EGF와 IGF-I의 첨가배양이 돼지 미성숙 난포란의 체외성숙과 배발달에 미치는 영향

백준종(Jun-Jong Baek),한만희(Man-Hye Han),박병권(Byung-Kwon Park),서길웅(Kil-Woog Seo),이규승(Kyu-Seung Lee) 충남대학교 농업과학연구소 2007 농업과학연구 Vol.34 No.1

본 연구는 돼지 난포란의 체외배양액과 배발달 배양액에 성장인자인 EGF와 IGF-Ⅰ을 각각 0, 1, 5 및 10 ng/㎖ 첨가배양함으로서 돼지 난포란의 체외성숙과 체외배발달에 미치는 영향을 구명하고자 실시하였다. 본 연구에서 얻어진 결과를 요약하면 다음과 같다. 1. 돼지 난포란을 체외성숙배양액인 NCSU-23 배양액에 EGF와 IGF-Ⅰ을 각각 0, 1, 5 및 10 ng/㎖ 첨가배양하여 성숙을 유기한 다음, 체외수정을 실시한 결과, 모든 처리구에서 핵성숙률, 정자침투율, 웅성전핵형성률, 다정자 침입률 및 평균침입정자수에서 유의적인 차이가 인정되지 않았다. 2. 체외수정을 실시한 후 , 배발달배양액인 NCSU-23에 7일간 배양한 결과, 배반포형성률은 EGF첨가군이 각각 11.2±1.5%, 15.0±0.8%, 16.8±2.8% 및 21.4±2.0%로서 10 ng/㎖의 첨가군이 유의적(P<0.05)으로 높은 결과를 나타냈으며, IGF-Ⅰ첨가군도 10.7±1.4%, 12.8±2.0%, 16.0±2.5% 및 21.9±2.4%로서 10 ng/㎖의 첨가군이 유의적으로 높은 결과를 나타냈다. 또한, 총세포수에 있어서도 EGF첨가군이 22.8±3.7개, 25.7±5.5개, 26.0±4.2개 및 35.1±4.7개, IGF-Ⅰ첨가군은 21.5±3.7개, 25.2±2.8개, 26.2±2.9개 및 33.2±3.6개로서 각각 10 ng/㎖ 첨가군이 유의적(P<0.05)으로 높은 결과를 나타냈다. 3. 돼지 난포란을 체외성숙 기본배양액인 함유된 NCSU-23 배양액에 44시간동안 체외성숙을 유기한 다음, 체외 수정용배양액인 mTBM에 정자와 같이 6시간동안 공배양함으로서 체외수정을 유기한 후, 배발달배양액인 NCSU-23에 EGF와 IGF-Ⅰ을 각각 0, 1, 5 및 10 ng/㎖ 첨가하여 7일간 배양한 결과, 배반포기 도달률은 EGF첨가군에서 각각 14.0±1.7개, 16.2±1.4개, 16.9±1.2개, 23.1±1.6개로서 10 ng/㎖ 첨가군이 유의적으로 높은결과를 나타냈고, IGF-Ⅰ첨가군도 각각 13.6±1.7개, 15.7±4.5개, 16.0±0.2개 및 25.0±0.8개로 10ng/㎖군이 유의적(P<0.05)으로 높은 결과를 나타냈으며, 총세포수에 있어서는 EGF첨가군이 각각 21.8±2.9개, 25.2±2.8개, 39.7±2.7개 및 46.2±3.6개로 10 ng/㎖첨가군이 유의적으로 높은 결과를 나타냈고, IGF-Ⅰ첨가군도 20.7±2.9개, 26.2±2.9개, 24.6±2.4개 및 46.1±3.5개로 10 ng/㎖ 첨가군이 유의적(P<0.05)으로 높은 결과를 나타냈다. 이상의 결과를 종합해 볼 때, EGF와 IGF-Ⅰ은 돼지 난포란의 체외성숙과 배발달과정에 영향을 미치며 특히, 10 ng/㎖의 농도에서 효과적인 것으로 조사되었다. The present study was carried out to examine the effect of EGF and IGF-Ⅰ in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU-23 maturation medium with 0, 1, 5 and 10 ng/㎖ EGF and IGF-Ⅰ (P>0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 1, 5 and 10 ng/㎖ EGF groups were 11.2±1.5%, 15.0±8.3%, 16.8±2.8% and 21.4±2.0%, also 0, 1, 5 and 10 ng/㎖ IGF-Ⅰ groups were 11.2±1.5%, 15.0±8.3%, 16.8±2.8% and 21.4±2.0%, respectively. In the total cells case, EGF groups were 22.8±3.7, 25.7±5.5, 26.0±4.2 and 35.1±4.7, also IGF-Ⅰ groups were 21.5±3.7, 25.2±2.8, 26.2±2.9 and 33.2±3.6, respectively. Both 10 ng/㎖ EGF group and 10 ng/㎖ IGF-Ⅰ group were significantly higher than those of other treatment groups (P<0.05). 3. The rates of blastocyst formation at day 7 in the NCSU23 culture medium of porcine IVF-produced embryos with 0, 1, 5, and 10 ng/㎖ EGF groups were 14.0±1.7%, 16.2±1.4%, 16.9±1.2% and 23.1±1.6%, also 0, 1, 5, 10 ng/㎖ IGF-Ⅰ groups were 13.6±1.7, 15.7±4.5, 16.0±0.2 and 25.0±0.8, respectively. And in the total cells case, EGF grups were 21.8±2.9, 25.2±2.8, 39.7±2.7 and 46.2±3.6, also IGF-Ⅰ groups were 20.7±2.9, 26.2±2.9, 24.6±2.4 and 46.1±3.5, respectively. Both 10 ng/㎖ EGF group and 10 ng/㎖ IGF-Ⅰ group were significantly higher than those of any other treatment groups (P<0.05). In conclusion, these results suggested that the addition of 10 ng/㎖ EGF and IGF-Ⅰ were effective on the blastocyst formation and total cells of blastocysts.

TGF- ${\beta}$ 첨가가 한우 난포란의 체외성숙에 미치는 영향

최선호,이혜현,연성흠,한만희,김현종,조상래,우제석,백광수,류일선,손동수,Choi, Sun-Ho,Lee, Hye-Hyun,Yeon, Seong-Heum,Han, Man-Hye,Kim, Hyun-Jong,Cho, Sang-Rae,Woo, Jae-Seok,Baek, Kwang-Soo,Ryu, Il-Sun,Son, Dong-Soo 한국발생생물학회 2004 발생과 생식 Vol.8 No.2

소 체외성숙시 난포란에 영향을 주는 과립막세포와 난자의 핵성숙을 촉진하는 물질이 확인되지 않은 혈청내의 물질 뿐만 아니라 호르몬이나 생리활성인자 등에 의해 촉진됨이 밝혀졌다. 이에 따라 체외성숙 및 체외발달에 사용되는 배양액의 조성도 복합배양액에서 단순배양액으로 전환을 시도하고 있으며, 체내의 조건에 보다 더 접근하고자 하는 시도들이 수행되고있다. 본 연구는 한우 난포란의 체외성숙율 및 체외수정 후 배발달율을 향상시키기 위하여 TGF-${\beta}$의 첨가가 미치는 영향에 대하여 조사하였다. 한우 난포란의 체외성숙시 TGF-${\beta}$ 0.1, 1. 10ng/ml를 첨가하였을 때 24시간에 MetaphaseⅡ 도달율은 95.8%, 95.8%, 100%를 나타내었으며, 농도간의 유의적인 차이는 없었다. TGF-${\beta}$로 체외성숙된 난포란을 체외수정 후 TGF-${\beta}$가 체외성숙 배양액을 동일하게 체외발달을 유도하였으나, 0~0.8%의 배반포율을 보였으며, TGF-${\beta}$로 체외성숙된 난포란을 체외수정 후 TCM 199_10% FBS, 0.8% BSA, 0.1% PVA에 배양한 결과 12.4%, 12.8%, 8.5%의 배반포 발달율을 보였고, 무혈청 배양액인 IVMD, IVD에 배양한 결과 38.4%, 34.8%의 배반포 발달율을 보였으며, 유의적인 차이를 나타냈다(P<0.005). 결론적으로 TGF-${\beta}$는 한우 난포란의 체외성숙시 상당히 유용한 물질이나, 체외발달에는 만족할 수 없어, 이외의 생리활성 인자들간의 상호관계에 대하여 더 많은 요구가 요구된다. It is well known that unidentified factors in sera, hormones and growth factors promote the proliferation of granulosa cells and nuclear maturation of bovine COCs in vitro. Attemps had been developed the simple composition of culture media and similar system to in vivo conditions has been applied. In the present study, we investigated the effect of TGF-${\beta}$ on in vitro maturation and in vitro development of Hanwoo COCs. When the COCs were matured in TCM 199 containing 0.1, 1 or 10 ng/ml TGF-${\beta}$ for 24 hrs, metaphaseⅡ of COCs were obtained 95.8%, 100% of matured COCs, respectively and there were no differences among the concentrations of TGF-${\beta}$. Matured COCs with TGF-${\beta}$ cultured in maturation medium after in vitro fertilization, developmental rate to blastocyst were 0~0.8%. Matured COCs with TGF-${\beta}$ were cultured in TCM 199+10% FBS, 0.8% BSA, 0.1% PVA, blastocyst formation were showed in 12.4%, 12.8%, 8.5% of those and cultured in IVMD or IVD without serum were 38.4%, 34.8%, respectively. There were significant differences among the media (P<0.05). TGF-${\beta}$ is available for i vitro maturation of bovine COCs, but further investigation would be need for finding the synergistic autocrine/paracrine fashion of other growth factors in early bovine development.

토주실험에서 동물용 의약품의 이동 특성

황선영 ( Sun Young Hwang ),한만희 ( Man Hye Han ),조재영 ( Jae Young Cho ) 한국응용생명화학회(구 한국농화학회) 2012 Journal of Applied Biological Chemistry (J. Appl. Vol.55 No.4

Veterinary antibiotics can enter the soil ecosystem and then may be transported into groundwater via leaching process. The main aim of this study is to investigate the distribution and mobility of tetracycline, amoxicillin and sulfathiazole in soil. The adsorption of veterinary antibiotics were applied to the Freundlich adsorption isotherms. Adsorption coefficient (KF) was indicated oxytetracycline > amoxicillin > sulfathiazole. Oxytetracycline concentration was highly detected in soil than in leachate. It is assumed that oxytetracycline was strongly absorbed by divalent cations such as Ca2+ in soil. However, amoxicillin and sulfathiazole were shown higher mobility due to the lower distribution coefficient.

한우에서 중복임신 사례

손동수 ( Dong Soo Son ),김종대 ( Chong Dae Kim ),조창연 ( Chang Yeon Cho ),진현주 ( Hyun Ju Jin ),연성흠 ( Seong Heum Yeon ),최창용 ( Chang Yong Choe ),최선호 ( Sun Ho Choi ),김현종 ( Hyun Jong Kim ),한만희 ( Man Hye Han ),김영근 한국임상수의학회 2006 한국임상수의학회지 Vol.23 No.2

EFG와 IGF-I의 첨가배양이 돼지 미성숙 난포란의 체외성숙과 배발달에 미치는 영향

백준종,한만희,박병권,서길응,이규승 충남대학교 농업과학연구소 2007 농업과학연구 Vol.34 No.1

The present study was carried out to examine the effect of EGF and IGF-I in vitro maturation (IVM) of porcine oocytes and development of porcine IVM/IVF embryos. The results were summarized as follows : 1. The rates of nuclear maturation, penetrated oocytes, pronuclear formation, polyspermic oocytes and mean numbers of the penetrated sperm were not different in NCSU-23 maturation medium with 0, 1. 5 and 10 ng/ml EGF and IGF-I (P>0.05). 2. The rates of blastocyst formation at day 7 after in vitro fertilization in 0, 1, 5 and 10 ng/ml EGF groups were 11.2±1.5%, 15.0±8.3%, 16.8±2.8% and 21.4±2.0%, also 0, 1, 5 and 10 ng/ml IGF- I groups were 11.2±1.5%, 15.0±8.3%. 16.8±2.8% and 21.4±2.0%, respectively. In the total cells case, EGF groups were 22.8±3.7, 25.7±5.5, 26.0±4.2 and 35.1±4.7, also IGF-I groups were 21.5±3.7, 25.2±2.8, 26.2±2.9 and 33.2±3.6, respectively. Both 10 ng/ml EGF group and 10 ng/ml IGF-I group were significantly higher than those of other treatment groups (P<0.05). 3. The rates of blastocyst formation at day 7 in the NCSU23 culture medium of porcine IVF-produced embryos with 0, 1, 5, and 10 ng/ml EGF groups were 14.0±1.7%, 16.2±1.4%, 16.9±1.2% and 23.1±1.6%, also 0, 1, 5, 10 ng/ml IGF-I groups were 13.6±1.7, 15.7±4.5. 16.0±0.2 and 25.0±0.8, respectively. And in the total cells case, EGF grups were 21.8±2.9, 25.2+2.8, 39.7±2.7 and 46.2±3.6, also IGF-I groups were 20.7±2.9, 26.2±2.9, 24.6±2.4 and 46.1±3.5, respectively. Both 10 ng/ml EGF group and 10 ng/ml IGF-I group were significantly higher than those of any other treatment groups (P<0.05). In conclusion, these results suggested that the addition of 10 ng/ml EGF and IGF-I were effective on the blastocyst formation and total cells of blastocysts.

6-Dimethylaminopurine 및 Cycloheximide의 처리가 돼지난포란의 단위발생에 미치는 영향

김종화,박병권,한만희,이규승 충남대학교 농업과학연구소 2007 농업과학연구 Vol.34 No.1

This study is aimed at finding an optimum density for 6-Dimethylaminopurine (6-DMAP) and cycloheximide which have an effect on the revitalization of porcine oocytes. The results were summarized as follows: 1. When 6-DMAP was treated with 2 mM for 2 hours. It showed a significantly (P<0.05) different high result in activation rate, cleavage rate and blastocyst growth rate of 51.2%, 52.7% and 25.2% respectively. 2. When Cycloheximide was treated with 5 ug/ml for 6 hours, It showed a significantly (P <0.05) different high result in activation rate, cleavage rate and blastocyst growth rate of 47.7%, 46.8%, and 27.3% respectively. 3. When it was cultured in the culture medium, NCSU, for 7 days after inducing activation with 6-DMAP and cycloheximide, it showed no differences in the number of inner cell mass (ICM) and total cell of blastocysts. To conclude, it has been examined for porcine oocytes to be suitable when 6-DMAP was treated with 2 mM for 2 hours, Cycloheximide with 5 ug/ml for 6 hours.