Offspring born from chimeras reconstructed from parthenogenetic and in vitro fertilized bovine embryos

Chimeric embryos were produced by aggregation of parthenogenetic (Japanese Red breed) and in vitro fertilized (Holstein breed) bovine embryos at the Y amaguchi Research Station in Japan and by aggregation of parthenogenetic (R ed Angus breed) and in vitro fertilized (Holstein breed) embryos at the St. Gabriel Research Station in Louisiana. After embryo reconstruction, live o ffspring were produced at each station from transplanting these embryos. Th e objective of this joint study was to evaluate the developmental capacity of reconstructed parthenogenetic and in vitro fertilized bovine embryos. In experiment I, chimeric embryos were constructed: by aggregation of four 8- cell (demi-embryo) parthenogenetic and four 8-cell stage (demi-embryo) IVF- derived blastomeres (method 1) and by aggregation of a whole parthenogeneti c embryo (8-cell stage) and a whole IVF-derived embryo (8-cell stage) (meth od 2). Similarly in experiment II, chimeric embryos were constructed by agg regating IVF-derived blastomeres with parthenogenetic blsatomeres. In this experiment, three categories of chimeric embryos with different parthenogen etic IVF-derived blastomere ratios (2:6; 4:4, and 6:2) were constructed fro m 8-cell stage bovine embryos. In experiment III, chimeric embryos composed of four 8-cell parthenogenetic and two 4-cell IVF-derived blastomeres or e ight 16-cell parthenogenetic and four 8-cell IVF-derived blastomeres were c onstructed. Parthenogenetic demi-embryos were aggregated with sexed (male) IVF demi-embryos to produce chimeric blastocysts (experiment IV), in the bl astocyst stage, hatching and hatched embryos were karyotyped. In experiment V, chimeric embryos that developed to blastocysts (zona-free) were cryopre served in ethylene glycol (EG) plus trehalose (T) with different concentrat ions of polyvinylpyrrolidone (PVP; 5%, 7.5%, and 10%), in experiment I, the aggregation rate of the reconstructed demiembryos cultured in vitro withou t agar embedding was significantly lower than with agar embedding (53% for 0% agar, 93% for 1% agar, and 95% for 1.2% agar, respectively). The aggrega tion was also lower when the aggregation resulted from a whole parthenogene tic and IVF-derived embryos cultured without agar than when cultured with a gar (70% for 0% agar, 94% for 1% agar, and 93% for 1.2% agar, respectively) . The development rate to blastocysts, however, was not different among the t reatments. In experiment II, the developmental rates to the morula and blas tocyst stages were 81%, 89%, and 28% for the chimeric embryos with partheno genetic:IVF blastomere ratios of 2:6, 4:4, and 6.2, respectively. In experi ment III, the developmental rate to the morula and blastocyst stages was 60 % and 65% for the two 4-cell and four 8-cell chimeric embryos compared with 10% for intact 8-cell parthenogenetic embryos and 15% for intact 16-cell p arthenogenetic embryos, To verify participation of parthenogenetic and the cells derived from the male IVF embryos in blastocyst formation, 51 embryos (hatching and hatched) were karyotyped, resulting in 27 embryos having bot h XX and XY chromosome plates in the same sample, 14 embryos with XY and 10 embryos with XX. The viability and the percentage of zona-free chimeric em bryos at 24 hr following cryopreservation in EG plus T with 10% PVP were si gnificantly greater than those cryopreserved without PVP (89% vs. 56%). Pre gnancies were diagnosed in both stations after the transfer of chimeric bla stocysts. Twin male (stillbirths) and single chimeric carves were delivered at the Yamaguchi station, with each having both XX and XY chromosomes dete cted. Three pregnancies resulted from the transferred 40 chimeric embryos a t the Louisiana station. Two pregnancies were lost prior to 4 months and on e phenotypically-chimeric viable male calf was born. We conclude that the I VF-derived blastomeres were able to stimulate the development of bovine par thenogenetic blastomeres and that the chimeric parthenogenetic bovine embry os were developmentally competent. Mol. Reprod. Dev. 53:159-170, 1999, (C) 1999 Wiley-Liss, Inc.