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Parthenogenetic Activation of Marmoset: MATERIALS AND METHODS(2)

MATERIALS AND METHODS(2)

Marmoset Oocyte Collection

Oocytes were collected by follicular aspiration as previously described. Briefly, female marmosets were anesthetized with alphadolone and alphaxalone (Saffan, ~2.5 ml/kg BW; Centaur). The ovaries and uterus were exteriorized by midline laparotomy. Follicles larger than 2 mm in diameter were aspirated with a pulled 1.5-mm-di-ameter glass capillary, broken off at 0.7- to 0.8-mm diameter, which was attached to a micrometer syringe. The follicular contents were drawn out and expelled into a 35mm sterile Petri dish containing alpha modified minimum essential medium (aMEM; Merck, Lutterworth Leics, UK) buffered with 25 mM Hepes, and supplemented with 0.05 mg/ml streptomycin sulfate, 0.06 mg/ml penicillin (Sigma Chemical Co., Poole, Dorset, UK), 1 IU/ml heparin (Mon-oparin; CP Pharmaceuticals Ltd., Wrexham, UK), and 1% heat-inactivated marmoset serum. Cheap Diskus Advair

Evaluation of Oocytes

Oocytes were incubated briefly in 0.1% hyaluronidase (Sigma Chemical Co.) in Medium 2 and gently pipetted with a flame-polished Pasteur pipette to remove cumulus cells. After cumulus cell removal it was possible to visualize the first polar body. Only oocytes that had extruded a first polar body and so were presumably in meiotic metaphase II were used in this study. Cumulus-free marmoset oocytes were incubated in MEM supplemented with 10% heat-inactivated marmoset serum in a humidified atmosphere of 5% CO2 in air, at 37°C, until activation stimulus, by electrical pulses or ethanol exposure, was applied.