Novel approach to cell sampling from preimplantation ovine embryos and itspotential use in embryonic genome analysis

The major obstacle in the extensive analysis of the embryonic genome is the small number of cells typically obtained after the embryo biopsy. The obje ct of the present study was to develop a simple approach that would allow t he collection of a sufficient number of cells from a single embryo for use in further analyses. A micromanipulator was used to make a hole in the zona pellucida of 28 compacted morulae, 27 early blastocysts and 31 expanded bl astocysts. After further culture, the trophoblastic cells, which herniated through this hole, were cut and cultured in vitro for different periods and used for embryo sexing. The results showed that biopsies can be taken succ essfully from 96.3% of early blastocysts, compared with 67.7% of expanded b lastocysts and 71.4% of compacted morulae. The trophoblastic vesicles conta ined 20.8 +/- 6.7 cells (mean +/- SEM) and, when cultured, formed a conflue nt monolayer. The sex of cells cultured was assayed by PCR and the 12 lambs born after transfer of biopsied embryos confirmed its 100% accuracy. Moreo ver, no significant differences were found in the viability rates in vitro among blastocysts vitrified immediately after biopsy (77.8%), blastocysts b iopsied and vitrified after 24 h culture (76.9%) and blastocysts vitrified without manipulation (88.5%). In experiments in vivo, the lambing rate of b iopsied and vitrified blastocysts was significantly (P < 0.05) lower (40.0% ) compared with vitrified control embryos (68.7%). This new approach to the biopsy of preimplantation embryos is a useful good model in the assisted r eproductive technologies of domestic, wild and human species.