• 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.

• Reproductive and Developmental Biology
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• v.30 no.1
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• pp.65-70
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• 2006

This study investigated apoptosis and in vitro development of parthenogenetic preimplantation porcine embryos. In vitro matured oocytes for $42{\sim}44h$ were used. Apoptotic cell death was analyzed by using a terminal deoxynucleatidyl transferase mediated deoxyuridine 5-triphosphate nick-end tabling (TUNEL) assay. In experiment 1, oocytes were activated with two electric pulses (CH) of 1.2 kV/cm for $30{\mu}sec$ (E), E + 6-dimethylaminopurine (6-DMAP) or E + cycloheximide (CH) and cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$. In experiment 2, oocytes were activated by E and cultured in PZM-3 or NCSU-23 under a gas atmosphere of 20% $O_2$ ($5%\;CO_2$, in air) or 5% $O_2$ $(5%\;CO_2,\;5%\;O_2\;90%\;N_2)\;at\;38.5^{\circ}C$. Oocytes activated with E+6-DMAP or E+CH showed higher blastocyst rates (36.3% and 32.5%) compared to E alone (27.7%). The frequency of apoptosis according to treatments were 5.3%, 7.7% and 7.1% respectively. Oocytes activated with E alone showed lower (P<0.05) frequency of apoptosis compared to other groups. In experiment 2, parthenotes cultured in PZM-3 showed slightly higher blastocyte rates (28.2% and 29.7%) compared to NCSU-23 (22.6% and 24.4%) regardless of atmosphere. Blastocysts generated in PZM-3 showed lower (P<0.05) apoptosis rate under 20% $O_2$ (9.2% vs 16.9%), whereas those in NCSU-23 had slightly lower apoptosis rate under 5% $O_2$ (14.0% vs 18.4%). This result represents that activation method and culture condition could affect the frequency of apoptosis as well as in vitro developmental rate.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.124-124
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• 2003

The successful development of embryos cloned by nuclear transfer (NT)have been dependent on a wide range of known factors including cell cycle of donor and recipient ooplast, oocyte quality, NT procedure and oocyte activation. The present study compared the development of cloned porcine embryos following different activation treatments. Cumulus-oocyte complexes (COCs) were aspirated from 26 mm follicles of slaughterhouse ovaries and cultured for 22 h in NCSU #23 medium supplemented with 10% porcine follicular fluid, 0.57 mM cysteine, 0.5 g/mL LH, 0.5 g/mL FSH and 10 ng/mL EGF. The COCs were further cultured for an additional 22 h in the same medium at $39{\cird}C$ in an atmosphere of 5% $CO_2$ in air, without hormonal supplements. Primary cultures of fibroblasts isolated from a female fetus on day 40 of gestation were established in DMEM + 15% FCS. For nuclear donation, cells at the 5th-6th passage were cultured in DMEM +0.5% FCS for 5 days in order to arrest the cells in G0/Gl. After enucleation, oocytes were reconstructed by transfer of donor cells and fusion with three DC pulses (1.4 KV/cm, 30 sec) in 0.28 M mannitol containing 0.01 mM $CaCl_2$ and 0.01 mM $MgCl_2$. Eggs were then divided into three treatment groups, control (without further treatment, Group 1), eggs cultured in 10 g/ml cycloheximide (CHX) for 5 h (Group 2), and eggs cultured in 1.9 mM 6-dimethylaminopurine (6-DMAP) for 5 h (Group 3). The eggs were then cultured in sets of 30 in 60 I drops of NCSU#23 supplemented with 4mg/ml BSA (essentially fatty acid free) until day 7 at $39{\circ}C$ in a humidified atmosphere of 5% $CO_2$. On day 4 the culture were fed by adding 20 I NCSU #23 supplemented with 10% FBS. Development rates into blastocysts were significantly higher (P<0.05) in Group 3 embryos compared to Group 1 controls ($27.6 \mu 2.7% vs. 20.1 \mu 4.1%$, respectively), but rates did not differ in Group 2 compared to control ($23.8 \mu 5.7%$). Total cell number in Group 3 blastocysts was however significantly higher (P<0.05) than in Groups 1 and 2 ($44.6 \mu 2.4 vs. 19.9 \mu 1.9 and 21.9 \mu 2.1$, respectively). These results suggest that 6-DMAP is more efficient than cycloheximide in the activation of electrically fused NT oocytes during in vitro production of cloned porcine embryos.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.26-26
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• 2003

Hyaluronic acid (HA)-binding sites have been shown the diagnostic potential fur assessment of sperm maturity, which is related to male fertility. This study was designed to evaluate chromosomal patterns in porcine embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) with non- or HA-binding sperm (HABS). For binding of sperm with HA, sperm incubated in 10 ${mu}ell$ drop containing HA (0.8 mg/ml)-agarose (0.8%) mixture for 15 min. IVF and ICSI with non- or HA-bound sperm examined with matured oocytes at 44 hr after in vitro maturation. Embryos were cultured in 50 ${mu}ell$ of NCSU 23 containing 0.5% BSA for 5 days and then in 50 ${mu}ell$ of NCSU 23 containing 10% FBS for 2 days. For the evaluation of chromosomal aneuploidies, chromosome 1 sub-metacentric specific probe was used in sperm and embryos by fluorescence in situ hybridization (FISH). The frequency of aneuploidy sperm for chromosome 1 was 6.25%. The significant differences following IVF and ICSI with non- or HA-bound sperm were not observed in blastocyst formation rates (18.6, 23.5, and 23.8%) and cell number (61.8 $\pm$ 12.5, 55.5 $\pm$ 7.3, and 59.3 $\pm$ 9.6). Moreover, the percentage of diploidy in 4-cell stage embryos was 57.1% (IVF), 68.8% (ICSI), and 76.3% (ICSI-HABS). These results suggest that HA-binding sites may be a material for selection of normal sperm for ICSI. Therefore HA selection of normal sperm may be reduce the loss to embryonic mortality prior to embryo transfer in pig.

• Journal of Embryo Transfer
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• v.25 no.4
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• pp.251-258
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• 2010

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 $39^{\circ}C$ in a 5% $CO_2$ atmosphere in North Carolina State University 23 (NCSU-23) supplemented with 25, 50, 75 or $100\;{\mu}M$ 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 $50\;{\mu}M$ 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 $50\;{\mu}M$ 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 $50\;{\mu}M$ and 17 h, respectively.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.132-132
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• 2003

멸종위험성이 높은 재래돼지를 유전자원으로서 안전하게 보존하고 유전적 다양성을 유지하기 위해 체내수정란의 동결보존 방법을 수행하였다. 재래돼지의 과배란유기는 altrenogest를 1일 20mg씩 18일 경구투여하고 PMSG 500~l,000IU 근육주사후 80시간에 hCG 500~750IU를 근육주사하였다. 발정이 관찰된 개체는 발정개시후 12시간과 24시간에 자연교배 또는 액상정액을 이용하여 2회씩 수정시켰다. 최종 수정후 5일째에 외과적으로 개복수술하여 FBS가 5% 첨가된 D-PBS 관류액으로 자궁으로부터 수정란을 회수하여 상실기, 배반포기, 확장배반포기의 수정란으로 구분하였다. 회수된 수정란은 FBS가 20% 첨가된 D-PBS의 0.4, 0.8, 1.4M glycerol 항동해제에 각 단계별로 10분씩 평형시킨후 수정란동결기(CL863, Australia)를 이용하여 18$^{\circ}C$부터 -7$^{\circ}C$까지 2$^{\circ}C$/min의 냉각속도와 -7$^{\circ}C$부터 -35$^{\circ}C$까지 0.5$^{\circ}C$/min의 동결속도(실험1), 1$^{\circ}C$/min의 냉각속도와 0.3$^{\circ}C$/min의 동결속도(실험 2)로 동결시켰다. 또한 FBS가 20% 첨가된 D-PBS의 ethylene glyco1(EG) 1.8M의 항동제에 15분간 평형시킨후 실험 1과 동일한 방법으로 동결시켰다(실험 3). 동결수정란의 융해는 37$^{\circ}C$의 항온수조에서 30초간 실시하였다. 항동해제로 glycerol을 이용한 수정란은 융해후 3가지 농도로 0.3M sucrose, 0.8M glycerol, 0.4M glycerol을 첨가한 D-PBS에 각각 10분씩 단계적으로 정치시킨 다음, 10% FBS 첨가 mNCSU-23으로 3회 세척했다. 항동해제로 EG를 이용한 수정란은 융해후 즉시 D-PBS에 각각 10분간 정치시킨 다음, 10% FBS 첨가 mNCSU-23으로 3회 세척했다. 항동해제가 제거된 수정란은 FBS가 10% 첨가된 mNCSU-23 배양액에서 39$^{\circ}C$, 5% $CO_2$ 배양기에 48시간 배양하면서 생존여부를 판단하였다. 실험 2에서 확장배반포배 수정란이 25.3%의 생존율을 나타내었으며, 실험 1과 실험 3에서는 수정란의 형태와 관계없이 생존성을 확인할 수 없었다. 이상의 결과로 보아 glycerol 완만동결에서는 확장배반포기 수정란 이상이 보존가능한 것으로 추정되나 더 추가적인 연구가 요구된다.

• Korean Journal of Animal Reproduction
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• v.20 no.1
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• pp.19-26
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• 1996

In the present study, we investigated devel-opmental ability and transgene expression of IVM/IVF derived porcine embryos following microinjection with SV40-LacZ. A total of 412 IVM/IVF derived embryos were used to examine developmental ability and transgene expression following DNA microinjection. After centrifugation, pronuclei were visible in 60.3% when examined between 18~21h after IVF. Development and transgene expression were assessed after 9 days in culture. The percentages of injected embryos reaching to the morula and blastocyst were significantly lower (P<0.05) than those of non-injected control embryos. However, the percentages of DNA microinjected embryos and non-injected embryos that developed to the blastocyst or hatched blastocyst stage in dual culture systems (NCSU23 and EMEM) were significantly higher (P<0.05) than those in NCSU23 medium alone. As the resuIt of X-gal staining, the proportion of positive embryos was 40~43% in morula and blastocyst stage embryos, however, mosaicism has been observed in the most putative transgenic morulae and blastocysts. In the PCR analysis, the percentages of embryos integrated gGH gene were 45.0 and 44.4% in morula and blastocyst stage, respectively. These results suggest that improved IVM /IVF system and culture condition increased the embryo viability and ex-pression of a microinjected transgene.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.81-81
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• 2002

$\beta$-Mercaptoethanol($\beta$-ME)은 일반적으로 황화합물(thiol compounds)의 일종으로, 배양액 중에서 이황화결합(disulfide bonds)을 분해하여 일정한 물질의 산화.환원반응에 관여하며, 특히 cysteine이 cystine으로 산화되는 것을 차단함으로서 cysteine의 이용능력을 증대시키고, GSH의 합성을 촉진 및 증대시키는 것으로 알려져 있고, 각종 활성산소로부터 세포를 보호하는 역할을 수행하는 것으로 보고되었다. 특히, 돼지수정란의 체외배양체계에 유의적인 영향을 미치는 것으로 보고되었다(Abebydeera 등, Theriogenol., 50:747-756, 1998). 따라서 본 실험에서는 돼지난포란의 체외성숙시 $\beta$-ME의 첨가배양이 체외수정과 배발달에 미치는 영향에 관하여 조사하였다. 돼지난포란을 10% PFF, 0.1mg/ml cysteine, 10IU/m1 PMSG, 10IU/m1 hCG 및 10ng/m1 EGF가 첨가된 NCSU23 배양액에 $\beta$-ME를 각각 0, 25, 50 및 100uM을 처리하여 22시간 동안 배양을 실시하고, 성선자극호르몬이 배제된 배양액에서 추가로 22시간을 배양하여 체외성숙을 유도하였다. 체외성숙이 유기된 난자는 난구세포를 제거하고, 2.5mM caffeine과 0.1% BSA가 첨가된 mTBM배양액에 정자를 1.25 $\times$ $10^{5}$cells/ml의 농도로 5-6시간 동안 공동배양을 실시하여 체외수정을 유도하였다. 체외수정후 일부의 수정란은 12시간에 난자 급속 염색방법으로 염색하여 다정자침입률 및 자.웅전핵형성률 등을 확인하였다. 그리고 나머지1-세포기의 수정란은 0.4mg/ml BSA가 함유된 NCSU23 배양액에 30 embryos/50ul 소적으로하여 38.8$^{\circ}C$, 5% $CO_2$의 탄산가스 배양기에서 각각 7일간 배양을 실시하였다. 조사된 결과는 SAS/STAT를 이용하여 통계분석을 실시하였다. 체외수정 12시간 후에 난자 급속 염색법으로 염색을 실시한 결과, 모든 처리구에서 핵성숙률(76.4~95.2%), 정자침투율(51.1~66.9%), 웅성전핵형성률(95.2~100%), 다정자침입률(18.2~25.6%) 및 평균침입정자수(1.2~l.4개)에서 유의적인 차이가 인정되지 않았다. 체외배양 48시간 난할률을 조사한 결과, 처리구별 차이(53.9~67.9%)는 인정되지 않았으나, 배양 7일째 배반포형성률은 각각 14.5, 25.4, 17.3 및 12.4%로서 25uM의 $\beta$-ME처리구가 유의적(P<0.05)으로 높은 배발달률을 나타내었고, 총세포수에 있어서는 대조구와 처리구간 유의적인 차이가 인정되지 않았다. 따라서 돼지 난포란을 성숙배양할 때, 25uM $\beta$-ME를 첨가배양하는 것이 양질의 돼지체외수정란을 생산하는 하나의 방법으로 조사되었다.다.

• Proceedings of the Korean Society of Embryo Transfer Conference
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• 2002.11a
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• pp.80-80
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• 2002

This study was carried out that to investigate the effects of amino acids supplemented with culture medium on development of porcine embryos cultured in vitro. Cumulus oocyte complexes (COCs) were cultured in the maturation medium containing hormones (0.5$\mu\textrm{g}$/$m\ell$ LH, 0.5$\mu\textrm{g}$/$m\ell$ FSH and 1$\mu\textrm{g}$/$m\ell$ estradiol-17${\beta}$) for 20-22 h at 39$^{\circ}C$ in an atmosphere of 5% CO$_2$in air. Subsequently, COCs were cultured in hormone-free maturation medium for 20-22 h. After maturation for 40-44h, oocytes were removed cumulus cells by pipetting and cultured with epididymal sperm for 5 h in the mTBM. Embryos obtained were divided in 4 groups (1) cultured in NCSU 23 containing 0.4% BSA to blastocyst stage(Control), (2) essential amino acids (EA), (3) non-essential amino acids (NA), (4) mixture of essential and non essential amino acid (EA+NA). All treated groups(2-4) were used a glucose free NCSU 23 medium supplemented with pyruvate (0.33 mM), lactate (4.5 mM) to morula stage. From morula to blastocyst stage embryos of all treated groups were cultured in NCSU 23 containing 0.4% BSA. The rates of cleaved oocytes at 48 h after IVF were from 82% to 88% in the groups of control, EA, NA and EA+NA, respectively. The in vitro developmental rates into blastocysts in the groups of EA and EA+NA were significantly (P<0.05) higher than those of group of control (35.1, 35.4 vs. 19.4%, respectively), however, no significant (P<0.05) between control and NA. In conclusion, supplemented with essential amino acid or mixture of essential and non essential amino acid in the culture medium at morula stage increased the rate of development to blastocyst on in vitro produced porcine embryos.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.62-62
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• 2003

This study was carried out to investigate the effects of activation agents on parthenogenetic activation of pig oocytes matured in vitro. The medium used for oocyte maturation was tissue culture medium (TCM) 199 supplemented with 26.19 mM sodium bicarbonate, 0.9 mM sodium pyruvate, 10 $\mu\textrm{g}$/ml insulin, 2 $\mu\textrm{g}$/ml vitamin $B_{l2}$, 25 mM Hepes, 10 $\mu\textrm{g}$/ml bovine apotransferrin, 150 $\mu$M cysteamine, 10 IU/ml PMSG, 10 IU/ml hCG, 10 ng/ml EGF, 0.4% BSA, 75 $\mu\textrm{g}$/ml sodium penicillin G, 50 $\mu\textrm{g}$/ml streptomycin sulfate and 10% pFF. After about 22 h of culture, oocytes were cultured without cysteamine and hormones for 22 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. Cumulus-free oocytes involving first polar body were activated by exposure to various concentrations of ethanol and exposure time of ethanol in Hepes-buffered NCSU23 medium. Also, oocytes were activated by electric pulse alone or combination with ethanol. For electrical activation, oocytes were rinsed twice in 0.3 M mannitol solution supplemented with 0.1 mM CaC1$_2$, 0.2 mM MgC1$_2$, 0.5 mM Hopes and 0.01% BSA, and transferred to a chamber consisting of two electrodes 1 mm apart which was overlaid with the same activation solution. Oocytes were activated with a single DC pulse of 1.3 ㎸/cm for 30 $\mu$sec. After activation treatments, oocytes were washed three times with Hepes-buffered NCSU23 medium and were washed twice with NCSU23 culture medium containing 0.4% BSA, and then cultured in 500 ${mu}ell$ of the same medium for 20 h at 38.5$^{\circ}C$, 5% $CO_2$ in air. The activation rates of oocytes were higher in 6, 7 and 8% ethanol concentrations compared with 0, 5, 9 and 10% ethanol concentrations. Significantly more oocytes (29.3~33.7%) were activated in the exposure for 8, 10, 12 and 15 min than those in the exposure for 0 and 5 min, but there was no difference due to exposure to 8% ethanol for 8 to 15 min. Electric pulse treatment followed by exposure to ethanol significantly improved the rate of oocyte activation (61.9%) compared with that of other 3 treatments. In conclusion, the optimal activation treatment of ethanol exposure alone for the in-vitro matured pig oocytes was 8% ethanol for 8 to 15 min. Electric pulse treatment followed by ethanol exposure significantly improved the rate of activation.n.