34 COMPARISON OF IN VIVO DEVELOPMENT DURING EARLY PREGNANCY OF CLONED FETUSES DERIVED FROM BOVINE FETAL FIBROBLASTS AT THE EARLY G1 AND G0 PHASES

Abstract

Enhanced development of bovine somatic cell nuclear transfer (NT) embryos to full term has been achieved using fibroblasts at the early G1 (eG1) phase instead of cells at the quiescent (G0) phase (Urakawa et al. 2004 Theriogenology 62, 714–728). The high abortion rate and abnormal placental development of NT embryos using G0 phase cells is related to the low formation rate of embryonic disks and the aberrant development of the trophectoderm in utero until Day 14 of gestation (Ideta et al. 2007 Cloning Stem Cells 9, 571–580). The purpose of this study was to examine the morphological development of conceptuses such as fetuses and fetal membranes in the early pregnancy of NT embryos using eG1 phase cells (eG1-NT embryos) and G0 phase cells (G0-NT embryos). Blastocysts derived from eG1-NT and G0-NT embryos were transferred to recipient heifers, and the conceptuses at Day 50 of gestation were retrieved nonsurgically using prostaglandin F2α (PGF2α) and oxytocin (Lavoir and Betteridge 1996 J. Reprod. Fertil. 106, 95–100). In vitro-fertilized (IVF), parthenogenetic and artificially inseminated (AI) embryos were used as controls. Data were analyzed using chi-square test and Student’s t-test. Pregnancy rates at Day 30 of recipient heifers carrying eG1-NT, G0-NT, IVF, parthenogenetic, and AI embryos were similar (57 to 100%; 4/7 to 8/8). Two recipient heifers carrying parthenogenetic embryos returned to estrus between Day 30 and 50 of gestation, whereas all other pregnancies remained viable. Most fetuses at Day 50 of gestation of all experiment groups (20/24) were recovered nonsurgically by several PGF2α and oxytocin treatments. The recovery rates of normal fetuses derived from eG1-NT embryos (83%, 5/6), IVF embryos (80%, 4/5), and AI embryos (88%, 7/8) were greater than those of G0-NT embryos (33%, 2/6) and parthenogenetic embryos (0%, 0/7). The amniotic fluid volume of G0-NT embryos was significantly greater than that of AI embryos (P < 0.05). But the amniotic fluid volume of eG1-NT embryos was the same as that of AI embryos (P > 0.05). The fetal weights of eG1-NT and IVF embryos were significantly greater than the fetal weight of AI embryos (P < 0.05). Our results suggest that efficient production of cloned offspring is possible by NT using donor cells that are in the early G1 phase.