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 compa re: (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 aspirati on, co-cultured with follicle shells in M199 based medium with or with out media changes at 38.5 degrees C in 5% CO2 under non-static conditi ons for 48 h and electroactivated using single or multiple pulses (cur rent strength 1.0 kV/cm for 50 mu s in 0.28 M inositol or mannitol bas ed media with 10 mM histidine) at different time intervals. The result s showed: (i) neither the pulse frequency nor the pulse interval influ enced rates of pronuclear formation but multiple pulse activation (3 p ulses at 5 min intervals) induced a higher incidence of development an d progression through the 4-cell block in contrast to one pulse activa tion; (ii) both the rate of nuclear maturation (88.6% vs. 77.6%) and p ost-activation cleavage (89.8% vs. 67.4%) were higher (P < 0.05) when oocytes were collected by follicle dissection rather than by aspiratio n; (iii) while changing to a hormone-free medium at 24 h was without e ffect 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) oocy tes 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 subsequ ent parthenogenetic development was higher in the older eggs. By contr ast, inositol-based medium supported development of young and old eggs equally well. Calcium and magnesium ions are, however, necessary in b oth mannitol and inositol media far activation of porcine oocytes matu red 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 activ ated at 48 h in inositol based medium using a three pulse activation s ystem. (C) 1997 Elsevier Science B.V.