84 PRODUCTION OF CLONED PIGLETS FROM NUCLEAR TRANSFER EMBRYOS AFTER VITRIFICATION

Cryopreservation of cloned embryos is expected to be beneficial in improving the efficiency of somatic cell cloning in pigs. We have already demonstrated that normal piglets can be produced from in vitro-matured and fertilized (IVM/IVF) embryos vitrified at an early cleavage stage after delipation (Nagashima et al. 2007 Biol. Reprod. 76, 900–905). In this study we utilized this technique in an attempt to produce piglets from cloned embryos reconstructed with IVM oocytes. Nuclear transfer (NT) embryos were reconstructed using oocytes matured in vitro in NCSU23 and preadipocytes as nuclear donors. The embryos were cultured in PZM-5 for approximately 98 h, and those that had developed to the morula stage were delipated using a noninvasive method described previously (Esaki et al. 2004 Biol. Reprod. 71, 432–437). The embryos were treated with 4% trypsin at 38°C for 1 to 4 min to induce a slight swelling of the zona pellucida, and then centrifuged (12 000g, 38°C, 23 min) with 7.5 µg mL–1 cytochalasin B to polarize cytoplasmic lipid droplets within the perivitelline space. The embryos were cultured for 1 h and vitrified by the minimum volume cooling (MVC) method using a MVC plate (Cryotop®; Kitasato Supply Co., Tokyo, Japan) in the presence of 15% ethylene glycol, 15% DMSO, and 0.5 m sucrose as cryoprotectants. Vitrified embryos were rewarmed by immersing the MVC plate diretly into rewarming solution containing 1 m sucrose and 20% calf serum at 38°C for 1 min, followed by stepwise dilution of the cryoprotectants. The rewarmed embryos were cultured for 2 days to the blastocyst stage, and then treated with 0.5% pronase to remove the zona pellucida before transfer to the uterine horn of recipients. A total of 103 vitrified blastocysts were transferred to 2 recipient gilts. Both gilts became pregnant and farrowed 2 and 4 piglets, respectively (6/103, 5.8%). These results demonstrate that cloned piglets can be produced from NT embryos that have been cryopreserved at the morula stage using noninvasive delipation and vitrification procedures. This study was supported by PROBRAIN.