In vitro and in vivo developmental potential of nuclear transfer embryos using bovine cumulus cells prepared in four different conditions.
We examined the effect of culture of donor cells on nuclear transfer efficiency using bovine cumulus cells treated with four different conditions: (1). group A, the cells removed from cumulus-oocyte complexes (COC) after aspiration of ovarian follicles; (2). group B, the cells removed from COC after in vitro maturation; (3). group C, the cells cultured in Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS) for 3 days after some subculture; and (4). group D, the cells cultured in DMEM with 0.5% FBS for an additional 5 days. Analysis of cell cycle using flow cytometry revealed that the relative proportion of donor cells at G0/G1 phase of cell cycle was 89.7% in group A, 89.5% in group B, 76.0% in group C, and 90.6% in group D. The developmental rates to blastocyst stage in groups C (45.3%) and D (46.4%) were significantly (p < 0.05) higher than in groups A (17.5%) and B (31.9%). After transfer of blastocysts produced in each group, nine of 24 recipients became pregnant on day 30. A total of five live calves were obtained from cumulus cells in all groups (group A [n = 1], group B [n = 1], group C [n = 2], and group D [n = 1]).
Akagi
Satoshi
S
Department of Animal Breeding and Reproduction, National Institute of Livestock and Grassland Science, Ibaraki, Japan. akagi@affrc.go.jp
Takahashi
Seiya
S
Adachi
Noritaka
N
Hasegawa
Kiyotoshi
K
Sugawara
Toru
T
Tozuka
Yutaka
Y
Yamamoto
Eiji
E
Shimizu
Manabu
M
Izaike
Yoshiaki
Y
D000818:Animals; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002453:Cell Cycle; D002478:Cells, Cultured; D019976:Cloning, Organism / Q000379:methods*; D017077:Culture Media, Conditioned; D004624:Embryo Transfer; D005314:Embryonic and Fetal Development; D005260:Female; D053652:Nuclear Transfer Techniques; D009865:Oocytes; D006080:Ovarian Follicle / Q000166:cytology; D011247:Pregnancy; D013997:Time Factors
D017077:Culture Media, Conditioned