Production of a cloned calf from a fetal fibroblast cell line.
The present study examined the in vitro and in vivo development of bovine nuclear-transferred embryos. A bovine fetal fibroblast culture was established and used as nucleus donor. Slaughterhouse oocytes were matured in vitro for 18 h before enucleation. Enucleated oocytes were fused with fetal fibroblasts with an electric stimulus and treated with cytochalasin D and cycloheximide for 1 h followed by cycloheximide alone for 4 h. Reconstructed embryos were cultured for 7-9 days and those which developed to blastocysts were transferred to recipient cows. Of 191 enucleated oocytes, 83 (43.5%) were successfully fused and 24 (28.9%) developed to blastocysts. Eighteen freshly cloned blastocysts were transferred to 14 recipients, 5 (27.8%) of which were pregnant on day 35 and 3 (16.7%) on day 90. Of the three cows that reached the third trimester, one recipient died of hydrallantois 2 months before term, one aborted fetus was recovered at 8 months of gestation, and one delivered by cesarian section a healthy cloned calf. Today, the cloned calf is 15 months old and presents normal body development (378 kg) and sexual behavior (libido and semen characteristics).
Mello
M R B
MR
Departamento de Reprodução Animal, Faculdade de Medicina Veterinária, Universidade de São Paulo, São Paulo, SP, Brasil. mrbmello@usp.br
Caetano
H V A
HV
Marques
M G
MG
Padilha
M S
MS
Garcia
J F
JF
Milazzotto
M P
MP
Assumpção
M E O A
ME
Lima
A S
AS
Nicácio
A C
AC
Mendes
C M
CM
Oliveira
V P
VP
Visintin
J A
JA
D000818:Animals; D002417:Cattle; D002460:Cell Line; D019976:Cloning, Organism / Q000379:methods / Q000662:veterinary*; D004624:Embryo Transfer / Q000662:veterinary*; D005260:Female; D005333:Fetus / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology*; D008297:Male; D053652:Nuclear Transfer Techniques; D009865:Oocytes / Q000254:growth & development; D011247:Pregnancy