INTRODUCTION

Cryopreservation technology applied to mammalian embryos of livestock such as cattle, goats, sheep, and other model animals has led to many successful developments since the 1980s. This, however, is not the case regarding cryopreservation of porcine embryos, which has faced many challenges over the years.

It has only fairly recently become feasible to produce progeny from frozen porcine embryos, although the highly variable cryotolerance of pig embryos over the developmental stages has limited applications for embryos at certain developmental stages. Porcine embryos at the perihatching stage are known to be most resistant to freezing, and their cryopreservation by the conventional freezing method has successfully led to offspring production. Conventional freezing, however, has been plagued by a low survival rate in that after thawing, even perihatching-stage embryos showed poor survival, at about 30%.

Vitrification has improved the survival rate of perihatch-ing-stage porcine embryos subsequent to cryopreservation. Moreover, technological improvements have led to higher survival rates in porcine morulae and early blastocysts, which are notoriously sensitive to damage by conventional freezing. This technique has been further improved to achieve more stable vitrification using the open pulled straw and minimum volume cooling (MVC) methods such that higher embryo viability is provided.

In an effort to improve survival after cryopreservation, alternative approaches have been directed at increasing cry-otolerance of porcine embryos. Nagashima et al. showed that delipation, which removes cytoplasmic lipid droplets in embryos, remarkably improves embryo viability following cryopreservation. And since then, the transfer of delipated porcine morulae and blastocysts was demonstrated to provide a practically feasible birth rate after cryopreservation.

These advancements in cryopreservation of porcine embryos have been successful only for in vivo-derived embryos, whereas in recent pig cloning research, an in vitro-matured (IVM) oocyte is usually employed, as is also the case with transgenic pig production and in vitro fertilization. Because improvements in cryopres-ervation technology of in vitro-produced (IVP) embryos will further facilitate studies on pig cloning and the establishment of a gene bank of transgenic pigs, it is necessary to establish cryopreservation technology for embryos derived from IVM oocytes. Successful cryopreservation of IVP embryos, however, remains elusive.

These considerations led to the present study, which describes a cryopreservation method for IVP porcine embryos. As a model, we used embryos that were parthenoge-netically developed from IVM oocytes. Postvitrification viability of embryos at the perihatching stage was first investigated due to their high resistance to cryopreservation, after which embryos were vitrified at the early cleavage stages with the future intent to transfer IVP embryos to the recipient’s oviduct. We also report on the effect of delipa-tion on cryotolerance of IVP porcine embryos, and on the successful vitrification of porcine IVP embryos, which were delipated by a newly developed noninvasive method.