70 RECONSTRUCTED BOVINE BLASTOCYSTS COMPRISING NUCLEAR TRANSFER-DERIVED INNER CELL MASS AND TROPHECTODERM FROM IVF EMBRYOS DO NOT IMPROVE IN VIVO DEVELOPMENT OF CLONES

Abstract

The cloning of cattle by somatic cell nuclear transfer (NT) is associated with a high incidence of abnormal placentation, excessive fluid accumulation in the fetal sacs (hydrops syndrome) and fetal overgrowth (Lee RSF et al. 2004 Biol. Reprod. 70, 1–11). Early embryonic loss in bovine NT pregnancies may also be due to immunological rejection (Hill JR et al. 2002 Biol. Reprod. 67, 55–63). As a means of overcoming placental abnormalities and improving pregnancy outcome in bovine NT, reconstructed blastocysts were produced by combining immunosurgically isolated inner cell masses (ICM) from Day 7 NT embryos with the trophectoderm (TE) of Day 7 IVF embryos. Oocytes for the production of NT and IVF embryos were obtained from abattoir-collected ovaries of dairy cows. The semen used for IVF was from the bull from which the cell line for NT was derived. The NT blastocysts were produced as described previously (Oback B et al. 2003 Cloning Stem Cells 5, 3–12) except that two one-cell embryos were aggregated together after NT (2NT). Blastocyst reconstruction was achieved using a modified procedure (Rorie RW et al. 1994 Vet. Record 135, 186–187). Embryos from four experimental groups were transferred individually to synchronized recipient heifers on Day 8 of culture: (1) ICM from 2NT embryos reconstructed with IVF TE (R-2NT, n = 15); (2) ICM from IVF embryos reconstructed with IVF TE (R-IVF, n = 15); (3) control 2NT (n = 10); and (4) control IVF (n = 10). Pregnancy rates were recorded and treatments compared using Fisher's exact test. After slaughter between Days 149 and 161 of gestation, morphometric measurements were determined for the fetuses, fetal organ weights, fluid volumes, and placentomes. Data were rank transformed; treatments were compared using Student's t-test with standard errors calculated from the pooled variation. Pregnancy rates on Day 35 were R-2NT (60%), R-IVF (47%), 2NT (90%), and IVF (10%). Pregnancy rates on Day 150 were R-2NT (40%), R-IVF (40%), 2NT (70%), and IVF (10%). The reason for the low IVF pregnancy rate was unknown. Previously, pregnancy rates using the same sire and cell line (but using Day 7 embryo transfer) on Day 35 were 63% (n = 40) and 69% (n = 42) for IVF and single, non-aggregated NT, respectively, and 50% and 33% for IVF and NT on Day 150. The single NT pregnancy rate was not significantly different from that for the 2NT embryos. There was no significant difference in pregnancy rates on Day 35 and Day 150 between R-2NT v. 2NT, R-2NT v. R-IVF, or 2NT v. R-IVF. The blastocyst reconstruction procedure did not have any impact on fetal development or influence pregnancy rates. All fetuses recovered were male. No significant differences were found between R-2NT and 2NT fetuses in terms of fetal weight, fluid volume, total placentome weight, and placentome numbers or in the relative and absolute weights of the brain, heart, liver, and kidneys. Thus, replacement of the TE in NT embryos with TE from IVF embryos did not overcome placental abnormalities or decrease fetal overgrowth prevalence.