• Reproductive and Developmental Biology
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• v.31 no.1
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• pp.43-48
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• 2007

This study was conducted to examine the characteristics of fresh and frozen semen, proliferating efficiency by AI, and the coat color of offsprings in Korean Native Striped Cattle (Bos namadicus Falconer, Chikso). Semen were collected from 6 heads of tiger-coated male Chikso. In vitro fertilization (IVF) was conducted with frozen-thawed semen and in vitro matured Korean native brown cattle (general Hanwoo) oocytes. Total 18 heads of Hanwoo and Chikso were inseminated using Chikso semen. Coat colors of total 40 offsprings produced by AI were evaluated. The characteristics of the fresh and frozen-thawed Chikso semen did not differ among individuals. In vitro fertilization rate of Chikso semen was not different from that of general Hanwoo semen. However, developmental rate to the blastocyst stage of IVF embryos was higher in Chikso semen (25.9%) than in general Hanwoo semen (p<0.05). There was no difference in conception rate after AI between Chikso and general Hanwoo. The coat colors of offsprings varied, only 42.5% (17/40 heads) of offsprings had tiger coat color. Futhermore, only 55% of offsprings produced from the tiger-coated recipients had tiger coat color. This result shows that proliferation of Chikso by AI is possible, but further research approaches may be needed to enhance the productivity of tiger-coated Chikso.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.142-142
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• 2003

TGF-$\beta$ super family는 난소를 포함하여 여러 기관 및 조직의 성장에 광범위하게 영향을 미치는 것으로 알려져 있으며, TGF-$\beta$ super family는 난자의 매우 초기에 영향을 미치며, 난포의 성장을 촉진하여 초기의 난자의 형태를 이루는데 중요한 역할을 하는 것으로 알려져 있다. 한편 TIMP-1은 난관상피세포로부터 분비되는 난자의 생리활성 촉진인자로 보고되고 있다. 따라서 본 연구는 한우난포란의 체외성숙에 난자의 생리활성 촉진인자를 이용하여 한우 체외성숙 및 체외수정에 미치는 영향을 구명하고자 실시하였다. 한우 난포란은 도축암소의 난소를 채취하여 회수하였으며, 체외성숙은 HP-TCM를 기본 배양액으로 TGF-$\beta$ 0.1, 1, 10 ng/ml를 첨가하여 실시하였고, TIMP-1은 0.05, 0.5, 5ng/ml를 첨가하여 6, 12, 24시간 동안 체외성숙시켰다. 체외성숙된 난자는 체외성숙율을 검사하기 위하여, 0.1% aceto-orcein으로 염색을 실시하여 핵의 변화를 검사하였다. (중략)

• 대한생식의학회:학술대회논문집
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• 2005.11a
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• pp.22-35
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• 2005

• Clinical and Experimental Reproductive Medicine
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• v.33 no.4
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• pp.219-227
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• 2006

Objective: To evaluate the impact of endometriosis on IVF-ET cycles and to compare IVF outcomes between stage I/II and stage III/IV endometriosis. Methods: We analyzed 697 patients (1,199 cycles) with endometriosis (stage I-II:638 cycles, stage III-IV: 561 cycles) and 325 pts (459 cycles) with tubal factor as controls between January 1994 and April 2004. Pts with endometriosis were diagnosed by laparoscopy and medical and surgical treatment were done in 353 cycles (55.3%) and 466 cycles (83.1%) of stage I-ll/stage III-IV endometriosis. Cycles with age>35 years or FSH>20 miU/mL or severe male factor infertility were excluded. Results: The number of retrieved oocytes ($9.97{\pm}7.2$ vs. $13.4{\pm}7.9$ (p<0.0001 )), total number of embryos ($6.5{\pm}4.8$ vs. $9.1{\pm}5.6$ (p<0.0001)), and good quality embryos ($2.43{\pm}1.6$ vs. $2.74{\pm}1.7$ (p=0.013)) significantly decreased in stage III-IV endometriosis than in control. But pregnancy rate of stage III-IV endometriosis was comparable with control (35.7% vs. 36.8%). Fertilization rate and number of total embryos were lower in stage I-II endometriosis than in control ($64.8{\pm}22.9$ vs. $70.8{\pm}20.8$ (p<0.0001), $7.6{\pm}5.0$ vs. $9.1{\pm}5.6$ (p<0.0001)). In patients with medical and surgical treatment of endometriosis, pregnancy rate and live birth rate was significantly lower in stage I-II than in stage III-IV endometriosis (29.2 vs. 36.2 (%), p=0.045, 23.9 vs. 31.5 (%), p=0.043). There was no difference in the mean age, but the duration of infertility was significantly longer ($56.5{\pm}26.3$ vs. $46.9{\pm}25.8$ (mon), p<0.0001) and fertilization rate was lower ($64.7{\pm}23.3$ vs. $70.5{\pm}22.7$ (%), p=0.001) in stage I-II than stage III-IV endometriosis. Conclusion: We suggest that IVF should be considered earlier in patients with minimal to mild endometriosis because of significantly decreased fertilization rates.

• Journal of Embryo Transfer
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• v.18 no.1
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• pp.27-33
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• 2003

The study was carried out to investigate the effects of morphology, reproductive cycle, incubation time and activation of oocytes in vitro maturation of canine oocytes and development of canine IVM/IVF embryos. The results were summarized as follows: 1. The developmental rates to 16 cells of fresh, salts and 4$^{\circ}C$-stored oocytes with and without cumulus cells were 14.3%, 5.0% and 7.5%, 2.8% and 5.7%, 0.0%, respectively. The rate of oocytes with cumulus cells(5.7%~14.3%) was higher than that of denuded oocytes(0.0%~5.0%). 2. The developmental rate to If cells of in vitro cultured oocytes recovered from ovaries collected at different stages of the reproductive cycle were 0.0%, 10.7%, 1.5%, respectively. 3. The developmental rate to 16 cells of fresh oocytes with cumulus cell cultured for 24, 32 and 48 hrs in $CO_2$ incubator were 0.0%, 5.3%, 11.8%, respectively. The rate of oocytes cultured for 48 hrs was higher than that oocytes cultured for 24 and 32 hrs. 4. The development to If cells treated activation and non-activation oocytes were 15.0%, 6.7%, respectively. The rate of oocytes treated activation was higher than that oocyte treat non-activation.

• Journal of Embryo Transfer
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• v.16 no.2
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• pp.73-78
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• 2001

This study was carried out to investigated developmental ability of IVM/IVF derived hanwoo embryos after DNA microinjection. Microinjected hanwoo embryos were cultured fur 7 days. The cleavage rates of DNA injected embrys(36.3%) was significantly lower than those of non-injected embryos(67.4%; p<0.05). The percentage of injected embryos reaching to the morulae and blastocyst was significantly lower than those of non-injected embryos(p<0.05). When injected embryo were cultured contaning L-ascorbic acid and $\alpha$-tocopherol for 168 hrs, the morulae and blastocyst rates were significantly higher than control(p<0.05). These results suggested that the addition of L-ascorbic acid and $\alpha$-tocopherol can enhanced development to the morulae and blastocyst of microinjected embryos and improved culture condition increased the transgenic hanwoo embryos.

• Journal of Embryo Transfer
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• v.13 no.3
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• pp.227-233
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• 1998

The aim of these experiments was to investigate the effects of oviduct epithelial cells on bovine in vitro fertilization. Oviduct epithelial cell monolayers (OEC) on the 4-well dish were prepared according to general procedures. Monolayers were formed within 5days. The medium for OEC culture (TCM199 with 10% FBS) was replaced with IVF-TALP 2h before each experiment. Macromolecules/proteins from oviductal conditioned medium (OM) were recovered by ultrafiltration, which desalted and concentrated macromolecules greater than 5kDa, and this OM were added to W medium (experiment 1). The cleavage rate in OM+OEC group was significantly higher than in OM group (p〈0.01). In this experiment 2, oocytes were inseminated on OEC with sperm which had been pre-incubated with OEC for 0 or 4h before insemination. In this experiment, oocytes were exposed to sperm only 8 h for clarifying the effect. After insemination, oocytes were cultured in CRlaa. At 42 h post insemination, oocytes were denuded and examined for evidence of cleavage. The cleavage rates of oocytes which were inseminated with OEC treated sperm for 4 h were significantly higher than those of the other group (p〈0.01). In conclusion, sperm released from OEC have more fertilizing ability than those before attachment.

• Clinical and Experimental Reproductive Medicine
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• v.24 no.2
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• pp.241-251
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• 1997

체외성숙과 수정된 돼지 난자의 체외발달능이 체외배발생 배양액인 NCSU 배양액에 0.4% BSA, 10% 혈청 혹은 아미노산 (2% BME 아미노산 용액과 1% MEM 아미노산 용액)을 첨가함으로서 조사되었다. 본 실험에 공시된 난자는 체외수정 추 30시간 (2-세포기)혹은 48 시간 ($2{\sim}4$-세포기)에 회수하였다. 실험I에서 0.4% BSA가 첨가된 NCSU 배양액에서 2-세포기 난자들의 배양경과시간에 따른 발달능을 조사한 결과, 배양 후 72 시간 (체외수정 후 102 시간)에 상실배기와 배반포기 배가 나타났으며, 배양 후 120 시간째 (체외수정 후 150 시간)에도 팽창된 배반포기 배까지만 발달하였다. 실험II는 체외수정 후 48 시간의 분할된 ($2{\sim}8$-세포기) 난자들의 핵과 외관적 분할구와의 수적 차이를 조사한 결과, $2{\sim}4$-세포기보다는 5-세포기 이상에서 핵과 분할구의 조화에 차이가 많았다. 실험III에서는 $2{\sim}4$-세포기 난자들을 배양후 5일째의 배반포들의 투명대의 두께, 난자 크기 그리고 inner cell mass (ICM)과 trophectoderm (TE)의 세포 배열을 조사한 결과, 난자의 크기가 커짐에 따라서 투명대가 얇아지고 전체 세포수가 증가하였지만, ICM의 비율은 차이가 없었다. 실험IV에서는 BSA, 혈청 혹은 아미노산이 첨가 혹은 무첨가된 배양액내에서 $2{\sim}4$-세포기 난자들의 배반포 후 부화능력을 조사한 결과, 모든 군에 있는 난자들은 팽창된 배반포기 배까지 발달할 수 있었던 반면, 난자의 부화는 아미노산 혹은 혈청이 포함된 배양액에서만 일어났다. 더우기 상실배기와 배반포기 시기에 혈청의 첨가는 부화 배반포기 배의 발달을 현저히 증가시켰다. 또한 아미노산과 혈청의 영향을 받은 팽창 배반포기 배는 얇은 투명대, 팽창된 난자의 크기 그리고 ICM과 전체 세포수의 증가를 보였다. 이상의 결과로 미루어 볼때, 배양액내에 대한 아미노산과 혈청의 첨가는 돼지 배반포기 배의 부화를 유도할 수 있다고 보며, 더우기 이들 요소들은 투명대의 두께, 난자의 크기 그리고 ICM과 전체 세포수에 영향을 미친다.

• Korean Journal of Animal Reproduction
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• v.18 no.3
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• pp.207-216
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• 1994

본 연구는 돼지 난포란으로부터 생산된 수정란의 체외발달에 미치는 아미노산, 우태아혈청 (FBS)과 Leukemia Inhibitry Factor의 영향을 조사하였다. 돼지난포란은 도살된 돼지의 난소로부터 회수하여 39$^{\circ}C$, 5% CO2 배양조건하에서 1$\mu\textrm{g}$/ml FSH-p, 1$\mu\textrm{g}$/ml Estradiol-17$\beta$와 10%FBS가 첨가된 TCM-199 배양액내에서 42시간동안 성숙시켰다. 사출된 정자의 수정능 획득은 45와 90% Percoll density gradient법을 통한 원심분리에 의해 얻었으며, 이들 수정능획득된 정자를 성숙된 난포란이 함유된 배양액에 3$\times$105/ml의 농도로 주입하여 10$\pm$1시간동안 배양함으로서 체외수정을 유도하였다. 수정된 난포란은 ; 1) 10% FBS가 함유된 TCM-199, DMEM, mKRB 또는 CR1aa 배양액, 2) 아미노산 또는 10% FBS가 첨가된 CR1 배양액, 3) STO 세포 또는 mLIF (1,000 unit/ml) 첨가된 CR1aa(10%FBS) 배양액, 4) mLIF (1,000 unit/ml)를 수정 직후 또는 8-세포기 이후에 첨가된 CR1aa(10%FBS)의 네가지 배양조건에서 각각 분리 배양하였다. 그결과 체외수정란의 배반포까지 발달율은 아미노산과 10%FBS가 포함된 CR1 배양액에서 다른 배양액에서보다 양호하였고, 특히 8-세포기 이후에 mouse LIF를 첨가한 CR1aa(10% FBS) 배양액에서는 다른 배양조건보다 현저히 높은 결과를 보였으며, 부화 배반포까지도 배발달을 유도할 수 있었다. 따라서 돼지수정란의 발달에 있어서 배양액내에 아미노산과 FBS 및 mouse LIF의 첨가는 효과가 있으며, 특히 8-세포기 이후에 있어서 mouse LIF의 첨가는 돼지의 수정란을 배반포 이후의 단계에까지 발달시킬 수 있었다.

• Journal of Embryo Transfer
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• v.19 no.1
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• pp.19-25
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• 2004

The objective of this study was to optimize the selection of sperm, optimal culture system of in vitro derived porcine embryos. The results obtained were summarized as follows: 1. When oocytes were inseminated with liquid sperm and frozen-thaw sperm, the cleavaged rate of liquid sperm (46.2%) was higher than that of frozen-thaw sperm (39.7%), however there were not show significant different each other. The blastocyst rates of liquid sperm (15.8%) was significantly higher than that of frozen-thawed sperm (9.3%)(P< 0.05). 2. When oocytes were inseminated with epididymal sperm after 1, 2 and 3 day storage, the cleavaged rate of epididymal sperm after 1, 2 and 3 day storage was 60.5, 61.0 and 56.8% respectively. The morulae (17.4, 19.9 or 17.3%) and blastocyst (8.7, 15.4, 11.3%) rate of epididymal sperm after 1, 2 and 3 day storage was no significantly respectively(P< 0.05). 3. In vitro developed to cleavaged rate of G1.3/G2.3 media used for culture was significantly(P< 0.05) higher as 62.1% compared with the results using the media NCSU23(52.8), however in vitro developed to blastocyst rate of NCSU23(11.6%) media was significantly(P< 0.05) higher than that'of G1.3/G2.3(4.7%). 4. When the fertilized oocytes were cultured with NCSU23 in addition to 1 mM glutathione(GSH), the cleavaged rate of treated groups of GSH(62.3%) was significantly higher than that of control(53.5%) respectively(P< 0.05). And in vitro developed to blastocyst rates of treated groups of GSH(15.6%) was higher than that of control(12.6%) however, there was no significant difference(P< 0.05).