Studies on fertilization in vitro and culture of mammalian eggs

Research Project

Project/Area Number	60480079
Research Category	Grant-in-Aid for General Scientific Research (B)
Allocation Type	Single-year Grants
Research Field	畜産学(含草地学)
Research Institution	Okayama University (1986) Kyoto University (1985)
Principal Investigator	NIWA Koji College of Agriculture, Okayama University, 農学部, 助教授 (40089115)
Co-Investigator(Kenkyū-buntansha)	YUHARA Masataka College of Agriculture, Okayama University, 農学部, 助教授 (20032980)
Project Period (FY)	1985 – 1986
Project Status	Completed (Fiscal Year 1986)
Budget Amount *help	¥6,900,000 (Direct Cost: ¥6,900,000) Fiscal Year 1986: ¥1,700,000 (Direct Cost: ¥1,700,000) Fiscal Year 1985: ¥5,200,000 (Direct Cost: ¥5,200,000)
Keywords	Cattle / Goat / Rat / In-vitro fertilization / Eggs / Oocytes / Spermatozoa / 受精能獲得
Research Abstract	FERTILIZATION IN VITRO IN THE RAT. Newly ovulated eggs from immature rats induced to superovulate were inseminated in vitro with preincubated spermatozoa. A total of 84 two-celled eggs about 35 hr after insemination were transferred to the oviducts of 8 pseudopregnant rats on the first day of diestrus. Five recipients which had received a total of 59 eggs became pregnant and 9 male and 7 female newborn young were obtained (27% of development). For the study of fertilization in vitro of rat oocytes matured in culture, oocytes with an intact germinal vesicle were cultured for 14-15 hr and exposed to preincubated spermatozoa. The proportion of the oocytes penetrated 21.5-46 hr after insemination was higher when they were cultured in a modified Krebs-Ringer bicarbonate (m-KRB) solution including fetal calf serum (FCS, 10 or 20%)(50-91%) than 4 mg bovine serum albumin (BSA)/ml (19-20%). The proportion of cleaved eggs 46 hr after insemination, however, was higher in the oocytes cultured in th … More e medium with BSA (90%) than FCS (50-52%). FERTILIZATION IN VITRO OF BOVINE FOLLICULAR OOCYTES. Frozen semen obtained from 6 different bulls were washed twice with Brackett & Oliphant's medium (BO medium) including 10 mM-caffeine and preincubated for 5 hr at 39゜C after thawing. Bovine follicular oocytes cultured for 20-22 hr in a Hepes buffered TC-199 medium supplemented with FCS (10%) were introduced into the preincubated sperm suspension. The proportion of the oocytes penetrated 20-24 hr after insemination varied greatly according to the different bulls: very high penetration rates (83% in average) were obtained in one bull (P-157), but in other 5 bulls penetration rates were considerably low (0- 25%). These variations in penetration rates in vitro were found to be independent of conception rates to artificial insemination using the same semen samples. When the frozen semen from a P-157 bull was used, higher penetration rate of the oocytes was obtained after insemination with spermatozoa which were washed BO medium (80%) than m-KRB solution (45%) including caffeine. The posibilities of fertilization in vitro of goat eggs and development in culture of cattle oocytes fertilized in vitro can be resolved by further experimentation. Less