Novel approach to cell sampling from preimplantation ovine embryos and its potential 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 object of the present study was to develop a simple approach that would allow the 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 blastocysts. 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 successfully from 96.3% of early blastocysts, compared with 67.7% of expanded blastocysts and 71.4% of compacted morulae. The trophoblastic vesicles contained 20.8 +/- 6.7 cells (mean +/- SEM) and, when cultured, formed a confluent monolayer. The sex of cells cultured was assayed by PCR and the 12 lambs born after transfer of biopsied embryos confirmed its 100% accuracy. Moreover, no significant differences were found in the viability rates in vitro among blastocysts vitrified immediately after biopsy (77.8%), blastocysts biopsied and vitrified after 24 h culture (76.9%) and blastocysts vitrified without manipulation (88.5%). In experiments in vivo, the lambing rate of biopsied 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 reproductive technologies of domestic, wild and human species.
Leoni
G
G
Department of Animal Biology, University of Sassari, 07100 Sassari, Italy.
Ledda
S
S
Bogliolo
L
L
Naitana
S
S
D000818:Animals; D001755:Blastocyst / Q000166:cytology*; D016009:Chi-Square Distribution; D015925:Cryopreservation; D004624:Embryo Transfer; D005260:Female; D016678:Genome; D008297:Male; D008846:Micromanipulation / Q000379:methods; D012732:Sex Determination Analysis; D012756:Sheep / Q000235:genetics*; D013048:Specimen Handling / Q000379:methods*