The Journal of Reproduction and Development Supplement

Ultrasound stimulation is a new oocyte activation protocol developed recently in our laboratory and we found that the ICSI and cloned embryos activated by ultrasound have the ability to develop into piglets after transfer to recipient females. However, the exocytosis patterns of cortical granules (CGs) of pig oocytes activated in vitro by ultrasound stimulation remain unknown. The objective of this study was to determine the effects of the artificial activation protocols [ultrasound and electricity with or without 2.2 ug/ml cytochalasin B (CB)] on nuclear activation status, CGs distribution and the fertilizing ability of pig oocytes matured and activated in vitro. The second polar body extrusion was significantly inhibited (P<0.05) and CGs centripetal migration observed when oocytes were treated with CB for 2h after artificial stimulation. However, the time courses of CGs exocytosis in oocytes activated in vitro did not differ between the artificial activation protocols. In oocytes activated by artificial stimulation, fluorescent clumps or spots were seen within the perivitelline space 2h after artificial stimulation. At 6 h following activation most oocytes activated by ultrasound remained CGs fluorescent spots of strong intensities while most of those activated by electricity decreased their intensities. The fertilization rate and the numbers of penetrated spermatozoa per oocyte were significantly decreased when the matured oocytes were inseminated in vitro following artificial stimulation compared with the control (P<0.05).

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To enable handy transport of vitrified embryos, we tried to develop a new vitrification method in which they can be kept at the dry ice temperature for several days by promoting the dehydration of embryos. Two-cell embryos obtained from ICR and C57BL/6 (B6) mice were vitrified with ethylene glycol (EG)-based solutions, EFSa solutions and EFSc solutions, which were mixtures of EG (30 to 50%) and FSa solution or FSc solution, respectively. FSa and FSc solutions were PB1 medium containing 30% Ficoll PM-70 plus 0.5 M sucrose or 1.5 M sucrose, respectively. The survival of ICR embryos vitrified with 30% or 40% EG (EFS30a, EFS40a, EFS30c, EFS40c) was high, but that vitrified with 50% EG (EFS50a, EFS50c) was quite low, regardless of the concentration of sucrose. When vitrified embryos were kept at -80°C for 4 days, none of embryos vitrified with EFS30a and EFS40a survived, whereas high proportions of embryos vitrified with EFS30c and EFS40c survived. The survival of embryos vitrified with EFS35c or EFS40c was maintained high even after being kept at -80°C for up to 10 days. When embryos derived from ICR and B6 mice were vitrified with EFS35c or EFS40c and then stored at -80°C for 4 days, the survival was maintained high even after being re-cooled in liquid nitrogen. In conclusion, we have developed a new vitrification method in which vitrified embryos can be kept at the dry ice temperature for several days. The method would enable handy transportation of vitrified 2-cell mouse embryos using dry ice.

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The objectives of the study were to determine the interval to post-injection oocyte activation and donor cell culture condition for reconstruction of sei whale iSCNT embryos. Slaughterhouse derived bovine COCs were matured in vitro and used as recipient cytoplasm. A 16 cm long and 51 g male sei whale fetus derived fibroblasts after serum starvation or confluency were used as donor nuclei. A single whole cell was injected into the recipient cytoplasm, activated (Ionomycin and 6-DMAP) within 30 min (immediate activation) or 4 h post-injection (delayed activation) and cultured. Parthenogenetic bovine embryos were used as control. In Exp. 1, pseudopronucleus (PPN) formation rate in iSCNT embryos did not vary between immediate (1.7%) and delayed (1.6%) activation although PPN formation rate varied significantly between iSCNT and parthenogenetic embryos (53.8%; P<0.05). In Exp. 2, rates of confirmed cleavage (stained nuclei) and blastocyst formation did not vary between serum starved (3.2 & 1.6%, respectively) and confluent (2.9 & 0.0%, respectively) cells derived iSCNT embryos. Parthenogenetic embryos had significantly higher cleavage rate (56.8%) than iSCNT embryos and higher blastocyst formation rate (9.5%) than confluent cell derived embryos (P<0.05). The only produced iSCNT blastocyst was negative for whale specific DNA and five parthenogenetic blastocysts were positive for bovine specific DNA when analyzed by PCR. In conclusion, this is the first attempt to produce sei whale cloned embryos using whale fetal fibroblasts as donor nuclei although the technique of sei whale iSCNT embryo production needs to be improved. The research work has been funded by the JSPS, Tokyo, Japan.

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