• 한국가축번식학회지
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• 제23권3호
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• pp.263-270
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• 1999

본 연구는 체외성숙배 양액에 L-ascorbic acid 와 selenium을 첨가 배양, 돼지 미성숙 난포란의 체외성숙, 체외수정 및 체외배발달에 미치는 영향을 검토코자 수행하였다. 배양액내 L-ascorbic acid를 0. 62.5. 100 그리고 30 $\mu$Ml 첨가 40~44시간동안 배양한 성적은 난핵포 붕괴율이 각각 86.8%, 92.9%, 91.7%, 그리고 92.6%였으며 핵성숙율은 각각 44.7%. 57.1%, 52.8%, 그리고 53.7%로 첨가구에서 유의적으로 높았다 (p＜0.05). 체외성숙배양액내 L-ascorbic acid와 selenium을 첨가 배양 후 체외 수정 유기 결과, 웅성전핵 형성율은 유의적으로 높았고 (p＜0.05) 다정자 침입율은 유의적으로 낮게 나타났으며 (p＜0.05), 체외수정 후 난할율, 상실배와 배반포배 발달율도 첨가구에서 유의적으로 높게 나타났다 (p＜0.05). 이러한 결과는 체외성숙 배양액내 L-ascorbic acid와 selenium을 첨가 배양했을 때 돼지 미성숙난포란의 체외 성숙율, 웅성 전핵 형성율 그리고 돼지 체외수정란의 배발달율을 향상 시킬 수 있으리라 사료된다.

• 한국수정란이식학회지
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• 제32권3호
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• pp.139-146
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• 2017

The objective of this study was to determine the effect of fructose that was supplemented to a chemically defined in vitro maturation (IVM) medium on oocyte maturation and embryonic development after parthenogenesis in pigs. The base medium for in vitro maturation (IVM) was porcine zygote medium (PZM) that was supplemented with 0.05% (w/v) polyvinyl alcohol (PVA) or 10% (v/v) porcine follicular fluid (pFF). In the first experiment, when immature pig oocytes were matured in a chemically defined medium that was supplemented with 5.5 mM glucose or with 1.5, 3.0 and 5.5 mM fructose, 3.0 mM fructose resulted in a higher nuclear maturation (91.5%) than 1.5 and 5.5 mM fructose (81.9 and 81.9%, respectively) but showed a similar result with 5.5 mM glucose (94.2%). However, there was no significant differences among groups in the embryo cleavage (89.4-92.4%), blastocyst formation (37.5-41.1%), and mean cell number of blastocyst (30.8-34.2 cells). Fructose at the concentration of 3.0 mM (1.08 pixels/oocyte) resulted in a higher intra-oocyte glutathione (GSH) content than 1.5 and 5.5 mM fructose (1.00 and 0.87 pixels/oocytes, respectively) while the cumulus cell expansion was not influenced. In the second experiment, effect of individual and combined supplementation of a chemically defined maturation medium with 5.5 mM glucose or 3.0 mM fructose was examined. No significant effect was found in the nuclear maturation (86.3-92.6%). Embryo cleavage was significantly increased by the combined supplementation with glucose and fructose (95.2%) compared to that with 3.0 mM fructose only (85.7%) while blastocyst formation (37.3-42.8%) and embryonic cell number (33.3-34.1 cells) were not altered. Effect of supplementation of pFF-containing medium with glucose and fructose + glucose was examined in the third experiment. No significant effect by the supplementation with glucose and fructose or glucose alone was observed in the nuclear maturation of oocytes (90.7-94.1%) and blastocyst formation (51.0-56.5%). Our results demonstrate that 3.0 mM fructose was comparable to 5.5 mM glucose in supporting in vitro oocyte maturation and embryonic development after parthenogenesis and could be used as an alternative energy source to glucose for in vitro maturation of pig oocytes.

• 한국가축번식학회지
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• 제20권4호
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• pp.413-421
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• 1997

체외에서 포유동물의 난자와 정자의 배양에 관한 연구는 난자의 성숙과정과 수정현상에 대한 많은 새로운 정보를 제공하였다. 동시에 체외수정의 연구로부터 얻은 결과는 또다른 의문을 제기하였다. 특히 동결융해정액을 이용한 돼지체외수정의 경우 낮은 정자의 침입율과 전핵형성율 및 높은 다정자침입(polyspermy)율은 아직도 해결해야할 문제점으로 남아있다. 돼지난자의 성숙에 관한 연구의 성과는 수정후 낮은 전핵형성율을 개선시켰으나 타동물종에 비하면 아직도 매우 낮게 나타나고 있다. 한편 동결정액의 처리를 위하여 caffeine이나 Ca2+와 같은 물질을 수정용 배지내에 첨가하는 등 수정능력획득의 유기를 위하여 여러 가지 방법이 연구되고 있지만 정자의 침입율은 아직도 낮고, polyspermy의 발생율은 높게 나타내고 있다. 따라서 정자의 침입율을 향상시키고 polyspermy를 억제하기 위하여 난관세포와의 공동배양, 난포액을 첨가한 배양액 내에서 정자의 전배양 및 정자농도의 조절은 매우 효과적인 방법으로 이용되어왔다. 그러나 수정란의 체외생산성 향상과 이와 관련된 연구를 보다 효과적으로 수행하기 위해서는 위에서 지적한 문제에 영향을 미치는 요인에 대한 보다 근본적인 이해가 요구된다.

• Reproductive and Developmental Biology
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• 제33권2호
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• pp.85-91
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• 2009

In this study, in vitro maturation system using fetal bovine serum (FBS) or porcine follicular fluid (pFF) was investigated to produce comparable oocytes to those derived from in vivo. Control group of oocytes was cultured in TCM 199 supplemented with 0.1% polyvinyl alcohol (PVA). Other three groups of oocytes were cultured in TCM 199 supplemented with 10% FBS, 10% pFF or 5% FBS + 5% pFF, respectively. After 44 h maturation, oocytes with the first polar body were activated with two electric pulses (DC) of 1.2 kv/cm for 30 ${\mu}sec$. Also, matured oocytes of four groups were reconstructed and fused. Reconstructed embryos were cultured in PZM-3 under 5% $CO_2$ in air at $38.5^{\circ}C$ for 6 days. The oocytes matured in the medium supplemented with FBS or/and pFF showed significantly higher maturation rates (64.0 vs. 73.9 to 85.2%). In PA embryos, cleavage rates (89.7 vs. 77.1 to 86.6%) and blastocysts rates (30.0 vs. 16.2 to 26.2%) were significantly higher in pFF group (p<0.05). In NT embryos, there was no difference among treatments in cleavage rate, but the blastocyst rates (28.5 vs. 15.5 to 24.6%) were significantly higher in pFF group (p<0.05). The apoptosis rate was significantly higher (p<0.05) in the control than other groups (10.8 vs. 4.9 to 8.2% for PA, 3.1 vs. 0.5 to 1.3% for NT). In order to select the comparable oocyte to in vivo oocytes, each group of oocytes was stained with Brilliant cresyl blue (BCB) after 42h maturation. The matured oocytes were separated according to color of cytoplasm; stained group (BCB+) and unstained group (BCB-). The oocytes matured in the presence of FBS or/and pFF showed significantly higher staining rates (70.3 to 72.7 vs. 35.1%) (p<0.05). To verify the fact that the supplementation of FBS or/and pFF can increase the maturation rates, cdc2 kinase activity, the catalytic subunit of MPF, was determined. The cdc2 kinase activity of the oocytes matured in the medium supplemented with FBS or/and pFF was significantly higher than control group (6.7 to 9.3 vs. 3.8). In conclusion, the supplementation of FBS or/and pFF can support in vitro maturation rate of porcine oocytes through the increment of cdc2 kinase activity level in the cytoplasm.

• 한국수정란이식학회지
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• 제22권4호
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• pp.245-249
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• 2007

The aim of present experiment was to examine hatching rate as in vitro indicator of viability of porcine embryos before early stage embryo transfer such as zygotes or 2-cell stage embryos. Cumulus-oocyte complexes (COCs) collected from ovaries were matured in North Carolina State University 23 (NCSU-23) containing 10% porcine follicular fluid (pFF), 10 ng/ml epidermal growth factor (EGF), $10{\mu}g/ml$ follicle stimulating hormone (FSH), $35{\mu}g/ml$ luteinizing hormone (LH), and 1mg/ml cysteine. After 24 hours, the COCs were transferred to the same medium without hormones. After 65h of maturation, oocytes were exposed to phosphate buffered saline (PBS) with 7% ethanol (v/v) for 7 minutes, and then the oocytes were washed and cultured in tissue culture medium (TCM) 199 containing 5 ug/ml cytochalasin B for 5h at $38.5^{\circ}C$ in an atmosphere of 5% $CO_2$ and 95% air with high humidity. After cytochalasin B treatment, the presumptive parthenotes were cultured in porcine zygote medium (PZM)-5 and cleavage of the parthenotes was assessed at 72h of activation, Normally cleaved parthenotes were cultured for an additional 8 days to evaluate their ability to develop to blastocyst and hatching stages. The fetal bovine serum (FBS) were added at Day 4 or 5 with concentrations of 2.5, 5 or 10%. The blastocyst rates were ranged within $39.1{\sim}70%$ in each treatment. However hatching rate was dramatically decreased in non-addition group. In this experiment, embryo viability in female reproductive tract may be estimated before embryo transfer with in vitro culture adding FBS by hatching ability.

• Journal of Animal Science and Technology
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• 제47권3호
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• pp.363-370
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• 2005

Experiments were conducted to determine the effects of beta-mercaptoethanol ($\beta$-ME) supplements to the in vitro maturation (IVM) medium on in vitro fertilization (IVF) and intracellular glutathione (GSH) concentration. Porcine cumulus-intact oocytes were matured in TCM-I99 medium containing porcine follicular fluid, sodium pyruvate, D-glucose, FBS, hormonal supplements, and $\beta$-ME (0, 25, 50 and 100 ${\mu}$M) for 36 to 46h. After culture, cumulus-free matured oocytes were co-incubated with epididymal spermatozoa for 18h. There were no significant differences in the maturation rate among treatment groups. However, increases (P < 0.05) in intracellular GSH concentration before and after. fertilization were observed in 50 ${\mu}$M $\beta$-ME supplements to the IVM medium. Also, increases (P < 0.05) in male pronuclear formation after IVF were observed in same treatment group. In conclusion, supplementing $\beta$-ME into the IVM medium increased intracellular GSH concentrations and increased fertilization in vitro.

• 한국수정란이식학회지
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• 제24권3호
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• pp.213-220
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• 2009

The objective of this study was to examine the effect of eCG and various concentrations (20, 40, and 80 ${\mu}g/ml$) of porcine FSH on nuclear maturation and intracellular glutathione (GSH) level of oocytes, and embryonic development after parthenogenetic activation (PA) and somatic cell nuclear transfer (SCNT) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones (10 IU/ml hCG and 10 IU/ml eCG or $20{\sim}80{\mu}g/ml$ FSH) for the first 22 h and then further cultured in hormone-tree medium for an additional 22 h. Nuclear maturation of oocytes ($85{\sim}89%$) was not influencem foreCG and various concentrations FSH. Embryonic development to the cleavage stage ($86{\sim}94%$) and mean number of cells in blastocyst ($33{\sim}37$ cells) after PA were not altered but blastocyst formation e-treignificaddlor(p<0.05) improvem forthe supplementation eith 80 ${\mu}g/ml$ FSHr(64%) compared to 47%, io8%, iand 47% in oocytes that were treated with eCG, 20,i and 40 ${\mu}g/ml$ FSH,i numectivelo. In SCNT, fusion ($78{\sim}83%$) of cell-cytoplast couplets and siosequent embryo cleavage ($82{\sim}88%$) were not influencem fordifferent gonadotropins but blastocyst formation tended to increase forthe supplementation eith 80 ${\mu}g/ml$ FSHr(25% vs. $11{\sim}18%$). Our nuults demonstrated that oocyte maturation and embryonic development after PA and SCNT e-frinfluencem fortype of gcem fortype of gits concentration. In this study, supplementation of maturation medium eith 80 ${\mu}g/ml$ FSHrimproved preimplantation development of PA and SCNT pig embryos, probably by increasing intracellular GSH concentration of matured oocytes.

• 한국수정란이식학회지
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• 제16권3호
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• pp.163-172
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• 2001

본 연구는 돼지 난포란의 체외성숙에 적합한 배양액을 조사하고, 아울러 산소농도가 돼지 난포란을 이용한 체외수정란의 생산에 미치는 영향을 구명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. NCSU-23과 TCM-199 배양액에 10% PFF를 첨가하여 5% 및 20% 산소조건하에서 체외성숙을 유기한 결과, 배양액 및 산소조건에 따른 난핵붕괴률 및 핵성숙률에는 유의적 (P>0.05)인 차이가 없었다. 2. NCSU-23이 TCM-199 배양액보다 다정자침입률 및 평균정자침입률에 있어서 유의적으로 낮은 결과를 나타냈고 (P>0.05), 5% 및 20% 산소농도에 따른 차이는 인정되지 않았다. 3. 체외수정을 실시한 후, 5% 및 20% 산소조건하에서 NCSU-23 배양액에서 배양하여 배발달에 미치는 영향을 조사한 결과, 배달달 2일째 배반포형성에 있어서는 NCSU-23 처리구가 TCM-199 처리구에 비해서 유의적 (P<0.05)으로 높은 결과를 나타냈다. 또한, 처리구별 배반포기의 총세포수를 조사한 결과 5% 및 20% 산소농도 사이에는 유의적인 차이가 인정되지 않았다. 따라서, 효과적으로 돼지 난포란을 이용하여 체외발달을 유기하기 위한 체외성숙조건은 20% 산소조건하의 NCSU-23 배양액이 적합한 것으로 사료된다.

• 한국수정란이식학회지
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• 제27권2호
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• pp.121-126
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• 2012

The objective of this study was to examine the effect of in vitro culture media on embryonic development of in vitro-matured (IVM) oocytes after parthenogenetic activation (PA) in pigs. Immature pig oocytes were matured in TCM-199 supplemented with porcine follicular fluid, cysteine, pyruvate, EGF, insulin, and hormones for the first 22 h and then further cultured in hormone-free medium for an additional 22~26 h. IVM oocytes were activated by electric pulses and cultured in porcine zygote medium-3 (PZM-3) and North Carolina State University-23 supplemented with essential and non-essential amino acids (NCSU-23aa). These media were further modified by supplementing 2.77 mM myo-inositol, 0.34 mM trisodium citrate, and $10{\mu}M$ ${\beta}$-mercaptoethanol (designated as mPZM-3 and mNCSU-23aa, respectively). Culture of PA embryos in mPZM-3 significantly increased development to the blastocyst stage than culture in NCSU-23aa (36.2% vs. 24.8%, p<0.05). Modified PZM-3 showed a significantly higher blastocyst formation than NCSU-23aa in both groups of embryos that were activated at 44 h and 48 h of IVM (51.0% vs. 35.5% and 49.0% vs. 34.2% in oocytes activated at 44 h and 48 h of IVM, respectively). Irrespective of the follicle diameter where oocytes were collected, embryonic development to the blastocyst stage was increased (p<0.05) by the culture in mPZM-3 compared to culture in NCSU-23aa (25.9% vs. 34.2% and 32.9% vs. 44.8% in embryos derived from small and medium size follicles, respectively). Our results demonstrated that culture media had significant effect on preimplantation development PA embryos and that mPZM-3 was superior to mNCSU-23 in supporting development to the blastocyst stage in pigs. This beneficial effect of mPZM-3 on embryonic development was not impaired by other factors such as time of oocyte activation and origin of immature oocytes (small and medium size follicles).

• Reproductive and Developmental Biology
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• 제35권3호
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• pp.221-225
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• 2011

This study was undertaken to evaluate the relationship between in vitro maturation and plasminogen activators (PAs) activity on porcine cumulus-oocytes complexes (COCs) exposed to oxidative stress. When COCs were cultured in maturation medium with hydrogen peroxide ($H_2O_2$), the proportion of the germinal vesicle breakdown (GVBD) and oocytes maturation were decrease with addition of $H_2O_2$, and were significantly (p<0.05) lower in medium with 0.1 mM $H_2O_2$ than control group. Also, the rate of degenerated oocytes was increased in as $H_2O_2$ concentration in eased. When COCs were cultured for 48 h, three plasminogen-dependent lytic bands were observed: tissue-type PA (tPA); urokinase-type PA (uPA); and tPA-PA inhibitor (tPA-PAI). PA activity was quantified using SDS-PAGE and zymography. When $H_2O_2$ concentration was increased, tPA and tPA-PAI activities also increased in porcine oocytes cultured for 48 h, but not uPA. In other experiment, embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 1.0 mM $H_2O_2$ and (3) control medium with 1.0 mM $H_2O_2$ along with catalase in concentrations of 0.01, 0.1, and 1.0 mg/ml, respectively. $H_2O_2$ decreased the rate of GVBD and maturation in porcine COCs but catalase revealed protective activity, against oxidative stress caused by $H_2O_2$. In this experiment, tPA and tPA-PAI activities were higher in media with 1.0 mM $H_2O_2$ alone. Increasing concentration of catalase decreased tPA and tPA-PAI activities in porcine oocytes. These results indicate that the exposure of porcine follicular oocytes to ROS inhibits oocytes maturation to metaphase-II stage and increase the oocytes degeneration. Also, we speculated that increased ROS level may trigger tPA and tPA-PAI activities in porcine oocytes matured in vitro.