한우 수정란의 채란과 젖소 이식 및 생식기 실습

박흠대,김종환,정덕수,김주현 한국수정란이식학회 1998 한국수정란이식학회 학술대회 Vol.1998 No.1

체내 수정된 Mouse 1-세포배의 체외발생에 미치는 혈관내피세포주(tHUE-2세포) 배양액의 영향

박흠대,이훈택,정길생,三井洋同 한국동물생명공학회(구 한국동물번식학회) 1995 Reproductive & developmental biology Vol.19 No.2

학술발표회 : 번식 ( 구두발표 ) ; mouse 난자의 체외발생에 미치는 혈관내피세포주배양액의 영향

박흠대,삼정양사 한국동물자원과학회 ( 구 한국축산학회 ) 1995 한국축산학회 심포지움자료집 Vol.- No.-

家兎卵子의 體外受精과 體外培養

朴欽大,李景廣,鄭吉生 한국동물생명공학회(구 한국동물번식학회) 1980 Reproductive & developmental biology Vol.4 No.1

Vitamin K1의 첨가가 돼지 체외 수정란의 발달과 생존율에 미치는 효과

박흠대,주역진,박용수 韓國受精卵移植學會 2014 한국동물생명공학회지 Vol.29 No.1

The in vitro production of porcine embryos was essential to increase of blastocyst development rate and select of high quality blastocyst in early stage. There were a lot of reports about in vitro porcine embryo development, but there was no report about the selection of high quality embryos. Therefore, in this study, we investigated the effect of vitamin K1 (vit K1) on the development and survival rate of porcine in vitro fertilized embryos. When vit K1 was treated for 24 hr at day 1 in vitro culture, blastocyst development rate in the control group (35.5 ± 3.2%) was significantly lower compared to 1.0 μM, 3.0 μM, or 6.0 μM groups (14.5 ± 4.3, 0.0, or 0.0%; p<0.05). The survival rates of blastocysts at day 8 in 1.0 μM, 3.0 μM or 6.0 μM of vit K1 treated groups (22.2 ± 2.9, 0.0 or 0.0%) were significantly lower than that of the control group (31.8 ± 2.6%; p<0.05). We were added at 1.0 μM, 3.0 μM or 6.0 μM vit K1 for different durations of time at day 1 in vitro culture. The development rate and survival rate in the group of 1.0 μM vit K1 for 6 hr was 26.5 ± 2.9% and 47.2 ± 2.8%, respectively, which were differed significantly in the group of 12 hr (p<0.05). In the group of 3.0 μM vit K1, the blastocyst development in control group was 36.4 ± 3.1% but, the survival rate 41.7 ± 3.2% in the group of 3.0 hr was significantly higher than that of the control group (p<0.05). In the group of 6.0 μM vit K1, the control group’s the blastocyst development was 32.0 ± 2.8% and the 0.5 hr supplement group’s survival rates was 42.9 ± 1.8% higher than other groups. We added vit K1 at day 1, day 2, day 4 and day 6 of in vitro culture, on the based the results of supplemented concentration and duration. In the group of 1.0 μM 6.0 hr addition, the blastocyst development rate of day 4 and the survival rate of day 2 were the highest in each group. In the groups of 3.0 μM 3.0 hr addition or 6.0 μM 0.5 hr addition, the blastocyst development (59.5 ± 4.1% and 50.0 ± 3.6%) and survival rates (72.7 ± 5.4% and 79.2 ± 4.0%) on day 4 were significantly higher than that of control and other experiment groups (p<0.05). Meanwhile, the number of cells in blastocysts that produced by vit K1 supplementation was 53.4 ± 5.8, 49.4 ± 3.8 and 51.5 ± 4.5 respectively, which were significantly higher than that of 40.2 ± 2.3 in the control group (p<0.05). There was no difference of the number of apoptotic cells between control and experiment groups. In addition, gene expression of survival blastocyst, the Bax mRNA expression was similar between the control and the experiment groups. However, Bcl-xL mRNA expression’s in the group of 6.0 μM 0.5 hr on day 4 was highest among control and experiment groups (p<0.05). In this study suggested that the control of concentration, duration and time was effective on the survival and cell number of porcine blastocyst derived from in vitro. We are not know what the exact reasons of the effect of vit K1 on embryo development and need to fur ther study. However, vit K1 might be using the selection of high quality porcine blastocyst. 돼지 수정란의 체외 생산 효율성 향상을 위해서는 배발생율과 더불어 고품질의 배를 조기에 선별해야 한다. 체외 배 발생율에 대한 보고는 많지만, 고품질의 배를 선별할 수 있는 기술에 대한 연구는 거의 없었다. 본 연구에서는 돼지 난포란 유래 수정란의 체외배양에 있어서 배반포로의 배 발달과 생존에 미치는 Vitamin K1(vit K1) 첨가 농도, 시기 및 시간의 효과를 검토하였다. 1.0 μM, 3.0 μM 및 6.0 μM vit K1을 배양 1일째 24시간 첨가한 결과, 배반포 발달율이 시험군이 14.5 ± 4.3, 0.0 및 0.0%로써 대조군의 35.5 ± 3.2%에 비하여 유의하게 낮았고(p<0.05), 배반포의 생존율도 대조군이 31.8 ± 2.6%로써 시험군의 22.2 ± 2.9, 0.0 및 0.0%에 비하여 유의하게 높았다(p<0.05). 상기 첨가 농도에서 첨가 시간을 달리한 결과, 1.0 μM 농도에서 6시간 처리군의 배반포 발달율과 생존율이 각각 26.5 ± 2.9% 및 47.2 ± 2.8%로써 가장 높았고 특히, 12시간 처리군보다 유의하게 높았다(p<0.05). 3.0 μM 농도에서는 대조군의 배발달율이 36.4 ± 3.1%로 가장 높았으나, 생존율은 3.0시간 첨가군이 41.7 ± 3.2%로 대조군에 비하여 유의하게 높았다(p<0.05). 6.0 μM 농도에서도 배발달율은 대조군(32.0 ± 2.8%), 생존율은 0.5시간 첨가군(42.9 ± 1.8%)이 가장 높았다. 각각의 vit K1 첨가 농도와 시간을 기준으로 서로 다른 배양 시기에 첨가한 결과, 1.0 μM 6시간 첨가군에서는 배반포 발달율은 배양 4일째 첨가군, 생존율은 배양 2일째 첨가군이 가장 높았다. 한편, 3.0 μM 3.0시간 및 6.0 μM 0.5시간 첨가군에서는 배양 4일째 첨가군의 배반포 발달율(59.5 ± 4.1% 및 50.0 ± 3.6%)과 생존율(72.7 ± 5.4% 및 79.2 ± 4.0%)이 대조군과 다른 시험군에 비하여 유의하게 높았다(p<0.05). 한편, vit K1 첨가에 따른 배반포의 세포 수를 조사한 결과, 첨가군(1.0 μM 6시간 배양 2일째, 3.0 μM 3.0시간 배양 4일째 및 6.0 μM 0.5시간 배양 6일째)이 53.4 ± 5.8, 49.4 ± 3.8 및 51.5 ± 4.5개로써 대조군의 40.2 ± 2.3개에 비하여 유의하게 많았다(p<0.05). 그러나 사멸세포 수는 시험군이 3.2 ± 0.9∼3.7 ± 2.1개로써 대조군의 4.2 ± 1.2개보다 적었으나, 유의차는 없었다. 세포 사멸 유도 유전자인 Bax mRNA 발현은 처리군과 대조군은 비슷하였으나, 세포 사멸 억제 유전자인 Bcl-xL mRNA 발현은 처리군이 대조군보다 높았고 특히, 6.0 μM 0.5시간 배양 4일째 첨가군이 가장 높았다. 이상의 결과로부터 돼지 미성숙 난포란 유래 수정란의 체외 배양에 vit K1의 첨가는 배반포의 생존율과 세포수 증가에 효과적이었다. 그 이유에 대해서는 아직 많은 부분이 밝혀져야 되겠지만, 고품질의 배반포 조기 선발에는 활용이 가능할 것으로 생각된다.

체외성숙 및 수정 시간이 한우 수정란 생산에 미치는 영향

박흠대,박용수 대구대학교 생명과학연구소 2003 생명과학연구 Vol.2 No.1

The present study was performed to investigate the effect of in-vitro maturation and in-vitro fertilization time in Korean Native Cow(KNC) oocytes on in-vitro development and to examine whether the duration of fertilization time of matured oocytes affects the blastocyst(BL) formation. There was no significant difference in cleavage and 8-cell stage rates among the treatment groups, but BL and BL/8-cell rates were significantly higher(P<0.005) in 18hr maturation(IVM18) group(31.0±5.7 and 82.0±5.1%) than 22 and 24hr maturation(IVM24) group. As the time of in-vitro fertilization time increased, the fertility rates was increased but the BL and BL/8cell rates were decreased. The proportion of BL formed on day 7 and 8 was significantly higher(P<0.05) in IVM18:IVF16 group(100%) than in IVM24:IVF24 group(55%). These results suggest that effective in-vitro production of KNC oocytes need to proper IVM and IVF times, and two conditions must be considered simultaneously.

체외에서 생산된 한우 배반포의 젖소 임신에 관한 연구

박흠대,윤상현,김종환,김은조,박경운,정덕수,이동칠,김주현 한국수정란이식학회 1997 한국수정란이식학회 학술대회 Vol.1997 No.2

본 연구는 체외에서 생산된 도살장 유래의 한우 배반포를 젖소의 자궁에 이식하여 임신율을 상승시킴으로써 농가의 수정란 이식의 이해 및 그 산업화 촉진의 계기를 마련하고자 실시하였다. 그 결과 배양기간을 단축시키면서도 한꺼번에 많은 양의 배반포를 생산할 수 있는 배양액의 개발이 시급하며, 수정란 이식의 산업화를 위해서는 질 좋은 배반포 생산은 물론이거니와 수란우의 선정을 위한 신기술의 개발 및 숙련된 수정란 이식사의 양성은 대단히 중요하다고 생각한다.

한우 난포란의 체외성숙 시 여러 가지 첨가물이 배 발생과 품질에 미치는 영향

박흠대,장미진,박용수 한국동물생명공학회(구 한국동물번식학회) 2006 Reproductive & developmental biology Vol.30 No.1

본 연구는 체외성숙 배지에 첨가하는 PVP의 농도, EGF, cysteine 및 PVP의 단독 또는 혼합첨가가 한우 체외수정란의 체외발생에 미치는 영향을 검토하였다. 체외성숙 배지에 PVP의 첨가농도(0.1~3.0%)에 따른 분할율은 차이가 없었으나, 배반포 발달율은 0.5% PVP 첨가군이 가장 높았다(P<0.05). PVP, EGF 및 cysteine의 단독 및 혼합 첨가에 따른 분할율은 cysteine 단독첨가군이 높았으나(P<0.05), 배반포 발달율은 차이가 없었다. Inner cell mass 수는 대조군과 cysteine 첨가군이 PVP 첨가군에 비하여 유의하게 높았고(P<0.05), 총 세포수도 cysteine 첨가군에서 가장 높았다. 수정란이식 결과는 대조군, EGF, cysteine 및 EGF+cysteine 군의 임신율은 46.1~63.6%로서 비슷하였으나, PVP 첨가군은 10%로서 다른 군에 비하여 유의하게 낮았다(P<0.05). 본 연구 결과는 체외성숙 배지에 PVP의 첨가로 배 발생은 가능하지만, 세포의 품질에는 악영향을 미치는 것을 보여준다. This study was examined the effects of concentrations of polyvinylpyrrolidone(PVP) and supplementation of EGF, cysteine and PVP during in vitro maturation on the development of bovine embryos. In experiment 1, 0.1 to 3.0% PVP was supplemented to IVM medium before IVF. The development rates to the blastocyst stage was significantly higher in 0.5% PVP group than 3.0% PVP group (P<0.05). In experiment 2, EGF, rysteine and PVP were supplemented to IVM medium. The hight cleavage rate was obtained from cysteine group, but blastocyst formation rates did not differ among groups. The highest total cell number and inner cell mass (ICM) cell number were observed in cysteine group. In PVP group, ICM cell number was significantly low than those of cysteine and control groups (P<0.05). After embryo transfer, pregnancy rate was significantly low in PVP group compared to other groups (P<0.05). These results indicate that the supplementation of PVP in IVM medium support the embryo development, but has a deteriorate effect on the blastocyst quality.

한우 난포란 유래 배반포의 체외생산을 위한 생물학적 요인들의 영향

박흠대,김재영,주재홍,공건오,윤산현,공일근,이상민,이상진,송해범 韓國受精卵移植學會 2000 한국동물생명공학회지 Vol.15 No.2

This study was carried out to investigate the effect of biological factors on the in vitro production(IVP) of bovine oocytes for development of simple culture methods and medium. Oocytes from the slaughterhouse ovaries were matured and fertilized using general protocol and this study was examined if there were necessary to co-culture, media change, media type and embryo density. This results were as follows: 1. The development rate according to co-culture with cumulus cells and non co-culture as drop culture was not significantly different in cleavage (68.9 vs 71.7%), 8-cell stage (41.2 vs 44.1%) and blastocyst stage (12.2 vs 13.8%), respectively (p<0.05) 2. The blastocyst development rates in YS and CRIaa were higher than that in TCM199 (12.4, 10.4 vs 3.7%), but the cleavage (69.0, 77.8 and 61.0%) and 8-cell stage (31.7, 37.0 and 35.7%) development accoring to YS, TCM199 and CRIaa ws not significantly different, respectively (p<0.05). 3. There was no significantly different in cleavage (62.6, 59.5 and 61.2%), 8-cell(34.7, 37.9 and 34.0%) and blastocyst (9.5, 11.6 and 12.8%) development among medium change time as control, Group I and Group II, respectively (p<0.05). 4. Blastocyst formation of 8-cell stage according to embryo density was not significantly different in 1, 10 and 25 embryos (27.3, 22.5 and 34.0%), respectively (p<0.05). These results indicated that simple culture system could reduce bovine IVP embryos as drop culture as non co-culture system, high density embryo (25 embryos/50 1 drop). YS defined medium and no medium change for whole culture period, although other biological factors need to examine in order to produce efficient IVP bovine embryos.

체외에서 양질의 한우 수정란 생산을 위한 배양조건의 설정 및 이식

박흠대,김종환,정덕수,이동칠,김주환,윤산현 韓國受精卵移植學會 1999 한국동물생명공학회지 Vol.14 No.1

To establish the optimal culture systems for production of transferable embryos in Korean Cattle, pregnancy rates of IVF-derived blastocysts according to different culture media, culture method and culture duration were compared. Development of IVF-derived embryos to blastocysts was most effective in YS medium group co-cultre with cumulus cells. Blastocysts cultured for 6 to 8 d in vitro showed higher hatching rate and good quality. Pregnancy rates after transfer of IVF-derived blastocysts cultured for 7 or 8 d were high. Through our experiments, it is considered that improvement of culture media and culture method is necessary for mass production of blastocysts with excellent of good quality in Korean Cattle.