Factors affecting electrical activation of porcine oocyte matured in vitro.
This work was undertaken to improve conditions for in vitro maturation and activation of porcine oocytes. Experiments were designed to compare: (i) electrical pulse frequency, (ii) methods of oocyte preparation, (iii) maturation conditions, and (iv) electrical poration medium on development. Oocytes were harvested by follicle dissection or aspiration, co-cultured with follicle shells in M199 based medium with or without media changes at 38.5 degrees C in 5% CO2 under non-static conditions for 48 h and electroactivated using single or multiple pulses (current strength 1.0 kV/cm for 50 microseconds in 0.28 M inositol or mannitol based media with 10 mM histidine) at different time intervals. The results showed: (i) neither the pulse frequency nor the pulse interval influenced rates of pronuclear formation but multiple pulse activation (3 pulses at 5 min intervals) induced a higher incidence of development and progression through the 4-cell block in contrast to one pulse activation; (ii) both the rate of nuclear maturation (88.6% vs. 77.6%) and post-activation cleavage (89.8% vs. 67.4%) were higher (P < 0.05) when oocytes were collected by follicle dissection rather than by aspiration; (iii) while changing to a hormone-free medium at 24 h was without effect on maturation (91.9% vs. 91.7%), rate of cleavage (81.6% vs. 72.3%, P < 0.05) at 24 h was enhanced by the medium change; and (iv) oocytes activated with 3 pulses 5 min apart in mannitol based medium at 48-49 h and at 53-54 h formed pronuclei at a comparable rate but subsequent parthenogenetic development was higher in the older eggs. By contrast, inositol-based medium supported development of young and old eggs equally well. Calcium and magnesium ions are, however, necessary in both mannitol and inositol media for activation of porcine oocytes matured in vitro. The present results suggest that optimal parthenogenetic activation and early development of IVM pig oocytes could be obtained if oocytes are harvested by dissection, cultured for 24 h in hormone-containing medium before being placed in hormone free medium and activated at 48 h in inositol based medium using a three pulse activation system.
Liu
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Department of Development and Signalling, Babraham Institute, Cambridge, UK. lliu@ansc1.cag.uconn.edu
Moor
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RM
D000375:Aging / Q000502:physiology; D000818:Animals; D002453:Cell Cycle / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques / Q000379:methods / Q000662:veterinary; D003470:Culture Media / Q000032:analysis / Q000494:pharmacology; D004558:Electric Stimulation; D005260:Female; D007294:Inositol / Q000032:analysis / Q000494:pharmacology; D008353:Mannitol / Q000032:analysis / Q000494:pharmacology; D008540:Meiosis / Q000502:physiology; D009865:Oocytes / Q000502:physiology*; D009866:Oogenesis / Q000502:physiology*; D010312:Parthenogenesis / Q000502:physiology*; D013552:Swine / Q000502:physiology*; D013997:Time Factors
D003470:Culture Media; D008353:Mannitol; D007294:Inositol