28 IMPROVED HATCH RATE AFTER PARTIAL DISSECTION OF ZONA PELLUCIDA IN CLONED PIG EMBRYO

For last 20 years, the efficiency of animal cloning has remained extremely low, despite many attempts to improve it. Although nuclear transfer experiments have been almost optimized, artificial holes are inevitably made in the zona pellucida (ZP) during nuclear transfer experiments, such as enucleation of maternal genome or injection of nuclear donor. Hatching from the ZP is a prerequisite for mammalian embryo implantation, and the condition of the ZP has a lot of influence on hatching. The present studies were performed to investigate the effects of artificial holes in the ZP, because of the nuclear transfer procedure, on hatching of clone embryos in pigs. All experiments were done in triplicate. Statistical analysis was performed using SPSS statistical software (SPSS Inc., Chicago, IL, USA). First, we made a slit in the ZP of porcine parthenote that was identical to the artificial holes of nuclear transfer experiment and compared in vitro development of Day 7 embryos with control group with intact ZP. Of slit blastocysts, 89.9% (80/89) were trapped at a slit, which looked like typical figure-eight shape, and did not complete the hatching process until Day 8, though 68.8% (64/93) of control blastocysts completed the hatching at Day 7. Then, to solve these abnormal hatchings caused by a slit in the ZP, we applied partial zona dissection (PZD) to porcine clone embryos and compared the hatching process with that of conventional clone embryos. Contrary to conventional clone blastocysts that were trapped at slit in the ZP (91.4%; 43/47), 89.5% (60/67) of clone blastocysts in PZD group were preferentially hatched through dissected hole at Day 7. These results suggest that trapping of conventional clone blastocyst in a slit of the ZP could be avoided by PZD. Through this study, we demonstrated that a slit in the ZP would hinder a blastocyst from hatching from the ZP and that partial dissection at the ZP could help clone blastocyst to hatch without trapping at a slit in the ZP. This assisted hatching in clone embryos would be useful for the successful hatching of clone blastocysts with a capacity of full-term development, so that the efficiency of animal cloning might be improved.

Intracytoplasmic sperm injection in the rat

We applied intracytoplasmic sperm injection (ICSI) to the rat comparing three different sperm injection techniques: conventional setup with a sharp needle bearing a spike (method 1), combination of partial zona dissection (PZD) needle and blunt pipette (method 2) and piezo-injection using a blunt pipette (method 3). We also investigated the timing of sperm pronuclear formation after injection. Survival rates after injection were 8%, 24% and 71% for the methods 1, 2 and 3, respectively. All surviving oocytes formed pronuclei by about 6 h after injection. Although the survival and activation rates following sperm injection using piezo-injection were high, the incidence of normal fertilisation, as evidenced by second polar body extrusion and formation of two pronuclei, was only 10%. The vast majority of the zygotes were multinucleated, although most of them subsequently underwent cleavage. Fixation and staining of injected oocytes at different times after injection revealed that replacement of sperm nuclear protamines by histones takes place by 15 min after injection, sperm head swelling occurs within 0.5–1 h after injection and pronuclei become fully developed by 7 h after injection. Although the rate of normal fertilisation in the rat following ICSI was low under the present experimental conditions, the results indicated that direct ICSI using a piezo-driven pipette would be a potentially valuable method of producing rat offspring.