• 한국수정란이식학회지
• /
• 제29권2호
• /
• pp.141-148
• /
• 2014

Quercetin and genistein, plentifully present in fruits and vegetables, are flavonoid family members that have antioxidative function and plant-derived phytoestrogen activity. The antioxidative effects of quercetin and genistein on boar sperm characteristics and in vitro development of IVF embryo were investigated. The sperm motility was increased by addition of genistein $50{\mu}M$ for 6 hr incubation compared to control (p<0.05). The sperm viability was increased by addition of quercetin 1 and $50{\mu}M$ and genestein 1 and $50{\mu}M$ for 3 hr incubation. In addition, the sperm viability seemed to be increased dose-dependantly by addition of quercetin or genistein 1 and $50{\mu}M$, respectively (p<0.05). The membrane integrities were not increased by quercetin or genistein treatments for 3 hr or 6 hr incubation period except for quercetin $1{\mu}M$ for 3 hr incubation. In mitochondrial activities, addition of quercetin $50{\mu}M$ for 6 hr incubation increased mitochondrial activity but decreased at $100{\mu}M$ concentration compared with control (p<0.05). When porcine IVF embryos were cultured in PZM-3 medium supplemented with low concentrations of quercetin ($1{\sim}10{\mu}M$), the developmental rates to morula and blastocyst increased but significantly decreased at high concentrations of quercetin ($25{\sim}50{\mu}M$). The highest developmental rate to blastocysts among all concentrations of quercetin was shown at quercetin $10{\mu}M$ (p<0.05). The developmental rates to morula or blastocysts at low ($0.01{\sim}1{\mu}M$) and high ($5{\sim}10{\mu}M$) concentrations of genistein were not significantly different among all treatment group and genistein did not affect on IVF embryo development. These results suggest that quercetin and genistein seem to have positive effects at certain concentrations on sperm characteristics such as motility, viability and mitochondrial activity. In addition, low concentrations of quercetin (1, 5 and $10{\mu}M$) in this experiment, seem to have beneficial effect on porcine IVF embryo development but genistein did not affect on it at all given concentrations ($0.01{\sim}10{\mu}M$).

• Reproductive and Developmental Biology
• /
• 제28권2호
• /
• pp.77-82
• /
• 2004

5% $CO^2와 5% O^2$ 농도 조건 하에서 NCSU23 배양액에 aesculetin을 0, 1, 5 및 10 $\mug/ml$를 첨가한 구에서 상실배기 이상 발육된 체외배양 성적은 10 $\mu$ g 첨가구(35.7%)가 여타구(0 $\mu$g, 30.2% ; 1$\mu$g, 29.5% ; 5$\mu$g, 29.2%)보다 통계적으로 유의하게 높은 성적을 얻었다(P<0.05). NCSU 23 배양액에 taurine을 0, 2.5 및 5.0 mM을 첨가, 체외배양을 실시한 결과 배반포기 이상 발육된 체외발육 성적은 각각 2.8%, 2.2% 및 7.0%였으며, 상실배이상의 체외 발육성적은 26.1%, 26.9% 및 31.7%로서 taurine 5.0 mM 첨가가 체외수정란의 체외발육 성적이 유의적으로 높은 것으로 나타났다(P<0.05). NCSU 23 배앙액에 melatonin을 0, 1, 5 및 10nM을 첨가하여 체외배양을 실시한 결과, 배반포기까지 발육된 체외 발육성적은 17.8%, 26.1%, 20.0% 및 16.3%로서 melatonin 1nM 첨가구가 여타구에 비해 통계적으로 유의하게 높은 성적을 나타냈으며(P<0.05), 상실배기 이상 발육된 체외발육 성적에서는 melatonin 1nM 첨가구가 39.1%로서 대조구 33.3%, 5 nM 첨가구의 33.3% 및 10 nM 첨가구의 27.9%보다 높은 발육율을 나타냈다(P<0.05). 한편 배반포기 수정란의 세포수 조사에서는 melatonin 10 mM 첨가구가 유의하게 낮은 것으로 나타났다.

• 대한생식의학회:학술대회논문집
• /
• 대한불임학회 1996년도 추계 학술대회 및 정기총회
• /
• pp.59-60
• /
• 1996

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.26-26
• /
• 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.

• Asian-Australasian Journal of Animal Sciences
• /
• 제22권1호
• /
• pp.26-34
• /
• 2009

Porcine embryonic stem (ES) cells have a great potential as tools for transgenic animal production and studies of regulation of differentiation genes. Although several studies showed successful derivation of porcine ES-like cells, these cells were not maintained long-term in culture. Therefore, this study was conducted to establish porcine pluripotent ES-like cells using in vivo fertilized embryos and to maintain these cells in long term culture. Porcine ES-like cells from in vivo embryos obtained by immunosurgery or whole explant culture were successfully cultured for over 56 passages. Morphology of porcine ES-like cells was flat-shaped with a monolayer type colony. These cells stained for alkaline phosphatase throughout the culture. Furthermore, porcine ES-like cells reacted with antibodies against Oct-4, SSEA-1, SSEA-4, Tra-1-60, and Tra-1-81, which are typical markers of undifferentiated stem cells. To characterize the ability of porcine ES-like cells to differentiate into three germ layers, embryoid body formation was induced. After plating of these cells, porcine ES-like cells were spontaneously differentiated into various cell types of all three germ layers. In addition, porcine ES-like cells were successfully derived from IVF blastocysts in media containing human recombinant basic fibroblast growth factor.

• 한국수정란이식학회지
• /
• 제17권3호
• /
• pp.239-249
• /
• 2002

Embryos derived from pig oocytes matured in mSOF are able to develop to blastocysts after IVF. Experiment 1 evaluated the effects of two maturation media (TCM-199 vs mSOF) on maturation rate, fertilization parameters, including penetration, polyspermy, male pronuclear formation, and the mean number of sperm penetrated per oocyte. Experiment 2 and Experiments 3 examined the effects of two maturation media on zona pellucida solubility and cortical granule distribution by transmissible electron microscopy, respectively. Experiment 4 assessed the effects of two maturation media on the in vitro embryo cleavage rate and development to blastocyst. Lastly, experiment 5 examined the cell number of blastocyst. An effect of media (P<0.05) was detected for mSOF on the mean number of sperm per oocyte. In TCM group, zona digestion time (196.5$\pm$15.5 vs 131.6$\pm$20.1 before IVF, 397.5$\pm$30.3s vs 185.3$\pm$16.4s after IVF, p<0.05) was higher in TCM-199 group. No significant effects of media was observed on cortical granule distribution between two groups by TEM. An effect (P<0.05) was observed on embryo development to blastocyst (16% vs 8%) but not on cleavage rates. No significant effects of media was observed on total cell number of blastocyst. We found that the high mean number of sperm penetrated per oocyte and the weaker zona pellucida on the basis of the digestion time was shown in pig oocytes matured in mSOF, however, porcine oocyte maturation with supplemented synthetic oviduct fluid medium (mSOF) resulted in blastocyst cell numbers comparable to those observed with Tissue Culture Medium 199.

• 한국가축번식학회지
• /
• 제21권2호
• /
• pp.139-146
• /
• 1997

소와 돼지의 난관 상피세포가 마우스 초기배의 발달 에 미치는 영향과 체외배양에 있어 최적의 배양조건을 알아보기 위하여 ICR 계통의 마우스에 PMSG 7.5 IU와 hCG 7.5 IU를 각각 복강주사하고 자연교미하여 48시간 경과 후에 난관에서 2-세포 초기배를 D-PBS로 관류하여 회수하였다. 회수된 배아는 소와 돼지의 난관 상피세포와 공배양하여 그 효사를 배반포 발달율과 핵의 수를 조사하였다. 또한 생체내와 실험 관내의 발육상태를 비교하기 위하여 hCG접종후 120 시간동안 생체내에서 발육시킨 신선 배반포배를 자궁 에서 채취하여 그 핵수를 계산하였다. 마우스 초기배는 TCM 199, Ham's F-10, Medicult IVF 배양액에서 소 난관 상피세포 또는 돼지 난관 상피세포와 공배양할 경우 91-97%의 높은 배반포 발달율을 보였으며 난관 상피세포간의 차이는 나타 나지 않았다. 각 배양조건에 따라 배양된 배반포의 핵수는 체내에서 자란 배반포에 비해 체외에서 배양한 배반포에서 유의적으로 적었다. 체외배양중 핵수는 공배양하지 않은 TCM 199, Ham's F-10, Medicult IVF medium 에서 각각 68.1${\pm}$6.00, 67.3${\pm}$4.49, 66.4${\pm}$5.64개로 나타났으며 BOEC와 공배양하였을 경우에는 94.3${\pm}$8.61, 92.5${\pm}$7.60, 92.1${\pm}$6.107B, POEC와 공배양하였을 때는 93.3${\pm}$5.80, 92.9${\pm}$6.53, 92.3${\pm}$7.35개로 체내에서 배양된 배반포의 경우의 107.2${\pm}$7.43개보다 적었다. 이상의 결과로 난관 상피세포인 BOEC와 POEC는 마우스 초기배야와의 체외공배양시 배아의 발달과 분화에 이로운 영향을 주어 발달율과 부화율를 향상시키나 핵수 증가에서는 체내조건보다 미홉한 것으로 사료된다.

• Reproductive and Developmental Biology
• /
• 제28권2호
• /
• pp.83-87
• /
• 2004

본 연구는 돼지의 난포란을 체외에서 성숙, 수정시킨 체외수정란의 체외배양 체계를 확립하고 그 기작을 규명하기 위하여 체외배양액에 항산화제인 melatonin의 첨가 및 melatonin과 sodium nitroprusside(SNP)의 첨가배양이 체외수정란의 체외발육에 미치는 영향을 검토하고자 실시하였다. NCSU 23 배양액에 melatonin을 0, 1, 5 및 10nM을 첨가하여 체외배양을 실시한 결과, 배반포기까지 발육율은 17.8%, 26.1%, 20.0% 및 16.3%로서 melatonin 1nM 첨가구가 여타구에 비해 통계적으로 유의하게 높은 성적을 나타냈으며(P<0.05), 상실배기 이상 발육 성적에서도 melatonin 1 nM 첨가구가 39.1%로서 대조구 33.3%, 5 nM 첨가구의 33.3% 및 10 nM 첨가구의 27.9%보다 높은 발육율을 나타냈다(P<0.05). NCSU 23 배양액에 SNP를 0, 50 및 100 $\muM을 첨가하여 체외 배양한 결과, 상실배 이상 발육성적은 각각 41.9%, 25.6% 및 28.4%로서 SNP 첨가구가 대조구보다 유의적으로 낮은 성적을 나타내었다(P<0.05). NCSU 23 배양액에 대조구, SNP 50 $\muM, SNP 50 $\muM에 melatonin 1, 5 및 10nM을 혼합첨가하여 체외 발육율을 조사한 결과, 배반포기 발육율은 각각 2.5%, 1.2%, 9.9%, 5.1% 및 3.7%로서 SNP 50$\mu$M + Mel. 1nM 첨가구가 여타구 보다 높은 성적을 나타냈으며, 상실배기 이상 체외 발육율은 31.3%, 34.1%, 39.5%, 29.4% 및 39.5%로서 SNP 50 $\mu M + Mel. 1 nM 첨가구와 SNP 50 $\muM + Mel. 10 nM 첨가구가 여타구보다 높은 발육율을 나타냈다. 모든 처리구에서 배반포까지 발육된 체외수정란의 세포수는 커다란 차이가 인정되지 않았다.

• 한국가축번식학회지
• /
• 제27권2호
• /
• pp.125-133
• /
• 2003

본 연구는 돼지 난포란의 체외배양액에 황화합물인 $\beta$-mercaptoethanol($\beta$-ME)을 첨가배양함으로서 돼지난포란의 체외성숙과 체외배발달에 미치는 향을 구명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. 돼지난포란을 체외성숙배양액인 NCSU-23 배양액에 $\beta$-ME를 각각 0, 25, 50 및 100$\mu$M 첨가배양하여 성숙을 유기한 다음, 체외수정을 실시한 결과, 모든 처리구에서 핵성숙률, 정자침투율, 웅성전핵형성률, 다정자침입률 및 평균침입정 수에서 유의적인 차이가 인정되지 않았다 P>0.05). 2. 체외성숙 및 수정을 실시한 후, 배발달 배양액인 NCSU-23에 7일간 배양한 결과, 배반포 형성률은 25$\mu$M의 $\beta$-ME 처리구가 유의적(P<0.05)으로 높은 결과를 나타냈고, 총세포수에 있어서는 대조구와 처리구간 유의적인 차이가 인정되지 않았다. 3. 배발달배양액인 NCSU-23에 $\beta$-ME를 각각 0, 12.5, 25 및 5$\mu$M 첨가하고 5 및 20%산소조건 하에서 7일간 배양한 결과, 처리구별 산소농도에 따른 배발달률 및 총세포수에 있어서는 유의 적 인 차이가 없었으나, 25$\mu$M $\beta$-ME 처리구에서 유의적으로 높은 결과를 나타냈다(P<0.05). 그러나 총세포수에서는 처리구 및 대조구간 유의성이 인정되지 않았다. 결론적으로 돼지난포란의 체외성숙 및 배발달시 황화합물인 $\beta$-ME의 첨가는 25$\mu$M이 가장 적합하며, 산소농도에 따라서는 영향을 미치지 않는 것으로 조사되었다.

• Reproductive and Developmental Biology
• /
• 제36권1호
• /
• pp.71-77
• /
• 2012

The objective of this study was to examine the effect of thymidine treatment during $in$ $vitro$ maturation (IVM) of porcine follicular oocytes on blastocyst development. Porcine oocytes were treated with thymidine (10 mM, 20 mM and 30 mM) for 2 or 6 hr in the preiods of IVM I and/or II. The survival rates of the blastocysts in the 6 hr treatment groups of 10 mM and 20 mM during IVM I period were significantly higher than those of control group ($p$<0.05). However, the survival rate of the blastocysts in the 2 hr treatment group of 20 mM during IVM II period was significantly higher than control group ($p$<0.05). Furthermore, the survival rate of the blastocysts in the 6 hr treatment group of 30 mM during IVM II period was significantly lower than control group ($p$<0.05). Consistent with the previous result, blastocyst development of both IVM I and II treatment group was also showed as similar pattern. Total and apoptotic cell numbers of blastocysts derived from thymidine treated porcine oocytes were examined by using Tunel assay. The results showed that there was no significant differences in total cell number of blastocysts between thymidine treated and untreated groups. However, apoptosis-positive cells in the thymidine treated group (6 hr IVM I) were significantly lower than those of other groups ($p$<0.05). Taken together, these results indicate that high quality oocytes were selected by DNA synthesis mechanism according to high concentration thymidine treatment during porcine oocyte maturation. Therefore, we concluded that presumptive selected oocytes by thymidine treatment during maturation periods improved the further embryo development and embryonic quality of IVF embryos by decreasing the incidence of apoptosis in preimplantation porcine embryos.