1992 Fiscal Year Final Research Report Summary
Development of cryopreservation method of the farm animal ovary
| Project/Area Number |
02660274
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
畜産学(含草地学)
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
MIYAMOTO Hajime Faculty of Agriculture, Professor Kyoto University, 農学部, 教授 (00026618)
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| Co-Investigator(Kenkyū-buntansha) |
SATO Eimei Institute of Medical Science, Univ of Tokyo Associate Professor, 医科学研究所, 助教授 (80093243)
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| Project Period (FY) |
1990 – 1992
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| Keywords | cryopreservation / rat ovary / goat ovary / vitrification / slow freezing / cryoprotectant / estrous cycle / histological examination |
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| Research Abstract |
Seven day-old rat ovaries were cooled by slow freezing in 2 M glycerol or by the vitrification method using modified VS1, warmed and diluted by stepwise method or in 0.5 M sucrose-PBS. In each cryopreservation process, they were evaluated histologically. Viability of vitrified ovaries were confirmed by organ culture. Viability of vitrified and sucrose-diluted ovaries were also assayed by orthotropic transplantation into spayed adult rats. Pieces of goat ovarian tissue (0.7-1 mm cubes) were cooled slowly in 2 M glycerol or 1.5 M DMSO, or cooled rapidly by vitrification method, warmed, diluted and evaluated histologically and cytologically. In cryopreservation of rat ovaries,vitrified ovaries showed better histological preservation, with mild cell shrinkage and pyknosis caused by osmotic and chemical effects of cryoprotectants and mild cell rupture by osmotic effect and ice formation. In ovaries cooled slowly, expansion and rupture of granulosa cells, and severe destruction and shrinkage of oocytes were noted. After 4 day-culture, growing follicles with 2 or more layers of granulosa cells were observed in vitrified ovaries (24.7*28.8 follicles/ovary). Endocrine activity of ovaries and/or growth of unilateral or bilateral ovaries were observed in 12/17 (70.6%) rats with grafts, but most showed abnormality in morphology and function. A part of them were mated and most became pseudopregnant, but none yielded progenies. These results indicate that rat ovaries cryopreserved by vitrification method have kept partial viability. In cryopreserved pieces of goat ovarian tissue, damage observed histologically were similar to those in rat ovaries. Pieces cooled slowly in 1.5 M DMSO showed better preservation. Pieces cooled slowly in 2 M glycerol or vitrified and diluted by stepwise method were severely damaged. In vitrified and sucrose-diluted pieces, cytological structure was well preserved in some of follicles, however, most showed mild damage.
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