• 한국수정란이식학회지
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• 제31권3호
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• pp.185-190
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• 2016

During the ovary preservation in low temperature, the cumulus oocyte complexes(COCs) lose their developmental competences after in vitro fertilization. We used phosphate-buffered saline (PBS) as a basic solutions of at various temperatures (25, 15 or $5^{\circ}C$) and supplemented them with 1mM glucose and 0.5mM glutamine as a source of carbohydrate metabolites. After recovery of COCs and in vitro fertilization, a significantly higher number of oocytes developed into blastocysts. The developmental competence of embryos that were originated from ovaries preserved at $15^{\circ}C$ was increased compared to those of 25 or $5^{\circ}C$. The maturation rate of oocytes was not differed between 24 and 36 h at $15^{\circ}C$ but showed lower than control group (71% versus 78%). In vitro-fertilized oocytes from ovaries stored at $25^{\circ}C$ for 24 h or at $5^{\circ}C$ for 24 h had a significantly decreased developmental potentials, but at $15^{\circ}C$ did not (27% versus 29% of blastocysts to develop into day 8). With these results, bovine ovaries can be preserved at $15^{\circ}C$ for 36 h without decreasing developmental capacity of in vitro-fertilized oocyte at least to the blastocyst stage. This information provides valuable information of preserving ovaries for embryo transfer or in vitro embryo production.

• 한국수정란이식학회지
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• 제24권1호
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• pp.39-45
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• 2009

This study was carried out to evaluate the nuclear, cytoplasmic maturation and developmental potential of bovine oocytes selected by brilliant cresyl blue (BCB) as indirect measurement of oocytes growth phase. Cumulus-oocyte complexes (COCs) were collected from 2 to 8 mm follicles from slaughterhouse Hanwoo ovaries. The COCs were divided into stained cytoplasm to blue (BCB+) and unstained (BCB-) according to their ooplasm BCB coloration stained by $26{\mu}m$ of BCB after 90 min. Selected COCs were cultured in a TCM 199 for 18 to 26 h. Nuclear maturation and total cell number was evaluated after in vitro maturation (IVM) or in vitro culture (IVC) using $10{\mu}g/ml$ Hoechst 33342, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay before (0 h) and after (24 h) IVM. The oocyte diameters were not differed significantly between BCB+ ($157.4{\pm}5.8{\mu}m$) and BCB+ ($149.0{\pm}31.0{\mu}m$) groups (p>0.05). However, the proportion of metaphase II oocytes in BCB+ group was significantly higher than BCB- group after IVM (p<0.05). GSH content of BCB+ group oocytes was significantly higher than that of BCB- group just after collection ($7.3{\pm}0.6$ vs. $4.8{\pm}0.6\;pmol/oocyte$, p<0.05), but not varied after IVM($13.1{\pm}0.9$ and $12.6{\pm}2.5\;pmol/oocytes$ for BCB+ and BCB- respectively; p>0.05). The proportion of blastocyst formation and total cell number in BCB+ group (23.5% and $105.5{\pm}28.6$) was significantly higher than that in BCB- (9.8% and $72.4{\pm}26.1$; p<0.05). The results indicate that BCB+ group oocytes may provide a cellular and functional basis for the greater developmental competence in Korean Native Cow (KNC) oocytes.

• 한국수정란이식학회지
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• 제32권4호
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• pp.311-317
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• 2017

Cryopreservation of oocytes by vitrification technique may contribute a lot in the field of reproductive biotechnology. The objectives of the present study were to evaluate the effectiveness of two cryo-devices for vitrification of immature oocytes of indigenous zebu cows. Slaughter house derived immature cumulus-oocyte-complexes (COCs) of cows were vitrified using 15% dimethyl sulphoxide (DMSO) as cryoprotective agent (CPA) with 0.5 mol sucrose in TCM 199 supplemented with 20% FBS. Vitrification of COCs was completed after immediate plunging of COCs loaded cryotop or French mini straw into the liquid nitrogen ($LN_2$). Then the COCs containing cryotop or French mini straws were warmed in 0.25 mol sucrose and 20% FBS supplemented TCM 199 followed by in vitro culture in $50{\mu}l$ droplets of bicarbonate buffered TCM 199 supplemented with 10% FBS, pyruvate, FSH and oestradiol for 24 hrs at $39^{\circ}C$ with 5% CO2 in humidified air. After maturation culture, oocytes were denuded and examined under inverted microscope for presence of polar body as the indication of maturation. Denuded oocytes were also stained by whole mount technique using 1% orcein to examine the maturation by presence of MII chromosomes. The in vitro maturation rate was significantly (p<0.05) higher in oocytes vitrified and warmed using crytop ($47.1{\pm}6.9%$) than that of French mini straw ($15.9{\pm}12.5%$). Moreover, in vitro maturation rate was significantly (p<0.05) highe r in control oocytes (not vitrified) ($84.5{\pm}14.2%$) than that of vitrified oocytes. In conclusion, cryotop is better than French mini straw as cryo-device for vitrification of bovine immature oocytes.

• 한국수정란이식학회지
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• 제15권3호
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• pp.271-277
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• 2000

This study was conducted to establish the optimal culture conditions for in vitro production of bovine embryos derived from slaughter house ovaries. Cumulus-oocyte- complexes (COCs) collected by aspiration from follicles of 2~7 mm in diameter were matured in Ham's F-10 medium supplemented with 0.01 $\mu\textrm{g}$/m1 epidermal growth factor (EGF) at 39$^{\circ}C$ in a humidified atmosphere of 5% $CO_2$in air. After 24 hrs of culture, the oocytes were co-cultured with epididymal sperm selected off by Percoll-density gradient in TALP medium for 24 hrs. The presumptive zygotes were cultured in HECM-6 medium for 3 d post-insemination, and followed by cultured in TCM199 medium until 7 to 10d post-insemination. The cultures were compared of their cleavage and development into later stage in culture medium by additions of different protein sources (PVA, BSA and BCS) and by different embryo density. The rates of cleavage and development rates into blastocyst were not significantly (P<0.05) different among the culture media containing with BSA (75.0% and 40.5%), BCS (76.7% and 38.0%) and PVA (72.5% and 42.2%), respectively. Significantly (P<0.05) higher blastocysts rates were obtained in culturing of 30 and 40 embryos in each 50$\mu$l droplets of culture medium than in 5, 10 and 20 embryos. These results indicate that the optimal density of embryos is 30~40 embryos in a 50$\mu$l droplet of culture medium. Furthermore there is no effect of different protein sources on early embryonic development.

• 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.

• 한국수정란이식학회지
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• 제26권2호
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• pp.123-128
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• 2011

In vitro maturation and activation of oocytes are primary steps towards biotechnological manipulation in embryology. The objectives of the present study were to determine the oocyte recovery rate per ovary, in vitro maturation rates of oocytes and rates of parthenogenetically activation of matured oocytes in Black Bengal goats. All visible follicles were aspirated to recover follicular fluid from individual ovaries (number of ovaries = 456). The immature cumulus oocyte complexes (COCs; n = 1289) were cultured in tissue culture medium (TCM)-199 supplemented with 10% (v/v) fetal bovine serum (FBS) for 27 hours at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The matured oocytes (n = 248) were activated with 5 ${\mu}M$ ionomycin for 5 minutes followed by treatment with 2 mM 6-dimethylaminopurine (6-DMAP) for 4 hours. After activation, oocytes were cultured for another 14 hours in TCM-199 supplemented with bovine serum albumin (BSA) at $39^{\circ}C$ with 5% $CO_2$ in humidified air. The pronucleus formation in activated oocytes was determined by staining with 1% orcein (whole mount technique). Matured oocytes (n = 176) without activation stimuli were used as control. The mean number of oocytes recovered per ovary was $3.5{\pm}0.5$. The proportion of oocytes matured in vitro, confirmed by the presence of first polar body, was $42.1{\pm}4.7%$. Parthenogenetic activation, evidenced by formation of pronucleus, occurred in $37.2{\pm}15.8%$ of matured oocytes. No pronucleus formation was observed in control oocytes. In conclusion, a combination of ionomycin and 6-DMAP induces activation in one third of Black Bengal goats' oocytes.

• 한국수정란이식학회지
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• 제32권3호
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• pp.201-207
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• 2017

소 난자의 체외 성숙 및 발달과정에서 항산화제의 첨가는 발생과정에 생성될 수 있는 ROS를 조절하여 체외발생에 도움을 주는 것으로 알려져 있으나 이에 대한 연구는 아직 미흡하다고 판단된다. 본 연구에서는 소 수정란의 성숙과정과 발생과정에서 ROS에 대한 방어 기작에 필요한 물질로 -SH기(thiol group)을 함유하고 있는 NAC, NACA, GSH 및 CYS를 첨가하여 COCs의 난자 성숙율과 체외 수정 후 수정란의 발생율을 조사하였다. 도축장 유래 난소의 성숙율은 항산화제 처리군과 대조군에서 차이를 보여주지 않았으나(p>0.05), 배반포 형성율은 0.1 mM CYS을 처리한 실험군에서 $32.3{\pm}5.0%$로 유의적으로 높게 관찰되었다(p<0.05). 항산화제 0.3 mM NAC, 0.2 mM NACA 또는 0.5 mM GSH를 처리하는 실험군에서 배반포 형성율은 각각 $18.8{\pm}3.7%$, $21.2{\pm}3.9%$ 및 $26.5{\pm}5.0%$로 조사되었다. 그러므로, 항산화 물질인 NAC, NACA, GSH 및 CYS을 난자의 성숙 및 수정란 배양과정에 첨가하면 난자의 성숙에 영향이 없으나, CYS처리군이 배반포형성율에 효과가 있음을 밝혔다(p<0.05).

• 대한수의학회지
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• 제38권3호
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• pp.652-658
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• 1998

The efficiency of nuclear transfer using donor embryos originated from ovum pickup(OPU) and activated recipient cytoplasts were examined for induction of twinning in Korean native cattle(KNC). After aspiration of follicle by OPU, regardless of the vacuum applied, we obtained same result in proportions of recovered cumulus-oocyte complex (COCs) with compact cumulus. Under electric stimulation(1.0kV/cm DC for $40{\mu}s$), most of activated oocytes proceed to anaphase II/telophase II within 3h(84.7%). In the treatment of oocyte activation, the preactivation which was performed before fusion had significant effect on the developmental rates to morula/blastocyst stage(9.4 vs 4.0%). In embryo transfer of nuclear transferred embryos, we obtained 2 twins from KNC recipients and 1 twin from a Holstein recipient. Our results showed that it is possible to obtain twins using nuclear transfer technique in KNC.

• 한국가축번식학회지
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• 제24권1호
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• pp.69-76
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• 2000

수정란이식에 필요한 다량의 수정란을 생산하는 수단인 배양액과 적정 수정란의 수는 수정란의 체외발달에 많은 영향을 미치므로 이들 관계를 조사하여 수정란의 체외배양체계를 확립하고자 본 연구를 실시하였다. 도축장에서 채취한 난소에서 미성숙 난자를 채란하여 10% FBS가 첨가된 TCM -199 에 LH(10 $\mu\textrm{g}$/$m\ell$), FSH(35 $\mu\textrm{g}$/$m\ell$), estradiol-17 $\beta$(1 $\mu\textrm{g}$/$m\ell$)가 첨가된 체외성숙배양액에서 24시간 동안 배양 후, 동결정액은 Percoll-density gradients(45 vs. 90%)을 이용하여 700 g 에서 30분 동안 처리한 다음, 체외수정배양액 (IVF-Fert)에서 체외수정을 유도하였다. 수정이 확인된 수정란은 50 ${\mu}\ell$ 배양액에 1, 25 또는 50개의 수정란을 난구세포와 공배양을 하여 9일 동안 배양하였다. 일정한 배양액에서 수정란의 수가 체외수정란의 발달에 미치는 요인을 분석하고, 개별배양 또는 그룹배양 시 난구세포와의 공동배양 효과를 조사한 결과는 다음과 같다. 1. 50 ${\mu}\ell$ 배양액에 1개, 25 또는 50개의 수정란을 수정 후 9일 동안 배양한 결과, 25와 50개의 수정을 배양했을 경우에는 발달율이 36.5 와 26.5%를 보여 1개의 수정란을 배양했을 경우에서의 발달율 6.2% 보다 유의적으로 높은 발달율을 얻었다(P＜0.05). 2. 1, 25, 50개의 수정란을 배양시 수정 후 6일째 발달율은 1.0~3.5%였으나, 수정 후 7, 8, 9 일째의 발달율은 1개의 수정란을 배양하는 것보다 25, 50개의 수정란을 배양한 처리구에서 유의적으로 높은 발달율을 얻었다 (P<0.05). 3. 일정한 배양액에서 1, 25 및 50개의 수정란을 배양 시 수정 후 8일째의 배반포 수정란의 세포수를 조사한 결과, 1개의 수정란을 배양시 배반포 수정란의 수는 93.0개였으나, 25, 50개의 수정란을 배양시는 각각 112개의 세포수를 얻어 유의적으로 높은 세포수를 나타내었다 (p＜0.01). 4. 일정한 배양액에 1개 또는 25개의 수정란을 난구세포와 공배양을 하거나, 하지 않았을 때의 발달율은 각각 15.0와 3.7% 또는 34.5와 9.0%로서 난구세포와 공배양을 하는 것이 유의적으로 높은 발달율을 나타내었다 (p＜0.01 수정 후 8 일째의 배반포 수정란의 세포수도 각각 96.1와 82.0개 또는 116.4 와 96.5개로서 난구세포와 공배양을 한 처리구에서 유의적으로 높은 세포수를 나타내었다 (p＜0.01). 이상의 결과를 요약하면, 수정란의 수가 체외수정란의 체외발달에 중요한 영향을 미친다는 것을 알 수 있었으며, 다량의 체외수정란을 생산하여 수정란 이식에 이용하기 위해서는 체외성숙 / 수정된 체외수정란을 일정한 배양액 (50${\mu}\ell$) 에 25, 50개의 수정란을 난구세포와 공배양하는 것이 높은 배 발달율과 세포 수를 얻 을 수 있을 것으로 사료된다.

• 한국가축번식학회지
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• 제23권4호
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• pp.337-345
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• 1999

본 연구는 소 체외수정란의 체외배양 시 공동배양배지 및 첨가되는 단백질원에 따른 소 체외수정란의 체외발육능을 검토하였다. 소의 미성숙 난포란을 체외에서 성숙, 수정시킨 후, BSA 또는 FBS를 첨가한 TCM-199 또는 CR1aa 배양액으로 단순배양 또는 난구세포, 소 난관상피세포 (BOEC) 및 Buffalo rat 간세포 (BRLC)와의 공동배양 후, 체외수정란의 분할율 및 발육능을 검사하였다. 소 성숙 난포란을 체외수정 후, 분할율은 배양액의 종류에 관계없이 BSA를 첨가한 경우에 유의적으로 높았다 (P＜0.01). 분할된 수정란을 BSA 또는 FBS가 첨가된 TCM-l99 또는 CRlaa 배양액 내에서 단순배양한 결과, 배반포 발육율은 단백질원에 관계없이 CR1aa 액에서 배양한 경우가 유의적으로 높았다 (P＜0.05). 분할된 수정란을 난구세포 또는 BOEC와 공동배양 시, TCM-l99 배양액에서는 FBS에 비하여 BSA 첨가구가 높은 배반포 형성율을 보였으나 (P＜0.05), CRlaa 배양액에서는 BSA와 FBS 첨가구 모두 높은 발육율이 얻어졌다. 한편, 분할된 수정란을 BRLC의 단층세포와 공동배양 시에는 배양액의 종류와 관계없이 BSA에 비하여 FBS가 수정란의 발육율을 향상시켰다 (P＜0.05). 본 실혐의 결과는 배양액 중에 BSA 첨가가 소 체외수정란의 분할을 촉진할 수 있으며, 체외수정란을 체세포와 공동배양 시, 수정란의 발육율이 배양액 및 첨가 단백질원의 종류에 따라 영향을 받아, TCM-199 액에서 난구세포 또는 BOEC와 공동 배양하는 경우에는 BSA 첨가가 효과적일 수 있음을 수 있음을 보여준다.