• Journal of Embryo Transfer
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• v.18 no.3
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• pp.269-274
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• 2003

This experiment was carried out to investigate the effects of different IVM-IVC culture media factors, such as development rates according to the maturation media, collecting times from slaughter to initiation of incubation and with cumulus cells, on in vitro maturation of oocytes collected from 3∼5mm diameter follicles of the swine abattoir The development rates significantly(p<0.05) higher when the oocytes were matured TCM-199 media than NCSU-23 media. In comparing with TCM199 medium in presence of Earle's salts and Hank's salt, there were no significantly differences between each salt balance in cleaved rate and in number of morulae plus blastocyst. Among 1,455 immature oocytes, 999(68.6％) of oocytes were cleaved. The number of development to the morulae and blastocysts were 617(61.8％) include 62 balstocysts(6.2％).

• Journal of Embryo Transfer
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• v.19 no.2
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• pp.165-172
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• 2004

This study was carried out to examine the effects of maturation media on in vitro maturation (IVM) of porcine immature oocytes, and on subsequent in vitro fertilization (IVF) and development (IVD). Cumulus-oocyte complexes (COCs) were collected from antral follicles of porcine ovaries collected from abattoir, and were matured in vitro in modified NCSU-37 (mNCSU-37), modified NCSU-23 (mNCSU-23), or TCM-199 supplemented with 10% porcine follicular fluid (pFF). Oocytes matured in vitro, were fertilized in vitro in modified Tris-buffered medium(mTBM) with the final motile sperm concentration of 1${\times}$105 sperm/mL, and subsequently putative embryos were developed in vitro in NCSU-23. The results are as follows. 1. In the result of IVM, the rate of germinal vesicle breakdown (GVBD) and of nuclear maturation were not significantly different among the media, though numeric value of them were slightly lower in TCM-199 than in mNCSU-37 or in mNCSU-23. 2. In the result of IVF, though the rate of sperm penetration was not significantly different among the maturation media, the percentage of oocytes with male pronucleus (MPN) of ones matured in mNCSU-37 (88.0%) was significantly higher than in TCM-199 (71.1%) (p<0.05). 3. In the result of IVD, the percentage of cleaved oocytes of ones matured in mNCSU-37 (52.3%) or in mNCSU-23 (53.7%) was significantly higher than in TCM-199 (43.1%) (p<0.05), but the rate of blastocysts at day 6 was not significantly different among the maturation media, though putative embryos from oocytes matured in mNCSU-37 or in mNCSU-23 were developed more than in TCM-199. These results suggested that mNCSU-37 or mNCSU-23 was more appropriate than TCM-199 as IVM medium for porcine immature oocytes.

• Reproductive and Developmental Biology
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• v.39 no.3
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• pp.89-95
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• 2015

The addition of growth factors and cytokines to in vitro culture (IVC) media could affect embryo development and the quality of the resulting blastocysts. The present study was performed to investigate the effect of porcine induced pluripotent stem cell (piPSC)-culture conditioned medium (CM) on the in vitro maturation (IVM) and development of parthenogentic embryos (parthenotes) in pigs. Cumulus-oocyte complexes (COCs) or activated oocytes were cultured in IVM or IVC medium supplemented with 0 (control), 25, or 50% of stem cell medium (SM) or CM, respectively. The maturation rate of CM-25% group was significantly improved when compared with control group (p<0.05), but that was not different among SM or CM groups. Blastocyst formation rate was significantly higher in CM-25% group (29.2%) than that of control (20.7%), SM-50% (19.6%) and CM-50% (23.66%, p<0.05). Cell number and the apoptotic cell index in blastocysts was significantly lower in SM-25% than in CM-25% group (p<0.05). The embryo quality related genes, OCT4, KLF4, TERT and ZFP42, were significantly increased in CM-25% group compared with control (p<0.05). In conclusion, the addition of 25% of CM to IVM and IVC medium positively influences not only the developmental potential also quality of parthenotes in pig.

• 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.

• Korean Journal of Animal Reproduction
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• v.20 no.4
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• pp.413-421
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• 1997

In vitro culture has provided new information on the mechanisms involved in fertilization how sperm and oocyte fuse together. At the same time, results obtained in vitro have led to new questions. Techniques for In vitro maturation of porcine oocytes have progressed such that the problem of the low rate of pronucleus formation with in vitro matured oocytes after in vitro fertilization has been nearly improved. On the other hand, porcine spermatozoa have been shown to be capacitated if the fertilization medium contains caffeine and Ca$^2+$, but the incidence of polyspermy in IVM-IVF oocytes is still high. To prevent polyspermy, co-culture with oviductal cells, sperm preincubation with porcine follicular fluid or control of sperm concentration, have been examined with significant effects but still remarkably high rates of polyspermy. The under standing of these influences is a prerequisite to enhancing in vitro production of porcine em bryos.

• Journal of Embryo Transfer
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• v.7 no.2
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• pp.97-110
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• 1992

돼지난포란의 안정된 체외배양 체계를 위하여 도살장에서 재취된 난소로부터 난포 및 난자난구세포체의 혀태적인 선정기준을 설정하고 이의 이론적 배경을 확립하였다. 난소를 난포상태 및 분포나 황체 존재 여부에 따라 A,B 및 C 의 세 가지 형태로 분류하고 각 type의 난소에서 직경 3-5mm인 난포로부터 난포란을 회수하였다. 회수된 난포란을 난구세포 부착상태에 따라 Good, Fair 및 Poor 의 세 가지 형태로 분류하여 각각을 호르몬이 첨가된 M16+FCS 배양액으로 35시간 동안 체외배양하여 성숙율을 비교 검토 하였다. 난소의 형태에 따라 회수된 난포란 중 Good 또는 Fair 형태는 Type A 및 C 난소에서 85%를 차지한 반면, Type B 난소에서는 53%에 불과하였다. 또한 이들 난포란을 체외배양한 결과 Type A 및 C 에서 회수된 난포란은 90 및 85%의 높은 성숙율을 보인 반면, Type C 의 난포란은 33%의 저조한 성숙을 나타냈다. 한편 핵형 분석 및 조직학적 분석에서도 Type C 난소의 경우 난포란의 핵형이 대부분 GVBD 및 퇴화 형태를 보였으며, 폐쇄포난포의 비율도 Type A 난소의 53%에 비해 월등히 높은 85%를 나타내어 성숙율 비교실험의 결과와 일치되는 경향을 나타내었다. 따라서 본 실험의 선별기준에 의한 돼지 난소 및 난포란의 형태적 분류 작업에 의해 난포란의 체외배양 성적 향상 및 안정된 배양체계의 확립이 가능하였다.

• 대한생식의학회:학술대회논문집
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• 2001.06a
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• pp.55-56
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• 2001

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.131-138
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• 2017

In most mammals, metaphase II (MII) oocytes having high maturation promoting factor (MPF) activity have been considered as good oocytes and then used for assisted reproductive technologies including somatic cell nuclear transfer (SCNT). Caffeine increases MPF activity in mammalian oocytes by inhibiting p34cdc2 phosphorylation. The objective of this study was to investigate the effects of caffeine treatment during in vitro maturation (IVM) on oocyte maturation and embryonic development after SCNT in pigs. To this end, morphologically good (MGCOCs) and poor oocytes (MPCOCs) based on the thickness of cumulus cell layer were untreated or treated with 2.5 mM caffeine during 22-42, 34-42, or 38-42 h of IVM according to the experimental design. Caffeine treatment for 20 h during 22-42 h of IVM significantly inhibited nuclear maturation compared to no treatment. Blastocyst formation of SCNT embryos was not influenced by the caffeine treatment during 38-42 h of IVM in MGCOCs (41.1-42.1%) but was significantly improved in MPCOCs compared to no treatment (43.4 vs. 30.1%, P<0.05). No significant effects of caffeine treatment was observed in embryo cleavage (78.7-88.0%) and mean cell number in blastocyst (38.7-43.5 cells). The MPF activity of MII oocytes in terms of p34cdc2 kinase activity was not influenced by the caffeine treatment in MGCOCs (160.4 vs. 194.3 pg/ml) but significantly increased in MPCOCs (133.9 vs. 204.8 pg/ml). Our results demonstrate that caffeine treatment during 38-42 h of IVM improves developmental competence of SCNT embryos derived from MPCOCs by influencing cytoplasmic maturation including increased MPF activity in IVM oocytes in pigs.

• Journal of Embryo Transfer
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• v.14 no.1
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• pp.17-22
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• 1999

To investigate the maturational and development competece of porcine oocytes of different diameter groups, oocytes were obtained by aspiration from slaughterdhouse ovaries. After washing three times in NCSU23 medium, each cumulus-oocyte complex was transferred into a $8{mu}ell$ drop of the maturation medium (one oocyte per drop) under paraffin oil. The diameter without zona pellucida of oocytes was measured with micor-calibrator (Mikrometer, E. Leitz) on a screen connected to a VCR on an inverted microscope $(200\times)$. After being measured, the oocytes were divided into 6 groups according to their diameter size : <105, 105 to < 110, 110 to < 115, 115 to < 120, 120 to < 125 and > $125{\mu}{\textrm}{m}$, and in vitro maturation (IVM), fertillzation (IVF) and production (IVP) of oocytes / embryo was performed. The rates of in vitro maturation on oocytes in the greater 105 ${\mu}{\textrm}{m}$ size groups(91.8~100%) were significantly (P<0.05) higher than in the < 105 ${\mu}{\textrm}{m}$ group(66.7%). The rates of sperm penetration were significantly (P<0.05) low in < $105{\mu}{\textrm}{m}$ group (50.0%) than others groups (81.6~85.5%). But the plyspermic fertilization rate was significantly (P<0.05) higher in < $110{\mu}{\textrm}{m}$ oocytes groups than in the $110\leq{\mu}{\textrm}{m}$ size groups. The rates of cleavage and development to blastocysts rose as oocytes diameter increased, however, while oocytes over $120{\mu}{\textrm}{m}$ in diameter failed to develop to blastocysts. There results suggest that porcine oocytes have acquired full meiotic competece at a diameter of $105{\mu}{\textrm}{m}$ but not yet attended full development competence to blastocyst and that oocytes have acquired full development competence at a diameter of $110{\mu}{\textrm}{m}$.

• Proceedings of the KSAR Conference
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• 2003.06a
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• pp.72-72
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• 2003

대부분의 동물에서 체내 또는 체외수정란의 체외 배양 시 일정한 발육 단계까지 발달한 후 발육지연이나 정지가 되는 체외 발육억제 현상이 나타나게 된다. 특히 돼지에서는 타 가축들과는 달리 4 세포기에서 체외 발육억제 현상이 일어나기 때문에 체외에서의 발육율이 매우 낮아 수정란 생산이 제한되고 있다. 따라서 본 연구는 이러한 돼지 체외발육 억제 현상을 극복하고 돼지 체외 수정란의 체외배양 체계의 기초 자료를 얻고자 돼지 미성숙 난자를 체외에서 성숙, 수정시킨 뒤, 체외 수정란의 배양 시 성장인자와 6탄당의 첨가에 따른 체외 발육율을 검토하였다. 난자의 핵 성숙과 세포질 성숙 및 세포 기능에 영향을 미치는 것으로 알려져 있는 성장인자로는 Insulin-like growth factor-I(IGF-I)과 Epidermal growth factor(EGF)를 사용하였고, 여러 종의 번식기관에 존재하여 배반포 형성을 촉진시키는 것으로 알려진 glucose, mannose, galactose 및 fructose가 6탄당으로 사용되었다. 체외수정란의 발육을 위한 기본 배양액인 NCSU-23에 각각 0, 1, 5, 10 및 20ng/ml의 IGF-I과 EGF를 각각 첨가하여 농도의 차이에 따른 발육율을 검토하였다. 또한 5.56mM의 glucose, mannose, galactose 및 fructose에 5ng/ml의 IGF-I 또는 10ng/ml의 EGF 첨가 유, 무에 따른 초기배 발육율을 검토하였다. 마지막으로, 각각의 6탄당에 위와 같은 농도의 IGF-I와 EGF 공동 첨가 유, 무에 따른 초기배 발육율을 검토하였다. 그 결과 돼지 체외 수정란의 체외 발육 시 배양액 내에 서로 다른 농도의 IGF-I과 EGF를 첨가하였을 때 IGF-I은 5ng/ml(12%)에서, EGF는 10ng/ml(10%)의 실험구에서 가장 높은 배반포기 배의 발육율을 나타냈다. 또한 각각의 6탄당과 IGF-I 또는 EGF 유, 무에 따른 초기배 발육율을 검토한 결과 IGF-I과 EGF 모두 glucose 첨가 시 타 첨가구에 비해 초기 발육 단계의 수정란 발육뿐만 아니라 배반포까지의 배발육(10～11%)이 타 첨가구(3～8%)에 비해 높게 나타났다. 한편, 각각의 6탄당이 첨가된 배양액 내에 IGF-I파 EGF 공동첨가 유, 무에 따른 초기배 발육율을 검토한 결과 모든 실험구에서 EGF와 IGF-I 첨가 시 무첨가보다 높은 초기 배 발육율을 나타냈으며 특히 초기 분열단계 수정란에서는 발육의 차이가 크게 나타났다. 본 연구 결과 성장인자와 6탄당의 첨가는 돼지 수정란의 체외배양 시 초기배 배발육에 효과적인 영향을 미치는 것으로 사료되며, 이는 체외 발육율이 타 가축에 비해 낮은 돼지의 수정란 생산에 있어 체외배양체계의 개선을 위한 기초자료가 될 수 있을 것이라 기대된다.