• 한국수정란이식학회지
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• 제21권2호
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• pp.121-128
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• 2006

본 연구는 고품질의 돼지 체외 수정란을 생산하기 위하여 체외 성숙 배지에 첨가하는 polyvinylpyrrolidone(PVP)의 분자량, 첨가 농도 및 시간(실험 1)과 체외 성숙 수정 배양 단계에서 PVP의 첨가(실험 2)가 배 발생과 세포수에 미치는 효과를 검토하였다. 돼지 미성숙 난자의 체외 성숙은 NCSU 23 용액, 체외 수정은 mTBM 용액, 체외 배양은 PZM 3 용액을 이용하였다. 체외 성숙용 배지에서 PVP의 분자량, 농도 및 첨가 시간에 따른 수정율과 배반포 발달율은 차이가 없었다. 그러나 배반포의 부화율은 분자량 40,000(30.1%), 0.5%(28.6%) 및 $0{\sim}44$시간(37.5%) 첨가군이 다른 시험군에 비하여 유의하게 높았다(p<0.05). 분자량 40,000 0.5% PVP를 체외성숙, 수정 및 배양 배지에 각각 첨가(W) 또는 미첨가(W/O)한 결과, 수정율은 체외성숙 수정 배양에서 W-W/O-W 군이 69.3%, 배반포 발달율과 부화율은 W-W/O-W 및 W-W/O-W/O 군이 각각 12.6과 30.0% 및 14.0과 37.5%로서 다른 군에 비하여 유의하게 높았다(p<0.05). 총 세포 수와 Trophoblast 수는 처리군 간에 유사한 경향이었으나, Inner cell mass 수는 W/O-W/O-W 군이 평균 7.4개로서 가장 높았다 (p<0.05).

• 한국수정란이식학회지
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• 제32권3호
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• pp.123-130
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• 2017

This study was conducted to investigate the effects of alpha-lipoic acid (aLA) as an antioxidant that decrease the reactive oxygen species (ROS) in bovine embryonic development. Slaughterhouse derived bovine immature oocytes were collected and 4 different concentrations (0, 5, 10 and 20 mM) of aLA was supplemented in bovine in vitro maturation (IVM) medium. After 20 hrs of IVM, maturation rates, levels of ROS and glutathione (GSH), and further embryonic development after parthenogenetic activation (PA) and in vitro fertilization (IVF) was investigated according to aLA concentrations. Maturation rate was significantly higher in 10 mM group than other groups (80.5% vs. 62.9, 73.9, 64.2%; P<0.05). In the levels of ROS and GSH in matured oocytes as an indicator of oocyte quality, significantly better results were shown in 5 and 10 mM groups compared with other 2 groups. After IVM, significantly higher rates of blastocyst formation were shown in 10 mM groups in both of PA (27.9% vs. 18.8, 22.3, 14.2%; P<0.05) and IVF (32.6% vs. 23.9, 27.3, 16.2%; P<0.05) embryos. In addition, significantly more cell total cell number and higher inner cell mass ratio in 10 mM PA and IVP blastocysts showed developmental competence in 10 uM groups. Therefore, based on the entire data from this study, using $10{\mu}M$ of aLA confirmed to be the optimal concentration for bovine oocyte maturation and embryonic development.

• Asian-Australasian Journal of Animal Sciences
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• 제32권12호
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• pp.1844-1853
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• 2019

Objective: We investigated how pituitary adenylate cyclase-activating polypeptide (PACAP) affects embryonic development during pre-in vitro maturation (pre-IVM) using porcine oocytes isolated from small follicles. Methods: We divided the follicles into the experimental groups by size (SF, small follicles; MF, medium follicles) and treated with and without PACAP and cultured for 18 hours (PreSF[-]PACAP; without PACAP, Pre-SF[+]PACAP; with PACAP) before undergoing IVM. The gene expression related to extracellular matrix formation (amphiregulin, epiregulin, and hyaluronan synthase 2 [HAS2]) and apoptosis (Bcl-2-associated X [BAX], B-cell lymphoma 2, and cysteine-aspartic acid protease 3) was investigated after maturation. The impact on developmental competence was assessed by the cleavage and blastocyst rate and total cell number of blastocysts in embryos generated from parthenogenesis (PA) and in vitro fertilization (IVF). Results: Cleavage rates in the Pre-SF(+)PACAP after PA were significantly higher than SF and Pre-SF(-)PACAP (p<0.05). The cleavage rates between MF and Pre- SF(+)PACAP groups yielded no notable differences after IVF. Pre-SF(+)PACAP displayed the higher rate of blastocyst formation and greater total cell number than SF and Pre-SF(-)PACAP (p<0.05). Cumulus cells showed significant upregulation of HAS2 mRNA in the Pre-SF(+)PACAP compared to the SF (p<0.05). In comparison to other groups, the Pre-SF(+)PACAP group displayed a downregulation in mRNA expression of BAX in matured oocytes (p<0.05). Conclusion: The PACAP treatment during pre-IVM improved the developmental potential of porcine oocytes derived from SF by regulating cumulus expansion and apoptosis of oocytes.

• 한국수정란이식학회지
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• 제19권3호
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• pp.275-282
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• 2004

본 연구는 체외성숙/체외수정 유래의 돼지 난자를 이용하여 체외발달시 배양액의 종류나 교체에 따른 영향을 구명하고자 수행하였다. mNCSU-23에서 체외성숙시킨 다음 mTBM에서 체외수정시킨 난자를 목적에 따라 두 가지로 나누어 실험한 결과는 다음과 같다. 1. 체외성숙/체외수정란을 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 CZB에서 4일 배양한 다음 Pig-MEM으로 옮겨서 나머지 3일간 배양한 결과, 난분할율은 배양액간 차이를 보이지 않은 반면, 추정수정란대 배반포 발달을(P<0.05)과 분할란대 배반포 발달율은 NCSU-23에서 배양된 것이 유의적으로 높았다(P<0.01). 그러나 배 반포의 ICM 세포수, TE 세포수 및 총세포수에서는 모두 차이가 없었다. 2. 체외성숙/체외수정란를 NCSU-23에서 배양액 교체없이 7일 동안 배양하거나 체외배양 5일째에 신선한 동일 배양액이나 0.4％ BSA를 10％ FBS로 대체한 배양액(mNCSU-23F)으로 완전히 교체하여 배양한 결과, 난분할율, 배반포발달율, 배반포의 ICM 세포수, TE 세포수 및 총세포수 모두 처리간 유의적인 차이를 보이지 않았다. 결과적으로, NCSU-23이 CZB/Pig-MEM보다 체외성숙/체외수정 유래의 난자를 체외발달시키는데 적합한 것으로 사료되며, 체외발달배양 과정에 신선한 배양액이나 일부 변경된 배양액으로의 교체에 대해서는 더 많은 연구가 필요할 것으로 사료된다.

• 한국발생생물학회:학술대회논문집
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• 한국발생생물학회 2003년도 제3회 국제심포지움 및 학술대회
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• pp.128-128
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• 2003

The aims of this study are 1) to test oocytes and embryos collected from in-vivo and in-vitro to achieving the valuable protocol by culturing, vitrifying and thawing of oocytes/embryos, and 2) to transfer them to recipient, and finally have resulted in pregnancies from recipient females after surgical or nonsurgical transfer. In vitro maturation and fertilization were performed according to the procedures of Funahashi et al. Fertilized oocytes were cultured in glucose-free NCSU 23 supplemented with 5 mM sodium pyruvate, 0.5 mM sodium lactate and 4 mg/ml bovine serum albumin for 2 days at 39$^{\circ}C$, and 10% fetal bovine serum was added to the culture medium thereafter. Embryos were treated with 7.5$\square$g/ml cytochalasin-B for 30 min, centrifuged at 13,000 ${\times}$ g for 13 min and then exposed sequentially to an ethylene glycol (EG) vitrification solution, aspirated into OPSs, and plunged/thawed into/from liquid nitrogen. In vivo embryos were surgically collected from three donors after Al. Forty-six embryos (18, 9 and 19 embryos, respectively) were washed 3 times in mPBS+10%FBS, followed treatments : cultured, centrifuged, vitrified, recovered and transferred to recipients as in vitro prepared embryos. Three recipients received surgically 34(control), 188 and 184 embryos (derived from abattoir), respectively. Another three recipients were received nonsurgically 150, 100 and 150 embryos, respectively. All recipient sows exhibited delayed returns to estrus. To our knowledge, these results suggest that required an improved techniques, more vigorous embryos preparation and cleaner uterous condition(use gilt).

• 한국수정란이식학회지
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• 제19권1호
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• pp.1-10
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• 2004

본 연구는 1997년 IMF 이후 급격히 감소된 한우의 두수를 증가시키고, 최근 젖소 송아지 가격이 현저히 낮게 형성되고 있어 양축가의 사기가 떨어지고 있는데 가임 젖소 암소에 한우 수정란을 이식시켜 한우 송아지를 생산케 함으로서 농가 소득을 증대시키고 실시하였다. 1. 체외수정 후 6, 7, 8 및 9일째의 수정란을 이식하여 59.4%, 68.2%, 66.0% 및 100%의 수태율을 나타냈으며, 상실배기(20.0%) 수정란이 배반포기(61.1%∼69.5%) 수정란보다 낮은 수태율을 나타내었다. 2. 수란우의 영양상태는 과비가 되지 않고 약간 야윈 듯한 체형을 선발하는 것이 좋은 것으로 나타났다. 3. 황체가 형성된 자궁각에 수정란을 이식한 결과 수태율이 70.1%로서 황체가 형성된 반대편 자궁각에 이식한 수태율 62.5%보다 높은 성적을 나타냈다. 4. 수란우에 수정란을 이식하기 전 hCG 1500 IU 와 GnRH 5 $m\ell$ 투여한 결과 69.9%를 나타내어 무처리구 63.0%보다 다소 높은 성적을 나타내었다. 5. 수란우와 공란우의 발정이 일치할 경우(0일) 이식 후 수태율이 72.6%로서 여타구보다 높은 성적을 나타내었다. 6. 수란우 산차에 따른 수정란 이식 후 수태율은 미경산우가 59.1%로서 경산우 70.6%보다 낮게 나타났다. 7. 수정란 상태에 따른 이식 후 신선란 및 동결란 수태율은 각각 70.6%와 36.4%를 나타내어 신선란이 높은 수태율을 나타내었다. 본 연구의 결과를 요약하면 체외 수정란은 체외배양 7일에 생산된 배반포 및 확장배반포기 수정란을 공란우와 수란우의 발정을 정확히 동기화 시키고, 수란우의 황체가 뚜렷하게 형성된 경산우를 이용하며, 이식 전 수란우에 Hormone을 처리하는 것이 수태율을 향상시키는 것으로 나타났다.

• Asian-Australasian Journal of Animal Sciences
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• 제20권12호
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• pp.1820-1826
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• 2007

In this study, we conducted various experiments in order to develop enhanced cultural conditions for in vitro-produced porcine embryos. All embryos were produced by in vitro maturation (IVM) and fertilization (IVF) of immature oocytes from abattoir-derived ovaries. In experiment 1, we cultured IVF embryos in 4 different groups, namely, 0% bovine serum albumin (BSA), 3% BSA, 0.05% Polyvinyl alcohol (PVA), and 0.5% Polyvinylpyrrolidone (PVP) added to the basal fluid cultural medium, Porcine zygote medium 3 (PZM-3). The rates of embryo development were higher in the group where the PZM-3 media had been supplemented with 3% BSA than the other groups. While not statistically significant, the percent of blastocysts and hatched blastocytes were 6.9% and 25.0% in the 3% BSA group vs. 1.2-6.4% and 0-16.7% in the other groups, respectively. In experiment 2, we added 10% fetal bovine serum (FBS) to PZM-3 on day 0 of culture and observed the development rate of blastocysts per day of culture from days 0 to 5. The development rate of blastocysts was higher at 15.6% on day 4 than on any other day, and was significantly higher than on day 0 or day 1 (p<0.05). The development rate of hatched blastocysts was 26.7% on day 4, and was higher than on any other day. In experiment 3, we cultured IVF embryos with different fluid culture media, grouped as 1) PZM-3+0.3% BSA (day0-day7); 2) PZM-3+0.3% BSA${\rightarrow}$day-4) PZM-3+10% FBS; 3) PZM-3+0.3% BSA${\rightarrow}$PZM-3+0.3% BSA+(day-4) FBS 10%; and 4) PZM-3+0.3% BSA+10% FBS (day0-day7). The development rates of blastocysts and hatched blastocysts were 21.5% and 53.1% in group 3, respectively, which was significantly higher than group 4 with respect to blastocyst development (5.2%, p<0.05) but not hatched blastocysts (14.3%). The total cell number (TCN) of blastocysts in group 3 was higher at $37.8{\pm}16.1$ than the other groups at $16.8{\pm}4.4$ - $30.1{\pm}10.9$; however, this was not significantly different. The results of this study showed that PZM-3 containing 0.3% BSA and supplemented with FBS during the later stage of culture on day 4 resulted in better TCNs and an increased rate of hatched blastocysts.

• 대한수의학회지
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• 제33권4호
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• pp.757-762
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• 1993

The ovaries of Korean Native cows or heifers were obtained from an abattoir and kept on 20 to $25^{\circ}C$ and transported to laboratory within 2 hrs. The follicular oocystes were collected from 2~6mm follicles in diameter and classified into 3 grades by the morphology of cumulus cells attached. The oocytes were matured in vitro(IVM) for 24 hrs. in TCM-199 supplemented with $23{\mu}g/ml$ FSH, $10{\mu}g/ml$ LH, $1{\mu}g/ml$ estradio-17 ${\beta}$ and granulosa cells at $39^{\circ}C$ under 5% $CO_2$ in air. They were fertilized in vitro(IVF) by incubation for 12 hrs. of epididymal spermatozoa pretreated with heparin, and then the zygotes were co-cultured in vitro(IVC) with oviductal epithelial cells for 7 to 9 days. Assessment of maturation revealed that 93.0%(147/158) of grade I oocytes had expanded of cumulus cells, which was higher(p<0.05) than the 79.4%(85/107) of grade II oocytes. Compared to epididymal sperm(32.9%), the insemination with frozen and thawed sperm resulted in slightly lower(20.5%), but not significant, development to morulae and blastocysts from grade I oocytes. Co-culture of bovine IVF embryos with oviductal epithelial cells improved the development to transferable embryos significantly(38.1%), compared to co-culture with granulosa cells(20.0%). When VF bovine embryos were vitrified at blastocyst, the post-thaw survival rate was obtained higher resulf for 1 min. equilibration time(82.6%) or 2 min.(73.9%) than 3 min.(18.2%) in EFS solution.

• 한국수정란이식학회지
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• 제15권1호
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• pp.67-75
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• 2000

This study was conducted to establish the optimal conditions for in vitro embryo production using oocytes derived from follicles of slaughter-house ovaries. The ovaries of Hanwoo were obtained from a local slaughter-house. The oocytes were aspirated from visible follicles of 2~7mm in diameter. The recovered oocytes which were completely surrounded by at least 2 layers of cumulus cells and a homogeneous cytoplasmic pigmentation were used. The selected oocytes were washed 3 or 4 times with D-PBS containing 10% bovine calf serum (BCS) and matured in vitor (IVM) in Ham's F-10 supplemented with 10% BCS or 0.01 $\mu\textrm{g}$/ml epidermal growth factor(EGF) at 39$^{\circ}C$ under 5% CO2 in air for 24 hours. They were fertilizqed in vitro (IVF) with fresh sperm separated by Percoll density gradient or swim-up in TALP media. The zygotes were cultrued with or without bovine oviductal epitherial cells(BOEC) in media(HECM-6 supplemented with 11 amino acid and / or TCM-199 supplemented with 10% BCS) for 7 to 10 days. The results obtained were as follow: The cleavage rate and developmental rate to blastocyst after maturation and IVF were not significantly different between Ham's F-10 with EGF(76.0% vs. 44.0%) and BCS(75.9% vs. 43.6%)(P<0.05). The cleavage rate and development rate to blastocyst after fertilizing by swim-up or Percoll method were not signifciantly(P<0.05) different between swim-up (80.2% vs. 29.2%) and Percoll(81.9% vs. 26.5%) (P<0.05). The cleavage rate in TCM 199(80.5) was signficiantly higher than that in HECM-6 (72.0%) (P<0.05). However, developmental rate to blastocyst using TCM 199 following HECM-6 for 3 or 4 days (42.2%) was significantly higher than that in TCM-199 alone(26.7%)(P<0.05). The cleavage rate and development rate of embryos produced in vitro by exchange timing for HECM-6 media were not significantly different between in day 3(78.6% vs. 45.5%) and day 4(75.0% vs. 43.2%)(P<0.05). The cleavage rate and developmental rate to blastocysts according to co-culture system were not significantly different between with (74.2% vs. 41.4%) and without BOEC(73.95 vs. 43.5%) (P<0.05). The number of blastomere in blastocyst stage after co-culture with or without BOEC was not significantly different (106.7$\pm$5.1 and 96.6$\pm$4.0). In conclusion, the most transferable IVP embryos could be produced from Ham's F-10 medium for IVM, Percoll density gradient method for IVF sperm separation and in vitro culture in HECM-6 until day 3 or day 4, and then transferred into TCM-199 until day 9 within adequate embryo density in culture droplets after insemination.

• Clinical and Experimental Reproductive Medicine
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• 제38권4호
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• pp.234-237
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• 2011

Objective: During stimulated IVF cycles, up to 15% of oocytes are recovered as immature. The purpose of this study was to investigate the trend of oocyte maturity in repeated ovarian stimulation for IVF. Methods: One hundred forty-eight patients were selected who underwent two consecutive IVF cycles using same stimulation protocol during 2008 to 2010. Ovarian stimulation was performed with FSH and human menopausal gonadotropin and flexible GnRH antagonist protocol in both cycles. Oocyte maturity was assessed according to presence of germinal vesicle (GV) and the first polar body. Immature oocyte was defined as GV stage or metaphase I oocyte (GV breakdown with no visible polar body) and cultured up to 48 hours. If matured, they were fertilized with ICSI. Results: Percentages of immature oocytes were 30.8% and 32.9% ($p$=0.466) and IVM rates of immature oocytes were 36.2% and 25.7% ($p$=0.077), respectively. A significant correlation was noted between percentage of immature oocytes in the two cycles (R=0.178, $p$=0.03). Women with >40% immaturity in both cycles (n=21) showed lower fertilization rate of $in$ $vivo$ matured oocytes (56.4% vs. 72.0%, $p$=0.005) and lower pregnancy rate (19.0% vs. 27.1%, $p$=0.454) after the second cycle when compared with women with <40% immaturity (n=70). In both groups, female age, number of total retrieved oocyte and embryos transferred were similar. Conclusion: In repeated ovarian stimulation cycles for IVF, the immature oocyte tended to be retrieved repetitively in consecutive IVF cycles.