[In vitro separation and development of sheep blastomeres].
Four cell embryos collected by laparatomy from Sardinian breed ewes superovulated with FSH-p (16 mg Sigma), were divested of their zonae pellucidae (ZP) by micromanipulation or chemical methods (pronase 0.5%, tyrode pH 2.2). The blastomeres were separated by pipetting using a flame polished pasteur pipette in a Ca free medium (PBS. Sigma) and were inserted into previously evacuated Z.P. using a Leitz micromanipulator. The Z.P. were removed either mechanically or with acid tyrode; pronase was unable to digest them after incubation at 30 degrees C for 120 minutes. The single blastomeres were cocultured on a monolayer of ovine oviductal epithelial cells in TCM 199 + 10 FCS at 38 degrees C in 5% CO2 for 60 hours. No developments were observed in blastomeres obtained by acid digestion of the ZP while 50% of the other blastomeres continued their development until the 16 cell stages. Our results suggest that coculture with oviductal epithelial cell monolayers can support in vitro development of single ovine blastomeres.
Loi
P
P
Istituto di Fisiologia Veterinaria, Università di Sassari.
Ledda
S
S
Filia
F
F
Gallus
M
M
Cappai
P
P
Naitana
S
S
D000818:Animals; D001757:Blastomeres; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000601:surgery; D005314:Embryonic and Fetal Development; D004847:Epithelial Cells; D009924:Organ Culture Techniques / Q000379:methods*; D010057:Oviducts / Q000166:cytology; D012756:Sheep / Q000196:embryology*
Separazione e sviluppo in vitro di blastomeri di ovino.