Alain Delval

Assessment of nuclear totipotency of fetal bovine diploid germ cells by nuclear transfer

Nuclear transfer was used to study nuclear reprogramming of fetal diploid bovine germ cells collected at two stages of the fetal development. In the first case, germ cells of both sexes were collected during their period of intragonadal mitotic multiplication at 48 days post coïtum (d.p.c.). In the second case, only male germ cells were collected after this period, between 105 and 185 d.p.c. Isolated germ cells were fused with enucleated oocytes. Reconstituted embryos were cultured in vitro and those reaching the compacted morula or blastocyst stage were transferred into synchronous recipient heifers. Of 511 reconstituted embryos with 48 d.p.c. germ cells (309 males and 202 females), 48% (247/511 ) cleaved; 2.7% (14/511 ) reached the compacted morula stage and 8 of them the blastocyst stage (1.6%). No difference was observed between sexes. All 14 compacted morulae/blastocysts were transferred into 6 recipients and one pregnancy was initiated. This recipient was slaughtered at Day 35 and an abnormal conceptus (extended trophectoderm and degenerated embryo) was collected. Its male sex, genetically determined, corresponded to that of donor fetus. Of 380 reconstituted embryos with male 105 to 185 d.p.c. germ cells, 72.1% (274/380 ) cleaved, 2.1% (8 380 ) reached the compact morula stage and 7 of these the blastocyst stage (1.8%). Three blastocysts and one morula were transferred into 4 recipients. Two became pregnant at Day 21 but only one at Day 35 which aborted around Day 40. Our results show that the nucleus of diploid bovine germ cells of both sexes can be reprogrammed. However, in the absence of further development of these reconstituted embryos, nuclear totipotency of bovine diploid germ cells remains to be evidenced.

Viability of cloned bovine embryos after one or two cycles of nuclear transfer and in vitro culture

We described an exclusively in vitro procedure for cloning and recloning bovine embryos. Embryos obtained by IVM/IVF/IVC developed to the morula stage were used as blastomere donors in cunjunction with IVM recipient oocytes. Reconstructed embryos were developed in vitro in co-culture using bovine oviductal epithelial cells. The resulting morulae were used as donors for recloning under the same experimental conditions. No significant difference was observed between cloning and recloning in terms of development (rates of blastocysts: 12.9 versus 14.9%), in the number of nuclei per blastocyst (63.8 versus 49.1), or in pregnancy rates (35.7 versus 33.3%). The high variability observed between replicates and the correlation between results in first and second cycle nuclear transfer may suggest an inherant potential of individual donor embryos to support development by cloning.

bPAG PROFILES IN RECIPIENT HEIFERS AFTER TRANSFER OF IVF AND NUCLEAR TRANSFER EMBRYOS

Bovine pregnancy associated gl continuously thoughout f estation wtt H coproteins (bPAG) are secreted by trophoblast concentrations increasing from implantation to the end of pregnancy (20 i et al., Biol. Reprod., 46, 83-92, 1992). Following the bPAG levels released after transfer of IVF and nuclear transfer (NT) embr OS during pregnancy may be useful for prediction of fetal well-bein and he1 to etect early placental abnormalities, embryonic mortality or abortion. he aim of thiixperiment was to determine bPAG levels in heifers bearing embryos and fetuses after non surgical transfer of two embryos IVF and transfer o P reduced in vitro (IVF) or by nuclear transfer. these emb OS were carried out in Denmark, NT and transfer of the cloned embryos were done in ‘2; twice monthly during pre Belgian laboratory actor d: nancy. A radioimmunoassa tng to the method described In the IVF group, no pregnancy loss was 64%. In the cloned group, 2 abortions ~180) and the calving rate was 21%. Data