초록 ▼

우수한 가축의 개량과 증식을 위하여 수정난이식 기술개발이 필수적인데 이를 위하여 수정난의 동결이 뒷받침되어야 한다. 본 연구는 수정난의 동결융해후 생존에 치명적 요인인 빙정형성이 없는 유리화방법(Vitrification)을 이용하여 Vitrification solution내의 내동제(耐凍劑)혼합이 동결융해후 생쥐 Morulae의 생존율에 미치는 영향과 유리화과정을 간편화 할 수 있는 적절한 Vitrification Solution을 개발하기 위하여 실행되었다. 연구방법은 과배란을 유기한 생쥐 Morulae를 채취한후 내동제의 독

우수한 가축의 개량과 증식을 위하여 수정난이식 기술개발이 필수적인데 이를 위하여 수정난의 동결이 뒷받침되어야 한다. 본 연구는 수정난의 동결융해후 생존에 치명적 요인인 빙정형성이 없는 유리화방법(Vitrification)을 이용하여 Vitrification solution내의 내동제(耐凍劑)혼합이 동결융해후 생쥐 Morulae의 생존율에 미치는 영향과 유리화과정을 간편화 할 수 있는 적절한 Vitrification Solution을 개발하기 위하여 실행되었다. 연구방법은 과배란을 유기한 생쥐 Morulae를 채취한후 내동제의 독성을 조사하기 위하여 10, 20, 30, 40, 50%의 DMSO, Glycerol, Ethylene glycol, Propylene glycol동결액에서 수정난을 동결하였다. 30% 단일용액과 혼합용액을 제조하여 빙정형성 유무를 조사하였으며, 빙정이 형성되지 않는 30% 혼합용액중 가장 효과적인 동결액을 선정하기 위하여 이 용액에서 수정난을 동결하였다. 가장 높은 생존율을 얻은 20G10E를 Vitrification Solution의 기본용액으로 하여 투과성 내동제인 Acetamide를 첨가한 동결액과 10, 20, 30의 Ficoll을 첨가한 동결액을 제조하여 동결융해후의 생존율을 조사하였으며, Sucrose와 평형시간이 수정난의 생존율에 미치는 영향을 조사하기 위하여, 가장 높은 생존율을 얻은 30% Ficoll을 첨가한 동결액에 10와 20% Sucrose를 첨가하여 각각 5, 10, 20분간 평형후 수정난을 동결하여 FDA-test로 생존율을 비교 조사하였다. 가장 좋은 새 Vitrification Solution을 택하여 수정란의 분할 상태별 그리고 난자의 크기와 종류가 다른 포유동물의 난포난을 채란하여 생존성 여부를 검토하였다.

Abstract ▼

Studies were carried out to select the optimal freezing meidea which give in single ( glycerol, ethylene glycol, dimethyl sulfoxide and mixture solutions ( glyerol + propylene glycol, glycerol + ethylene glycol ) of permeable cryprotectants. On this freezing media, acetimide and non-permeable cryop

Studies were carried out to select the optimal freezing meidea which give in single ( glycerol, ethylene glycol, dimethyl sulfoxide and mixture solutions ( glyerol + propylene glycol, glycerol + ethylene glycol ) of permeable cryprotectants. On this freezing media, acetimide and non-permeable cryoprotectants. On this freezing media, acetimide and non-permeable cryoprotectants (sucrose, Ficoll) were added to find the proper levels and equilibration time in vitrification solution on the survival of vitrified mouse morulae. 1. In toxicity test of permeable cryoprotectants, the higher FDA-score(4.1) of mouse morulae frozen in 20, 30 and 40% single solution of cryoprotectant (glycerol, ethylene glycol, DMSO) was obtained in 30% solution. The FDA score (4.1) of 30% glycerol was higher than 30% ethylene glycol(3.6) and DMSO (1.4) (P<0.05). 2. 20, 30 and 40% single solutions of permeable cryoprotectants containing m-PBS with 10% sucrose and 20% BSA did not crystallize during cooling, but crystallized during warming. However, the 30% mixture solution of the two permeable cryoprotectants did not crystallized both during cooling and warming. 3. When mouse morulae were frozen in 30% mixture solutions of two permeable cryoprotectants(glycerol and propylene glycol, glycerol and ethylene glycol), highest FDA-score(4.5) was obtained in a mixture solution of 20% glycerol and 10% ethylene glycol (20G10E) than other 30% mixture solution(10G20E, 15G15E, 20G10P, 15G15P, 10G20P) and there was significant difference between 20G10E and 10G20E(P<0.05). 4. When 10, 15 and 20% of acetamide were added to the new vitrification solution(20G 10E), FDA-scores of embryos were 4.4(control), 4.4(10%), 3.6(15, 20%), respectively. The addition of acetamide did not affect the survival of forzen-thawed morulae(P<0.05). 5. The survival between 5 min(3.5) and 10 min(4.6), 10 min(4.6) and 20 min(3.2) of equilibration in 10% sucrose, and 20 min(3.2) and 5 min(4.0), or 10 min(4.3) in 20% sucrose was significantly different (P<0.05), the highest survival (4.6) was obtained in mouse morulae equilibrated in VS(20G10E) containing 10% sucrose for 10 minutes. 6. FDA-score of morulae frozen in the new vitrification solution containing 0, 10, 20 and 30% Ficoll was 4.5, 4.2, 4.4 and 4.6, respectively and had no significant effect among concentrations of Ficoll(P>0.05). The development rate after culture(24h) was 89%(20% Ficoll) and 93% (30% Ficoll), respectively. 7. When embryos were frozen according to the developmental stage, the survival rate(FDA score) of mouse embryo was 62%(2 cell), 68%(4 cell) and the survival rate of rat embryos was higher in 2 cell(100%) and 4 cell(94%) than 16 cell(82%) (P<0.05). 8. The survival rate of ovarian oocytes was 74%(10% Ficoll) and 70%(30% Ficoll) in mouse, and 70%(30% Ficoll), respectively. However, the high survival rate of rabbit(78%) and pig(62%) was obtained in 20% Ficoll. 9. The optimal concentration of permeable cryoprotectant in freezing media was 30% and the highest survival of frozen-thawed mouse morulae was obtained after 10 minutes equilibration in 20% glycerol + 10% ethylene glycol + 10% sucrose without crystalization during cooling and warming among 30% mixture solutions. The optimal concentration of Ficoll was 10 and 30%. It is suggested that New vitrification solution(20G10E) can be used in freezing of mouse, rat and rabbit ovarian oocytes except pig.

목차 Contents

• 1. 서 론...7
• 2. 재료 및 방법...10
• 3. 결과 및 고찰...14
• 4. 적 요...19
• 5. 도 표...21
• 목 차...35
• 2. 재료 및 방법...37
• 3. 결과 및 고찰...41
• 4. 적 요...46
• 5. 도 표...48
• 목 차...62
• 1. 서 론...63
• 2. 재료 및 방법...65
• 3. 결과 및 고찰...68
• 4. 적 요...72
• 5. 도 표...73
• 6. 인 용 문 헌...77