Parthenogenetic development and cytoskeletal distribution of porcine oocytes treated by means of electric pulses and cytochalasin D

Oocyte activation is a key issue in current animal biotechnology. This study was designed to reveal a suitable condition for activation of the pig oocyte. In-vitro matured oocytes were activated by means of double DC pulses (150 V/mm for 60 jusec, 1 sec apart), the same treatment followed by cytochalasin D (CD, 5 micro M for 4 hrs), or two sets of the same electricity. Oocytes were examined for their chromosome configurations and the types ofpronuclear formation by fluorescence staining 8 hrs post-activation. High rates of pronuclear formation were obtained after each treatment (93-96%). A greater proportion of oocytes having one diploid nucleus or two haploid pronuclei were found by treatment with electric pulses plus CD (84%) than by treatment with one (33%) or two sets of DC pulses (41 %). Two sets of DC pulses caused partial loss of cortical microfilaments and a decrease in density of microfilaments and microtubules in the peripheral ooplasm. Oocytes treated with CD had a wavy layer of cortical microfilaments. The incidence of cleavage 48 hrs post-activation was similar for all treatments (69-78%), but the blastocyst formation 168 hrs post-activation was higher after the combined treatment with electric pulses and CD (24%) than the treatment with one (11%) or two sets of electric stimulation (5%). The results suggest that increased development to blastocysts after a combined treatment with double electric pulses and CD may depend on a higher incidence of diploid parthenogenotes, probably due to altered microfilament organization of the ooplasm caused by the CD treatment rather than the DC pulse itself.