3 from the Basic Research Program of the Korea Science & Engineering Foundation.

• Journal of Embryo Transfer
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• v.19 no.3
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• pp.275-282
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• 2004

This study was carried out to examine the effects of development media and those change on in vitro development (IVD) of porcine oocytes matured and fertilized in vitro. Putative embryos, which were matured in vitro in modified NCSU-23 (mNCSU-23) supplemented with 10％ porcine follicular fluid (pFF) and were fertilized in mTBM, were developed in vitro as the experimental scheme. The results are as follows. When porcine putative embryos were cultured in vitro in NCSU-23 or CZB/Pig-MEM, the percentage of oocytes cleaved was not different between two systems, but the percentage of blastocysts in NCSU-23 was significantly higher than in CZB/Pig-MEM (P<0.05). And when porcine putative embryos were cultured in vitro in NCSU-23 during 7 days with or without changing media at day 5, which was supplement with or without 10％ fetal bovine serum(FBS), the percentage of oocytes cleaved, blastocysts at day 6, and the cell number of ICM, TE and total of blastocysts at day 7 were not different among three treatments. As a result of this study, it is supposed that NCSU-23 be more favorable, to develop porcine embryos derived from IVM/IVF, than CZB/Pig-MEM, but that demonstration on the effects of changing medium with fresh one stand in need of the more experiments.

• Asian-Australasian Journal of Animal Sciences
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• v.17 no.2
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• pp.164-167
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• 2004

This study was carried out to investigate in vitro fertilization and development of in vitro matured pig oocytes inseminated with the Duroc boar sperm by different sperm washing media after thawing of the 5 ml frozen straws. Immature follicular oocytes (30-40) were transferred into each well of a Nunc 4-well multidish containing $500{\mu}l$ mTCM199 maturation medium. The sperm rich portion of ejaculates was collected into a 250 ml insulated vacuum bottle and gradually cooled 22 to $24^{\circ}C$ over a 2 h period. Semen was centrifuged at 800 g for 10 min and the seminal plasma discarded. Sperm were esuspended in a lactose-egg yolk and N-acetyl-Dglucosamine (LEN) diluent to contain $1{\times}10^{9}$ sperm/ml and cooled to $5^{\circ}C$ over a 2 h period. Immediately before freezing, semen was rediluted with an equal volume of LEN+4% glycerol and packed into 5 ml straws. After thawing of the 5 ml straw, the 5 ml semen was diluted with 20 ml Beltsville thawing solution (BTS) at room temperature. Oocytes were inseminated with untreated (unwashed and nonpreincubated) or treated sperm (washed two times in BTS, mTLP-PVA and mTBM media, respectively and nonpreincubated) with $2{\times}10^{7}$ sperm concentration. Oocytes were coincubated for 6 h in $500{\mu}l$ mTBM fertilization. At 6 h after IVF, oocytes were transferred into $500{\mu}l$ NCSU-23 culture medium for further culture of 6 h. Sperm penetration, polyspermy and male pronuclear formation of oocytes at 12 h after IVF and developmental ability of oocytes at 48 h after IVF were evaluated. Sperm penetration rate, male pronuclear formation and rate of cleaved embryos were higher in the BTS, mTLP-PVA and mTBM treatments than the unwashed treatment (p<0.05). The rate of blastocysts from the cleaved oocytes (2-4 cell stage) were higher in the mTLP-PVA treatment than in the unwashed, BTS and mTBM treatments. In conclusion, we recommend the washing of frozen-thawed sperm with mTLP-PVA medium before in vitro fertilization of oocytes in mTBM medium.

• Journal of Embryo Transfer
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• v.29 no.4
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• pp.361-368
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• 2014

This study was designed to evaluate the effect of bovine serum albumin (BSA) in a maturation medium on oocyte maturation and embryonic development in pigs. Immature pig oocytes were matured for 44 h in a medium supplemented with 0.4% (w/v) BSA, 0.1% (w/v) polyvinyl alcohol (PVA), or 10% (v/v) pig follicular fluid (PFF). After IVM, oocytes reached metaphase II stage were activated for parthenogenesis (PA) or used as cytoplasts for somatic cell nuclear transfer (SCNT). Nuclear maturation (89.5%, 90.7% and 91.3% for BSA, PVA and PFF, respectively) and intraoocyte glutathione contents (1.20, 1.16 and 1.00 pixels/oocyte for BSA, PVA and PFF, respectively) were not altered by the macromolecules added to maturation medium. IVM of oocytes in a medium containing BSA (21.4%) and PVA (20.7%) showed significantly lower blastocyst formation after PA than culture in medium with PFF (39.2%). After SCNT, oocytes matured in medium with BSA showed decreased embryonic development to the blastocyst stage (9.2%) compared to those matured in medium with PFF (28.9%), while 23.6% of SCNT oocytes matured in medium with PVA developed to the blastocyst stage. When the effect of BSA in a maturation medium during the first 22 h and the second 22 h of IVM in combination with PFF or PVA was examined, PVA-BSA showed a higher nuclear maturation (94.1%) than BSA-PFF (84.5%). However, there was no significant difference in the blastocyst formation among tested combinations (47.3, 52.2, 50.0, 44.4 and 49.0% for PFF-PFF, PFF-BSA, PVA-BSA, BSA-PVA and BSA-PFF, respectively). Our results demonstrate that BSA and PVA added to maturation medium can support oocyte maturation comparable to PFF-supplemented medium. However, maturation of oocytes in a BSA-containing medium decreases embryonic development after PA and SCNT when compared with the medium supplemented with PFF.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.27-34
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• 2004

This study evaluated the efficiency and compared with different materials of loading vessels for vitrification-plastic/glass, copper grid and nylon. The loading method, vitrification, cryop-reservation and warming method of the oocytes were examined. The loading samples prepared in manual or company-made and sterilized, loaded the COCs selected on each samples and cultured for maturation during 40 hours, and then exposed sequentially to ethylene glycol solution. Thawing method was reversely treated and exposed for warmed oocytes. After oocytes were thawed, fertilized and cultured in vitro for 3-4 hours, rates of development and morphological appearance were examined. The results were as summarized: ㆍOPS from company-made or hand-made of the hematocrit micropipettes, NLS from fishing line and EMG from company-made for EM were used for loading oocytes, respectively. ㆍThe efficiency of freezing method and loading convenience were orderly higher in OPS, NLS and EMG. The optimal capacity per vessel was orderly lowered in NLS, EMG and OPS, respectively. ㆍAfter oocytes were warmed, the recovery rate, morphology and rate of development were orderly higher in OPS, NLS and EMG, respectively. ㆍIn conclusion, OPS has the advantages of achieving a little more survival and preserving results than other two loading methods.

• Korean Journal of Veterinary Research
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• v.32 no.1
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• pp.135-142
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• 1992

This experiment was carried out to establish an effective technique of in vitro maturation of porcine follicular oocytes. Porcine ovaries were collected from an abbatoir and delivered to the laboratory in phosphate buffered saline in an hour. Immatured follicular oocytes were collected from the ovaries and divided into groups by the size of follicles and by the attachment of granulosa cells. The follicular oocytes were cultured in m-KRB solution supplemented with FCS(10%), follicular fluid(10%) or hormones of PMSG(10IU/ml), hCG(10IU/ml ) and $estradiol-17{\beta}(1{\mu}g/ml)$ for 48 hours at $39^{\circ}C$ under an atmosphere of 5% $CO_2$ in air. The results are as follows ; 1. The mean recoveration rate of follicular oocytes was 61.8%. 2. The maturation rate was significantly(p<0.05) higher when the oocytes were collected from large-sized follicles and under good state of granulosa cell attachment. 3. The maturation rate was significantly(p<0.01) promoted when the follicular oocytes were cultured in m-KRB solution supplemented with follicular fluid(74.8%) or hormones and fetal calf serum(70.6%).