• 대한생식의학회:학술대회논문집
• /
• 대한불임학회 2003년도 제45차 추계학술대회
• /
• pp.101-102
• /
• 2003

• 한국초지조사료학회지
• /
• 제38권4호
• /
• pp.320-324
• /
• 2018

본 연구는 티모시 건초와 농후 사료 위주의 사료를 급여한 한우 씨수소 정소상체 정자 체외수정 효율 조사를 통해 정자의 활용 가능성을 조사하였다. 농후 사료는 체중의 1.8%를 급여하고 양질의 티모시 건초를 자유채식 시킨 14개월령 거세우의 정소에서 분리된 정소상체 미부의 정자를 회수하고 동결 흉해 후 체외수정을 실시한 결과는 다음과 같다. 웅성전핵과 자성전핵이 형성(2PN)된 난자는 정상수정으로, 1개의 전핵(1PN), Expanded Sperm Head (ESH), Polyspermy 형태는 비정상적인 수정의 형태로 평가하였다. 정상적으로 수정된 난자의 비율은 정소상체 정자의 경우 전체 침투율은 49.7% 그리고 정상적인 2PN을 가진 난자는 18.5%를 보였으며, 대조구 정자의 전체 침투율은 54.4%로서 정소상체 정자 보다 높은 결과를 보였으나 유의적인 차이를 보이지는 않았다. 정상적으로 2PN을 형성한 비율은 36.7%로서 정소상체 정자를 이용한 정자 보다 높았으나 유의적인 차이는 없었다. 체외수정 후 발달률 조사에서 정소상체 정자의 분할률은 81.2%, 대조구 정자는 82.7%로 유사한 결과를 보였으나, 배반포 발달률은 정소상체 정자 24.4%와 대조구 정자 12.2%로 정소상체 정자를 사용한 난자의 발달에서는 유의적으로 높았다(p<0.05).

• 한국수정란이식학회지
• /
• 제10권2호
• /
• pp.131-138
• /
• 1995

Essential and non-essential amino acids supplemented to culture medium stimulate mammalian embryo development in vitro. Amino acids such as glycine, taurine and alanine are concentrated in the lumen of oviduct and uterus and it can he thought that these amino acids may have physiological role on fertilization and embryo development. Our aim of this experiment was to investigate the effects of essential and non-essential amino acids, taurine or glycine supplemented to fertilization medium on the cleavage and subsequent in vitro development of bovine oocytes matured and fertilized in vitro. Immature oocytes were obtained from slaughtered Holstein cows and heifers and matured in TCM199 containing 10% fetal calf serum, 2.5 $\mu$g /mL of FSH and LH and 1 $\mu$g / mL of estradiol with granulosa cells in vitro. After maturation, oocytes were coincubated with sperm in fertilization medium supplemented with Minimum Essential Medium (MEM) essential and non-essential amino acids, taurine (3.75 mM) or glycine (10 mM) for 30 hours in vitro. Inseminated oocytes were cultured in synthetic oviduct fluid medium (SOEM) containing MEM essential, non-essential amino acids and 1 mM glutarnine up to 8 days after fertilization.Supplementation of fertilization medium with MEM essential and non-essential amino acids lowered significantly (p<0.05 and p<0.001) the cleavage rate after 30 hours of IVF (53.3%) and at Day 3 (62.7%: Day 0: the day of I VF) compared to control (64.3% and 77.3%, respectively). Subsequent developmental rates to morulae (Mo) and expanding blastocysts (ExBL) also significantly decreased (p<0.001 and p<0.05 for Mo and ExBL) when oocytes were coincubated with sperm in the medium containing MEM amino acids. Taurine added to fertilization medium have not increased the cleavage rate over the control, whereas glycine showed significantly lower (p<0.01) cleavage rate at Day 3 than that of taurine, but there was no significant difference in the developmental rates to Mo and ExBL of bovine embryos irrespective of the supplementation of taurine or glycine to fertilization medium. In conclusion, supplementation of fertilization medium with essential and non-essential amino acids, taurine or glycine has no beneficial effect on in vitro cleavage and development of bovine oocytes matured and fertilization in vitro.

• Clinical and Experimental Reproductive Medicine
• /
• 제26권3호
• /
• pp.355-362
• /
• 1999

Objective: This study was performed to compare the clinical response to controlled ovarian hyperstimulation (COH) of in vitro fertilization and embryo transfer (IVF-ET) according to the size of baseline ovarian cyst. Method: From February 1992 to March 1999, a retrospective analysis was done of 272 cases who underwent COH using mid-luteal phase long protocol of gonadotropin-releasing hormone agonist (GnRH-a) for IVF-ET. These cases were divided into four group; group 1 (n=63) had cysts with mean diameters between 20.0 and 29.0 mm on their baseline ultrasound on cycle day 3, group 2 (n=57, $30.0{\sim}49.0mm$), group 3 (n=68, >50.0 mm) and control group (n=84). Cases were excluded according to the following criteria; pure male factor infertility, the presence of only one ovary, high CA-125 level and previous endometriosis. Results: There were no statistically significant differences between cases with baseline ovarian cyst <50.0 mm in diameter and control group in any of the parameters. However, cases with baseline ovarian cyst>50.0 mm in mean diameter needed more amount of human menopausal gonadotropin (hMG), showed significantly lower estradiol ($E_2$) level, the number of follicle >15.0 mm on the day of human chorionic gonadotropin (hCG) administration, the number of oocytes retrieved, the number of mature oocytes, and pregnancy rate compared with control group. Conclusion: This study suggests that cases with baseline ovarian cyst <50.0 mm in diameter do not adversely impact on IVF-ET outcome. However, cases with baseline ovarian cyst >50.0 mm in diameter had adverse effects on various parameters. Therefore, to improve the outcome of IVF-ET in these cases, ovarian cyst aspiration prior to initiating COH may be required.

• Clinical and Experimental Reproductive Medicine
• /
• 제24권3호
• /
• pp.361-368
• /
• 1997

A total of 55 patients with various etiologies of infertility particitated in a study comparing two regimens of controlled ovarian hyperstimulation (COH) with GnRH agonists and gonadotropins. Nineteen patients were given an ultra-short stimulation protocol when the agonist was administered for 3 day from Day 2 of the cycle. The remaining 36 patients were given a long stimulation protocol when the agonist was administered from the mid-luteal phase of the cycle preceding the stimulation cycle. The mean number of gonadotropins used per patient was not different between two groups. No significant differences were found in the mean number of oocytes recovered, fertilization rate and embryo cleavage rate between two groups. Pregnancy and delivery rates were higher in ultra-short protocol than in long protocol, but these were not significant. These results suggest that an ultra-short protocol is as effective as a long protocol in in-vitro fertilization and embryo transfer.

• 한국수정란이식학회지
• /
• 제24권3호
• /
• pp.145-150
• /
• 2009

This study was conducted to examine the effects of human follicular fluid and gonadotropin (FSH+HCG+rhEGF) on in vitro maturation, fertilization and development of human immature oocytes. Cumulus-oocyte complexes (COCs) were collected following for in vitro fertilization and embryo transfer (IVF-ET) cycles of the patients. At the time of oocytes collection, oocytes were classified into MII, MI and GV in accordance with their appearance (MII: Fully mature oocyte at metaphase II of meiosis; MI: Nearly mature oocytes at metaphase I of meiosis; GV: Immature oocytes at prophase I of meiosis). After controlled ovarian stimulation using gonadotropin(FSH) and human chorionic gonadotropin (HCG) in 70 ICSI cycles, 158 MI to MII matured oocytes were intracytoplasmic sperm injection (ICSI) ${\sim}4$ h after in vitro culture and 553 MII oocytes were ICSI after denudation. The aspirated MI and GV oocytes were cultured in culture medium containing 10% (v/v) serum protein substitute (SPS), 10% (v/v) human follicular fluid (hFF) and 10% (v/v) serum protein substitute (SPS)+1 IU/ml FSH+10 IU/ml HCG+10 ng/ml recombinant human epidermal growth factor (rhEGF). The maturation rate of immature oocytes was similar among the three group. When maturation medium was supplemented with 10% SPS, 10% hFF or gonadotropins, the fertilization rate of in vitro matured oocytes was higher in 10% SPS (80.0%), but there was no statistical significance (78.2%; hFF, 76.9%; gonadotropin, p>0.05). The development rate of human embryos developed to $6{\sim}8$ cells were not significant difference in the medium containing SPS, hFF and gonadotropins (65.6%, 65.9% and 66.7%). The results of these study suggest that human follicular fluid and gonadotropins supplemented in the culture medium was not effected on the in vitro maturation, fertilization and development of human immature oocytes.

• 한국동물번식학회:학술대회논문집
• /
• 한국동물번식학회 2003년도 학술발표대회 발표논문초록집
• /
• pp.57-57
• /
• 2003

수정란의 체외생산(IVP)기술은 동물생산기술의 적용과 생리학이나 세포생물학의 기본 연구에 새로운 biotchnologies의 발생에 있어 매우 중요하고 흥미로운 사건으로서 여러 종류의 포유류에 적용되고 있다. 이러한 기술은 3가지의 절차를 밟아야 하는데 In vitro maturation(IVM), In vitro fertilization(IVF) 및 In vitro development(culture)(IVD)가 그것이다. 그런데 돼지는 다태동물로서 난소의 난포가 성장할 때 난포간 meiotic competence가 난자에 의하여 진행되므로 난포내의 난포액에 의하여 난자의 발육이 좌우된다고 보고 있다 돼지WP에 사용되는 난자는 난포의 직경이 3~5mm에서 수집하고 cumulus-oocyte complexes(COCs)의 형태에 따라 선택하는 것이 보편화되어 있다. 실험목적은 돼지 난소의 antral follicles이 성장할 때 크기에 의하여 oocyte meiotic competence의 방출에 어떤 영향을 주는지를 난자의 체외성숙과 체외수정 및 체외발육 비율을 각각 조사하여 미성숙 난자의 체외배양 기술을 개선할 목적이었다. 수행내용은 도축돈 난소의 난포크기별 3mm<, 3~5mm, 5mm)로 나누어, 다시 말해서 preantral stage, antral stage, postantral stage으로 구분하여 채취한 COCs를 IVM, IVF, IVC 상태에서의 COCs 형태, cell cleaved rate, developmental rate 등을 조사하였다.

• Asian-Australasian Journal of Animal Sciences
• /
• 제14권8호
• /
• pp.1188-1195
• /
• 2001

A tetraparental chimeric bull was successfully produced by aggregating bovine IVF embryos of F1 (female Holstein${\times}$male Japanese Black) and F1(female Japanese Brown${\times}$male Limousin) and culturing in vitro without the zona pellucida at Yamaguchi Research Station in Japan. In the microsatellite genotyping, 12% (28/228) microsatellite primer sets ware potentially useful for this parentage analysis in the chimeric bull, 78.6% (22/28) of microsatellite present in the chimeric bull were uniquely contributed from the Japanese Black and 21.4% (6/28) from Limousin. This chimeric bull semen was used in producing IVF embryos. The chromosome preparations were made from peripheral lymphocytes. Based on chromosome analysis the Chimera had apparently normal chromosomes (29 acrocentric pairs, one large sub metacentric X chromosome and one small sub metacentric Y chromosome). The proportion of acrosome reacted spermatozoa after 1 h of incubation was higher (p<0.01) with the Chimera than with the Holstein and in Japanese Brown bulls. But did not differ from Japanese Black and Limousin bull sperm. Fertilization rates observed after 5 h of sperm-oocyte incubation with Chimera sperm were higher (p<0.05) than with Japanese Brown and (p<0.01) than with Holstein sperm, but did not differ from Japanese Black and Limousin sperm. The cleavage rates of IVF oocytes inseminated with Chimera sperm were also higher (p<0.001) compared with Holstein, (p<0.01) Japanese Brown and (p<0.05) Limousin, but did not differ from Japanese Black sperm. The blastocyst rates of IVM oocytes inseminated with sperm were higher (p<0.05) than in Limousin, Japanese Brown and Holstein, but did not differ from Japanese Black. Chimeric cattles were produced by aggregation of parthenogenetic (Japanese Brown) and in vitro fertilized (Holstein) bovine embryos at the Yamaguchi Research Station in Japan and by aggregation of parthenogenetic (Red Angus) and in vitro fertilized (Holstein) embryos at the St. Gabriel Research Station in Louisiana. The aggregation rate of the reconstructed demi-embryos cultured in vitro without agar embedding was significantly lower than with agar embedding. The aggregation was also lower when the aggregation resulted from a whole parthenogenetic and IVF-derieved embryos cultured without agar than when cultured with agar. The development rate to blastocysts, however, was not different among the treatment. To verify parthenogenetic and the cells derieved from the male IVF embryos in blastocyst formation, 51 embryos were karyotyped, resulting in 27 embryos having both XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zonafree chimeric embryos at 24 h following cryopreservation in EG plus T with 10% PVP were significantly greater than those cryopreserved without PVP. Pregnancies were diagnosed in both stations after the transfer of chimeric blastocysts. Twin male and single chimeric calves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes detected. Three pregnancies resulted from the transfer of 40 chimeric embryos at the Louisiana station. Two pregnancies were Jost prior to 4 months and one phenotypically chimeric viable male born.

• Reproductive and Developmental Biology
• /
• 제31권1호
• /
• pp.49-53
• /
• 2007

Astaxanthin is a kind of carotenoid compounds, having a antioxidant and anti-inflammatory activities. The antioxidative mechanism by which carotenoid scavenge free radicals has been clearly elucidated, but has not tried for the development of mammalian preimplantation embryo. This study was conducted to investigate the antioxidative effect of astaxanthin on in vitro development of porcine in vitro fertilized embryos. Porcine embryos derrived from in vitro fertilization (IVF) were cultured in 5% $CO_{2} in air at $38.5^{\circ}C$ in PZM-3 medium supplemented with different dosages of astaxanthin ($0,\;1,\;5\;and\;10{\mu}M$) and taurine (0, 1, 2.5 and 5 mM) as a positive control, and execute to compare the effects of various antioxidants such as taurine, melatonin and asculatin on in vitro development. The proportions of embryos developed to the blastocyst stage were increased when $1\;and\;5\;{\mu}M$ of astaxanthin (26.6 and 23.4%, respectively) and 1 and 2.5 mM taurine (25.8 and 26.4%, Respectively) were supplemented, compared to controls (p<0.05). Also, various antioxidant-treated groups were significantly higher rates of blastocysts (astaxanthin, 27.4%; taurine, 29.1%; melatonin, 26.8%; aesculetin, 27.9%, respectively) than control (18.8%). There was no difference in mean cell number of blastocysts between antioxidants and control. This result indicates that astaxanthin has an antioxidant feature when porcine IVF embryos were cultured in vitro.

• 한국수정란이식학회지
• /
• 제19권3호
• /
• pp.245-255
• /
• 2004

포유동물 난자의 체외수정은 외래유전자 도입에 의한 형질전환동물 생산과 우수한 형질을 가진 개체의 보존, 인간의 불임연구 등과 같은 수정란이식 기술로서 널리 이용되고 있다. 돼지 난포란을 이용한 체외수정란의 생산은 초기단계인 체외성숙 기술의 미확립, 그로인한 체외수정 시 높은 다정자 침입율과 불완전한 웅성전핵 형성 몇 체외발달능 정지현상(cell blocking) 등 어려움 때문에 아직도 다른 가축보다 양질의 수정란을 생산하기가 어려운 것으로 알려져 있다. 이와 같은 것을 해결하기 위하여 많은 연구자들은 배양액내에 hormon, growth factor, antioxdants 등과 같은 외인성 인자들을 첨가하고 있다. 이들 인자 중 antioxdant는 free radical을 소거하고 과산화물 생성을 억제하여 난자를 산화적 스트레스로부터 보호한다. 따라서 본 연구는 돼지 난포란으로부터 체외수정란의 생산에 있어서 배양액내 cysteine, catalase 및 glutathione의 첨가가 체외성숙, 체외수정 및 체외배양에 어떤 영향을 미치는지를 검토하였다. 실험 1은 체외성숙용 배양액인 TCM-199 용액에 catalase(100, 200, 500U/$m\ell$)와 glutathione(0.5, 1.0, 1.5mM/$m\ell$)의 첨가, 실험 2는 성숙된 난자의 체외수정용 배양액인 mTBM 용액에 cysteine(0.1, 1.6, 1.0mM/$m\ell$), catalase(100, 200, 500U/$m\ell$)와 glutathione(0.5, 1.0, 1.5mM/$m\ell$)의 첨가, 실험 3은 체외성숙 및 체외수정된 난자의 체외배양용 배양액인 NCSU-23 용액에 cysteine(0.1, 1.6, 1.0mM/$m\ell$), catalase(100, 200, 500U/$m\ell$)와 glutathione(0.5, 1.0, 1.5mM/$m\ell$)을 첨가하여 배반포로의 배 발달율을 관찰하였던 결과는 다음과 같다. 1. 체외성숙시 catalase의 경우는 500U 첨가군의 27.2%로 무첨가군의 15.4%보다 유의하게 높았다(p<0.05). 한편 glutathione의 경우 배반 포로의 배 발달율은 무첨가군과의 차이는 없었다. 그러나 1.0mM 첨가군에서 상실배까지의 배 발달율인 72%는 무첨가군의 53.9%보다 유의하게 높았다(p<0.05). 2. 체외수정시 여러 종류의 항산화제 첨가는 첨가하는 농도와 관계없이 배반포로의 배 발달율은 무첨가군과 차이가 없었다. 3. 체외배양시 여러 종류의 항산화제 첨가는 첨가하는 농도와 관계없이 배반포로의 배 발달율은 무첨가군과 차이가 없었다. 이상의 결과를 종합적으로 하면 돼지 난포란을 이용한 배반포의 체외생산에 있어서 배양액내 항산화제의 첨가는 체외성숙단계에서만 효과적이었다. 이것은 아마도 항산화제가 체외성숙 시 난포란 내에서 일어나는 여러 가지 생화학 반응의 처리시간과 관련하여 활성화시킴으로써 난포란의 생존력을 높인 것이라고 사료되기 때문에 앞으로는 돼지 난포란의 효율적인 체외성숙에 대해서 배양액내 첨가물질은 물론 나아가서 방법론적인 측면에서 더욱 연구되어져야 할 것이다.