The aim of this study was to activate unaged bovine oocytes matured 24 hr in vitro.
In the first experiment, we demonstrated that artificial activation of in vitro matured bovine oocytes, assessed by pronuclear formation, is age-dependent, and young oocytes matured 24 hr are difficult to activate compared with aged oocytes matured 32 or 42 hr.
In the next experiment, young oocytes were randomly assigned to either electrical stimulation or chemical treatment. Oocytes were electrically stimulated with two DC pulses of 1.0 kV/cm for 25, a s ec, in non-electrolyte medium containing 0.3 M mannitol (T1), electrolyte modified PBS (T2) or modified PBS with 0.01 mg/ml BSA (T3) . Oocytes were incubated for 5 min in modified PBS containing 7% ethanol (T4) or 50, E M calciumionophore (T5) . Alternatively, oocytes were incubated with 1 0μg/ml cycloheximide in 12.5 mM-Hepes TCM 199 for 10 hr (T6), 7% ethanol for 5 min followed by incubation in 10μg/ml cycloheximide for 10 hr (T7) or 50μM calcium ionophore for 5 min followed by incubation in 10μg/ml cycloheximide for 10 hr (T8) .
At 18 hr (T1-5) or 8 hr (T6-8) po-stactivation, the oocytes with one or more pronuclei were classified as activated oocytes. The activation rates of T2 (82.2%, 83/101) and T3 (71.7%, 43/60) were higher (P<0.05) than that of T1 (49.5%, 48/97) . The activation rates of T4, T5, T6, T7 and T8 were 46.7% (42/90), 26.7% (43/161), 66.0% (209/291), 92.6% (113/122) and 62.9% (56/89), respectively. The activation rate of T7, was higher than those of other chemical treatments (P<0.05) .
Young oocytes were parthenogenetically activated by T7 and 5 μg/ml cytochalasin D treatment, and incubated for development to blastocysts. By this treatment, 86.2% of the oocytes was activated, and 79.1% of the activated oocytes formed two pronuclei. Rates of treated oocytes that cleaved, developed to the morula≤andblastocyst≤stages were 49.2%, 7.5% and 3.0%, respectively.
The results suggest that unaged bovine oocytes matured 24 hr in vitro can be efficiently activated by electrical stimulation in electrolyte medium or by treatment with ethanol-cycloheximide, and activated oocytes can develop to the blastocyst stage.
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