• Journal of Embryo Transfer
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• v.19 no.3
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• pp.245-255
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• 2004

In recent years, an increasing number of studies on pig in vitro maturation(IVM) and in vitro fertilization(IVF) have been separated. the wide range of new technologies, including that in applied molecular genetics, has increased this interest. the production of viable porcine embryos in vitro is a prerequisites for the successful production of transgenic pigs to date. The efficiency of IVM/IVF techniques in the porcine is lower than that obtained in other species such as cattle and mouse. The several problems are generally thought to be the cause of poor results: the low rate of MPN formation derived from inadequate IVM of oocytes, the high incidence of polyspermy after IVF and cell blocking at 4 cell during embryos culture. For there reasons overcoming, many studies have been conducted to improve in vitro embryo-genic competence of oocytes. In the last several years, many maturation culture media have been evaluated and various exogenous factors such as hormones and grows factors have been tested to improve the efficiency of porcine in vitro system. In the study several antioxidants have been examined to improve in vitro fertilization and development of porcine oocytes. In this study, several antioxidants were examined to determine the effects on the development of oocytes to the cleavage, morula and blastocyst stage when added at the maturation(IVM) or in vitro fertilization(IVF) or in vitro culture(IVC) of porcine embryos. Porcine oocytes were matured, fertilized and embryos were cultured in defind conditioned medium in vitro with or without supplementation with the antioxidents of cysteine, catalase and glutathione. 1. Significant improvement of blastocyst rate (27.2％ versus 15.4％, p<0.05) were achieved when catalase(500U/$m\ell$) were added to TCM-199 medium and morula rate(72.0％ versus 53.9％, p<0.05) were significantly higher when glutathione(1.0mM/$m\ell$) were added to TCM-199 medium than those of control. 2. In mTBM medium for oocytes fertilization, the addition of cysteine, catalase and glutathione had no positive effect on embryonic development. glutathione had no positive effect on embryonic development. In conclusion, this study shows that addition of catalase, gluththione during IVM improved the rate of porcine embryo development.

• Journal of Animal Science and Technology
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• v.51 no.5
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• pp.413-420
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• 2009

The research has been aimed to investigate the effect of different sera including fetal bovine serum (FBS), male bovine serum (MS), female bovine serum (FS), and castrated-male bovine serum (C-MS) on cell proliferation, follicular maturation and ovulation in vitro. Established cell lines and primary cells were cultured in the culture media supplemented with different sera and cells proliferation was observed by cell counting and MTT assay. The results indicated that cell proliferation was significantly different for different serum source. Proliferation of bovine and human myogenic satellite cells was highest in MS. In contrast, proliferation of breast cancer cells and immune cells were the highest in FS and FBS, respectively. There was no difference in the rate of follicular growth, whereas the rate of ovulation was higher in FBS and C-MS. Finally, the wound healing effect and cell proliferation of 3T3-L1 cells showed that wound healing was fastest in FS and cell proliferation was higher in MS. These results suggest the importance of an optimal serum selection in the experiments involving cell culture system, and gender-specific Hanwoo sera may be used as a substitute to FBS.

• Journal of Embryo Transfer
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• v.12 no.2
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• pp.219-226
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• 1997

Follicular fluid influxed into the oviduct during ovulation may affect movement of sperm for fertilization Thus, in this study, the effect of follicular fluid, obtained from follicles of l0mm in diameter, on number and quality of sperm recovered by swim-up separation was investigated and sperm-movement stimulating components extracted from follicular fluid with methanol and isooctane were separated by gel filtration with Sepadex G-1O, G-25 and G-1OO gels, and were isolated by electrophoresis with SDS-PAGE mini gel. The results obtained were as follows; 1. Diluted follicular fluid stimulated sperm movement. 2. Sperm-movement stimulating factors were in methanol extract. 3. Sperm-movement stimulating effect of methanol extract appeared in fraction I among fractions recovered after gel filtration. And the fraction I contained proteins indicating 4 major bands as about 47, 43, 25 and 14 kilodaldons and 5 minor bands as about 67, 58, 23, 22 and 21 kilodaldons. 4. The fraction I recovered from G-100 gel showed significantly low percentage of motile sperm and had no protein indicating the band of 67 kilodaldons among the minor bands.

• Korean Journal of Animal Reproduction
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• v.25 no.2
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• pp.181-189
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• 2001

The objective of this study was to evaluate the development of porcine follicular oocytes fertilized by intracytoplasmic sperm injection (ICSI). Cumulus-oocyte-complexes (COCs) were collected by aspiration from follicles of 2~7 mm in diameter from a local slaughterhouse. Oocytes were matured in vitro for 40~44 h, and spermatozoa were prepared by swim-up in the presence or absence of 5 mM dithiothreitol (DTT) and then M II stages of the oocyte were either centrifuged or not centrifuged for the following injection of ooplasm. Injected oocytes were cultured in NCSU 23 medium for 6 to 8 days. The results obtained were as follows: 1. The rates of cleavage and development rates into blastocyst by ICSI were not significantly different between the with (53.0% and 19.7%) or without (48.3% and 23.8%) centrifugation, respectively (P＜0.05). 2. The cleavage and developmental rates to blastocyst after ICSI with or without 5 mM DTT treated-sperm were not significantly different (60.4% vs 16.4% and 45.5% vs 22.2%), respectively (P＜0.05). 3. The cleavage and the developmental rates to btastocyst were not significantly different between the zygotes obtained by IVF (51.8% vs. 22.4%) and ICSI (51.4% vs. 21.6%) (P＜0.05). 4. The number of blastomere in blastocyst stages after IVF or ICSI was not significantly different (46.7$\pm$2.9 and 41.9$\pm$4.6).

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.60-60
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• 2003

소의 난포란과 난구세포의 체외배양시 plasminogen activators(PAs)의 생산을 보고하였다 (Choi 등, 1998). 따라서 본 연구는 돼지 난포란 및 난구세포의 체외성숙시 PAs의 생산을 SDS-PAGE와 Zymogram을 이용하여 조사하였다. PCOCs는 도축암퇘지의 난소로부터 채취하여, 난구세포가 충실한 것만 선별하였으며, 실험구에 사용될 난구세포는 pipetting에 의해 분리하여 이용하였다. 돼지 신선정액은 D-PBS로 1,500 rpm, 5분간 2회 원심분리하여 정장물질을 제거하고, 3회째는 5mM caffein이 함유된 BO(Brackett과 Oliphant, 1985) 배양액으로 세정하였다. 처리한 돼지 정액은 1$\times$$10^{8}$ cells/$m\ell$로 조정하여 20${\mu}\ell$씩 분주하고 0, 1, 2, 3 또는 4시간 동안 39$^{\circ}C$ 5% $CO_2$, 95% 공기인 배양기에서 수정능획득을 유도하였다. 배양이 완료된 정액은 20${\mu}\ell$의 sample buffer(5% SDS, 20% glycerol, 0.0025% bromophenol blue 그리고 0.125M Tris HC1 buffer)에 넣어 -7$0^{\circ}C$ 동결기에 보관하였다. 전기영동은 4% stacking gel과 10% separating gel로 분리하였으며, 20 mA에서 90분간 실시하였다. Zymogram은 Choi 등(1988)의 방법에 따라 실시하여 PAs의 생산을 확인하였으며, 이상의 실험은 3반복을 실시하였다. 시험구 전체에서 urokinase type plasminogen activator(uPA)가 확인되었으며, 체외수정능 획득시간에는 차이가 없었다. 두 종류의 고분자량의 uPA의존성 영역이 나타났으며, 분자량은 65kD과 62 kD이었다. 이러한 결과로 볼 때 Hart 등(1986)이 uPA의 경우 다양한 영역의 분자량 변이를 확인할 수 있었다고 한 것과 동일하였으며, 돼지 정자가 체외수정능 획득시 uPA를 생산하는 것을 확인할 수 있었다.

• Proceedings of the KSAR Conference
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• 2001.03a
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• pp.27-27
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• 2001

본 연구는 돼지 난포란의 체외 성숙시 hyaluronic acid (HA) 및 Tocopherol첨가가 돼지 수정란의 배발달에 미치는 효과를 검토하기 위하여 실시하였다. 돼지 난포란은 0.1% PVA, 3.05mM D-Glucose, 0.91mM sodium pyruvate, 0.57mM cysteine, 0.5$\mu\textrm{g}$/$m\ell$ LH, 0.5$\mu\textrm{g}$/$m\ell$ FSH and 10ng/$m\ell$ EGF가 첨가된 TCM-199에서 HA와 Tocopherol을 농도별로 첨가하여 42~44시간 배양함으로써 체외성숙을 유도하였다. HA의 효과를 보기 위해 체외성숙시 0.1% HA가 첨가된 처리구에서 배발달율은 25.6%로 대조구 16.7% 보다 높았다.(Table Omitted). 또한 수정시 HA를 0, 0.02, 0.2, 2.0 로 처리한 결과 8.7%, 19.4%, 11.1%, 0%로 대조구(8.7%)에 비해 0.02% 처리구(19.4%)에서 배발달율이 가장 높았다. 수정후 control, TCM-199, TCM-199+0.1% HA가 첨가된 배양액에 washing해주었을 때 발달율은 TCM-199+0.1% HA 첨가구에서 배발달율(35.6%)이 유의적으로 높게 나타났다. Tocopherol을 0, 50, 100, 200mM로 처리한 결과 수정된 난자중 배발달율은 32.1%, 40.0%, 35.7%, 39.6%로 처리구에서 높았으며, 성숙배양 후반기 22h. 동안 Tocopherol 첨가한 후 수정전 HA로 washing한 결과 Tocopherol 무첨가구에서는 차이를 보이지 않았으나, Tocopherol 첨가구에서는 HA처리구에서(19.0%)에서 무처리구보다(14.3%) 높은 결과를 나타내었다. 따라서 배양액내에 HA, Tocopherol를 첨가함으로써 미성숙 난포란의 수정율 및 발달율을 높일수 있을 것으로 생각된다.

• Korean Journal of Animal Reproduction
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• v.26 no.1
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• pp.41-51
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• 2002

The present study was carried out to examine the effect of cysteamine in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF Embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU23 maturation medium with 0, 25, 50 and 100 $\mu$M cysteamine (P〉0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 25, 50 and 100 $\mu$M cysteamine were 17.9$\pm$6.1, 17.4$\pm$6.3, 24.2$\pm$1.9 and 16.9$\pm$2.0%, respectively. And the total cells were 30.7$\pm$2.4, 34.9$\pm$2.8, 39.6$\pm$2.3 and 36.8$\pm$3.6, respectively. Fifty $\mu$M cystealnine group was significantly higher than those of any other treatment groups (P＜0.05). 3. The ratios of ICM/total cells in 20~40% category were 20.5, 41.6, 19.5 and 31.5%, respectively. Twenty five $\mu$M cysteamine group was higher than those of other groups. 4. The rates of blastocyst formation at day 7 in the NCSU-23 culture medium of porcine IVF-produced embryos with 0, 25, 50, and 100 $\mu$M cysteamine were 16.0$\pm$0.2, 13.6$\pm$1.7, 25.0$\pm$0.8 and 15.7$\pm$4.5%, respectively. And the total cells were 27.0$\pm$3.7, 36.1$\pm$4.8, 34.0$\pm$3.8 and 25.2$\pm$4.4, respectively. Fifty $\mu$M cysteamine group was significantly higher than those of any other treatment groups (P＜0.05). 5. The ratios of ICM/total cells in 20~40% category were 53.8, 30.0, 16.6 and 11.1%, respectively. The addition groups of cysteamine were lower than those of control group. In conclusion, these results suggested that the addition of 50 $\mu$M cysteamine in the IVM medium and 25~50 $\mu$M cysteamine in IVC medium were effective on the blastocyst formation and total cells of blastocysts.

• Journal of Life Science
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• v.19 no.12
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• pp.1764-1768
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• 2009

This study was carried out to examine a concentration of steroid hormones and in vitro development of mouse embryos in culture supernatant of bovine granulosa cells (GC). To obtain the culture supernatant, granulosa cells were retrieved from mature follicles (6~15 mm diameter) and immature follicles (2~5 mm diameter) of bovine ovary and were cultured, respectively, in media of Ham's F-10 with 15% FCS for 16 days. Mature and immature granulosa cells formed their monolayers easily and showed similar growth patterns in culturing. There was no morphological difference between mature and immature granulosa cells. High levels of both progesterone and estradiol were detected in the culture supernatant of mature granulosa cells and immature granulsa cells, and the endocrine profiles of the two types of cells were similar. Progesterone secretion of granulosa cells was high in the late stage of culturing and estradiol secretion was high in the early stage of culturing. In vitro development rates of mouse embryos to morula, blastocyst and hatched blastocyst were significantly (p<0.05) higher in culture supernatant of mature granulosa cells (92.7%, 78.1% and 34.5%) and in culture supernatant of immature granulosa cells (96.4%, 78.5% and 26.8%) than in Ham's F-10 (86.7%, 41,7% and 13.3%). However, there was no difference between the culture supernatant of mature granulosa cells and the culture supernatant of immature granulosa cells in the development of embryos.

• Reproductive and Developmental Biology
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• v.28 no.2
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• pp.113-117
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• 2004

The present study was carried out to examine the effects of $O_2$ concentrations and culture media (North Carolina State University (NCSU)-23, porcine zygote medium(PZM)-3 or PZM-4) on in vitro development of porcine IVM/IVF embryos. Porcine oocyte-cumulus complexes were cultured in BSA-free NCSU-23 medium containing porcine follicular fluid (10%), cysteine (0.9 mM), $\beta$-mercaptoethanol (25 $\mu\textrm{g}$/$m\ell$), epidermal growth factor (10 ng/$m\ell$) and hormonal supplements (PMSG and hCG: 10 IU/$m\ell$) for 20∼22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20∼22 h. After culture, cumulus-free oocyte were coincubated with liquid boar spermatozoa for 5∼6h. Putative zygotes were transferred to NCSU-23, PZM-3 and PZM-4 medium under the condition of 5% $O_2$ or 20% $O_2$ concentrations. At 48 h, no mean differences were found in cleavage rates. However, the rates of blastocyst formation at day 7 after in vitro fertilization were significantly higher in PZM-3 medium under the condition of 5% $O_2$ concentration than other treatments (19.9$\pm$2.4 vs. 11.1$\pm$2.0 to 16.0$\pm$2.5%, P<0.05). The total cell numbers of blastocysts were significantly higher in 5% $O_2$ than in 20% O2 (P<0.05). However, no differences was found among the culture media within each $O_2$ concentrations. In conclusion, the use of PZM-3 medium in 5% $O_2$ concentration was effective on in vitro development of porcine IVM/IVF embryos.

• Korean Journal of Animal Reproduction
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• v.20 no.2
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• pp.111-117
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• 1996

This experiment was designed to evaluate the effect of transforming growth factor-$\beta$ (TGF-$\beta$) and insulin-like growth factor-I (IGF-I) in bovine oocyte maturation in the presence or absence of serum on subsequent fertilization and embryo development. In addition, various concent rations of these growth factors were evaluated for the ability to promote development of eight-cell stage embryos to the blastocyst stage. Cumulus-oocyte complexes were recovered from 2 to 6 mm follicles obtained from slaughterhouse ovaries and cultured at 38.5$^{\circ}C$ for 24 hours in TCM-199 (HEPES Modification) with or with out 20 % fetal bovine serum (FBS) to which the following growth factors were added TGF$\beta$ IGF-l or TGF $\beta$ + IGF-I, all at 10 ng/ml each. The matured oocytes were fertilized in IVF-TL medium with frozen-thawed semen at a concentration of 1 ${\times}$ 10$^6$ cells/ml of fertilization medium following Percoll separation. After 24 hours of sperm-egg incubation, the embryos were transferred to CZB medium without glucose for 48 hours and then cultured in TCM-199 with 20% fetal bovine serum (FBS) for 96 hours. The addition of growth factors to IVM medium in the presence of serum had no effect on cleavage and subsequent embryo devlopment to blastocyst. In the absence of serum, TGF- improved cleavage and development to blastocyst compared to control's(p<0.05) and no synergistic effeet of IGF-I + TGF-$\beta$ was observed. In the second experiment, eight-cell embryos obtained by in vitro maturation (IVM) in TCM-199 + 20% FBS without growth facrors and in vitro fertil-ization (IVF) were cultured in the in vitro cuiture (IVC) medium supplemented with 5, 10 ng/ml TGF-$\beta$ or 5, 10, 50, 100 ng/ml IGF-I. Cleavage rate and development to the blastocyst stage was observed during seven days of incubation. The supplementation of 10 ng/ml TGF-$\beta$ to lVC medium for eight-cell embryos improved development to blastocyst (p<0.05) compared to control. In conclusion, these data indicate that the supplementation of growth factors to IVM medium in the presence of serum does not influence cleavage and subsequent embryo development. However, significantly more oocytes matured in serum-free TCM-199 and eight-cell embryos cultured in lVC medium developed to blastocyst with supplementation of 10ng/ml TGF-$\beta$.