| Project/Area Number |
12660258
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied animal science
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| Research Institution | HIROSHIMA PREFECTURAL UNIVERSITY |
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Principal Investigator |
HORIUCHI Toshitaka HIROSHIMA PREFECTURAL UNIVERSITY, DEPARTMENT OF BIORESOURCES, ASSOCIATE PROFESSOR, 生物資源学部, 助教授 (70209279)
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| Co-Investigator(Kenkyū-buntansha) |
YAMADA Manabu HIROSHIMA PREFECTURAL UNIVERSITY, DEPARTMENT OF BIORESOURCES, PROFESSOR, 生物資源学部, 教授 (90106005)
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| Project Period (FY) |
2000 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2001: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2000: ¥2,700,000 (Direct Cost: ¥2,700,000)
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| Keywords | INTRACYTOPLASMIC SPERM INJECTION / BOVINE / FREEZE-DRIED SPERM / SPERM ASTER / FERTILIZATION / EMBRYONIC DEVELOPMENT / 精子再凍結 |
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| Research Abstract |
We examined fertilization and embryonic development of bovine oocytes injected intracytoplasmically with freeze-dried spermatozoa and obtained the following results :
1. The viability of bovine frozen-thawed spermatozoa at low temperature was decreased during 1-7 days.
2.The viability of repeated frozen-thawed spermatozoa pelleted in dry-ice in condition of thawing at 0 ℃ and 5-min equilibrium at liquid nitrogen (LN,) gas was higher than that in condition of thawing at 37 ℃ and 0-min equilibrium at LN2 gas (47 % VS. 7 %) The percentage of oocytes with two polar bodies, and cleavage and blastocyst rates after intracytoplasmic injection with repeated frozen-thawed spermatozoa was not significantly different from those after ICSI with frozen-thawed spermatozoa.
3. The percentage of bovine oocytes with the male pronucleus after injection of spermatozoa freeze-dried in CZB medium or Na-EGTA buffer was not significantly different, but the sperm aster formation rate in the group of Na-EGTA buffer was significantly higher than that in the group of CZB medium (39 % VS. 16 %). Cleavage and blastocyst rates in the group of Na-EGTA buffer were significantly higher than those in the group of CZB medium (65 % VS. 28 % and 6 % VS.0 %)
4. The treatment of 2.5-5 mM dithiothreitol or 0.01 % triton X-100 after rehy dration of freeze-dried bovine spermatozoa did not improve the formation of male pronucleus, sperm aster microtubles and embryonic development in vitro.
5. Cleavage and blastocyst rates following freeze-dried sperm injection in the group of 5uM ionomicin for 5 min and, after a 3-h interval, with 1.9mM 6-dimethylaminopurine for 3 h (72 % and 11 %) were significantly higher than those in the group of 7 % ethanol for 5 min at 4 h post-ICSI (59 % and 1 %).
The present study demonstrates tiiat the bovine oocytes fertilized by intracytoplasmic injection with freeze-dried spermatozoa can develop to blastocysts.
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