보고서 정보
주관연구기관 |
순천대학교 SunChon Natinal University |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2006-07 |
과제시작연도 |
2005 |
주관부처 |
농림축산식품부 Ministry of Agriculture, Food and Rural Affairs(MAFRA) |
과제관리전문기관 |
농림기술관리센터 Agricultural Research & development Promotion Center |
등록번호 |
TRKO201400023030 |
과제고유번호 |
1380002016 |
사업명 |
농림기술개발 |
DB 구축일자 |
2014-11-29
|
초록
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○ 연구결과
· 배란적기 판단: 질 상피세포의 변화보다 초음파진단에 의한 배란적기의 판단이 정확함. 초음파 진단방법은 개(진도개)의 난포가 6.7 mm 이상까지 조사되지 않아 graffian follicle의 최대 size로 판단된다. 즉, 약 6-7 mm 정도의 난포크기는 배란직전으로 판단됨.
· 발정유기는 GnRH (500 IU) + CPE + GnRH (500 IU)처리에 의하여 60% 발정유기율
· 과배란처리를 위하여 GnRH (1000 IU) + CPE + GnRH (1000 IU) 방법으로 실시한 결과 1
○ 연구결과
· 배란적기 판단: 질 상피세포의 변화보다 초음파진단에 의한 배란적기의 판단이 정확함. 초음파 진단방법은 개(진도개)의 난포가 6.7 mm 이상까지 조사되지 않아 graffian follicle의 최대 size로 판단된다. 즉, 약 6-7 mm 정도의 난포크기는 배란직전으로 판단됨.
· 발정유기는 GnRH (500 IU) + CPE + GnRH (500 IU)처리에 의하여 60% 발정유기율
· 과배란처리를 위하여 GnRH (1000 IU) + CPE + GnRH (1000 IU) 방법으로 실시한 결과 10두 중 9두에서 발정유기 가능하였으나, 과배란유기에는 효과가 저조함
· 이렇게 발정유기 및 과배란 처리된 개체로부터 난자를 회수하기 위하여 외과적 수술방법에 의한 채란방법을 정립
· 미성숙난자의 체외성숙을 위하여 다양한 체외성숙조건으로 체외배양을 실시한 결과 개의 종 특이성으로 MII 단계까지 안정적인 결과를 얻지 못하여 궁극적으로 완벽한 배양조건을 건립하지는 못하였다. 그러나 난소낭액의 첨가와 배양온도의 시기별 변화를 줌으로써 50% 이상의 발달율도 얻었으나, 반복된 결과를 얻지 못하였음
· 개 난소 수송 시 온도가 저온에서 개 난자의 생존율은 치명적인 영향을 미치는 것으로 판단됨
· 미성숙난자의 난관이식 후 성숙율도 체외성숙과 같이 전혀 개선되지 않았다. 이는 난자 자체의 활성화와 연관이 있다는 증거로 판단됨
· FSH 처리구에서는 과립막세포의 확장이 유의적으로 높았고, LH 처리구에서는 대조구와 차이가 없었다. 그러나 핵발발율에서는 처리구간 유의차가 없었음
· 난자의 크기는 체내 배란된 난자가 체외 회수된 난자보다 유의적으로 컸고, 체외난자 중에서는 발정기의 난자가 유의적으로 컸음
· 발정주기에 따른 난자의 GV단계를 조사한 결과 체내 배란된 난자는 100%가 GV-V 단계이며, 체외 채란된 follicular 단계의 난자는 57.4%로서 비발정기 및 황체기보다 유의적으로 GV-V단계로 발단된 난자가 많았음. 체외성숙 결과 체내 채란된 난자의 50%가 MII로 발달하였으나, 체외채란된 난자는 발정주기에 관계없이 발달율에 차이가 없었음
· 개 뇌하수체호르몬의 첨가에 의한 체외성숙율을 조사한 결과 40, 400 ㎍/ml 처리구에서 17.4, 15.4% MI-II 발달율을 보였음
· 이상의 결과를 분석하면 개의 발정유기는 인위적인 hormone 처리에 의하여 가능하나, 이들을 과배란처리에 의한 난자의 회수에는 한계가 있다고 판단된다. 체외성숙을 유도하기 위하여 다양한 배양조건으로 시도하였으나 만족할 만한 체외 성숙율을 얻지 못하였다. 그러나 난소낭액을 첨가하였을 때 50% 이상의 MII 단계의 발달율을 확인한 결과가 있었으나, 반복실험에서 성공하지 못하였다. 이는 발정 시 생체내액이 개 난자의 체외성숙조건에 필요하다고 판단된다.
Abstract
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This study was carried out to establish of estrus induction, superovulation and in vitro maturation of canine oocytes for appling of preservation of genetic materials and development of worldwide supeior dog, such as Jindo dog and Sapsaree dog. Jindo dog was designated already 53th of natural mounme
This study was carried out to establish of estrus induction, superovulation and in vitro maturation of canine oocytes for appling of preservation of genetic materials and development of worldwide supeior dog, such as Jindo dog and Sapsaree dog. Jindo dog was designated already 53th of natural mounment in Korea and 334th in FCI at 1995. Also Sapsaree dog was designated already 368th of natural mounment in Korea. To preservation of the genetic in jindo and sapsaree-dog, reproduction and breeding was systematically needed. Recently, although the bovine and other domestic animals has been approved and commercial application, frozen semen, aritfical insemination, induction of estrus and super-ovulation and in vitro maturation technology was not established in canine species. Therefore, this research objective was establishment of in vitro culture systems in Jindo and Sapsaree dog oocyte.
To confirm an ovulation time point, staining of vaginal epithelial cell and diagnosis of follicle size by ultrasonography was applied in estrus or induced estrus dog. Although the staining of vaginal epithelial cells can be applied, confirmation rate was not exact rather than ultrasonography, which can apply to measure of follicle size in estrus dog. In general, Maximan size of follicle in Jindo dog was measured to 6.7 mm and so it's size is graafian follicle, that mean, could be ovulate as soon.
To use of in vivo eggs, we tried to induced estrus and superovulation from anestrus dog. Anestrus dog was injected various hormones to induce estrus such as two combination methods, which was GnRH + CPE + GnRH and PGF2a + eCG + hCG. Both methods can be induced an estrus 9 out of 10 dogs, but superovulation was not improved in both treatments. To collect in vivo ovulated oocytes, the surgical collection method was developed in induced or superovulated dogs by surgery. However, the recovery rate was not improved in induced or superovulated groups. the developmental rate to MII in collected oocytes was not different in both treatment groups and also still low to 37.5%. The induced ovulated eggs were not ovulated same time and so did not found same developmental stage eggs derived from in vivo ovulated eggs.
To improve the in vitro maturation condition of oocytes in canine, the collected oocytes were matured in various culture conditions such as carious culture media, supplement of protein sources and follicle fluid, transport temperature, culture periods and in vivo transfer and culture temporally. However, there was not improved in vitro development derived from in vitro collected oocytes. This is related on oocytes sources not in vitro culture system, because in vivo transfer oocytes was not improved developmental rates. The oocytes was so sensitive to low temperature for transportation, even it was several hours. However, an addition of ovarian bursa fluid (OBF) in IVM media may be improved of IVM rate to MII, even the result was not replicated continuly.
Cumulus cells were connected with maturation and development of oocytes and so investigated expansion of cumulus cells and in vitro development depend on gonadal hormones. Expansion of COCs in FSH group were expanded more than in LH and control group. However, the developmental rate to MII was not improved in any hormone treatment groups. The size of in vivo ovulated oocytes were significantly bigger than that from in vitro collected oocytes, but there was not significantly different between estrus stage of in vitro collected oocytes. GV-V stage of in vivo ovulated oocytes was significantly more advanced than that in in vitro collected oocytes, but the oocytes of follicular stage were advanced rather than other in vitro oocytes. Also the development to MII from in vivo collected oocytes were significantly higher than those from in vitro collected oocytes, but in vitro collected oocytes was not different among estrus stages. Development rate in 40 and 400 ㎍/ml CPE groups were higher than those in other treatments.
Although estrus can be induced, efficiency of superovulation was still low and could not collected all of MII same time, because of ovulation at different time and specific reproductive physiology in dog. Even the oocytes were matured in vitro in various culture condition, the developmental efficiency was not improved. We need to examine the culture condition of in vitro maturation to apply the basic research and application of IVM oocytes in canine.
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