67 A HOLLOW FIBER VITRIFICATION METHOD ENABLES CRYOBANKING OF IN VITRO-MATURATION/IN VITRO-FERTILIZATION-DERIVED TRANSGENIC PIG EMBRYOS

We have recently developed a novel high-performance embryo cryopreservation method: the hollow fiber vitrification (HFV) method (Matsunari et al. 2012 J. Reprod. Dev., in press). In this study, we aimed to demonstrate the utility of the HFV method for the cryopreservation of transgenic pig embryos produced by in vitro oocyte maturation/fertilization (IVM/IVF). In vitro-matured oocytes were inseminated with cryopreserved epididymal sperm (Kikuchi et al. 1998 Theriogenology 50, 615–623) from a transgenic pig carrying the humanized Kusabira-Orange gene (Matsunari et al. 2008 Cloning Stem Cell 10, 313–323) and then cultured for 96 h. Morulae with normal morphology were divided into the vitrification and nonvitrification groups. The vitrification of embryos was performed by the HFV method using 20-mM HEPES-buffered TCM199 containing 20% calf serum as a base medium. Cellulose acetate hollow fibers (25 mm), each containing 10 to 20 embryos, were placed in an equilibration solution containing 7.5% ethylene glycol and 7.5% dimethyl sulfoxide for 5 to 7 min and were then placed for 1 min in the vitrification solution containing 15% ethylene glycol, 15% dimethyl sulfoxide, and 0.5 M sucrose. The embryos were then vitrified by immersion in liquid nitrogen and held there for 5 to 10 min. The embryos were warmed by immersing the hollow fiber in a 1-M sucrose solution at 38.5°C, followed by a stepwise dilution of the cryoprotectants using 0.5-M sucrose solution (3 min) and the base medium (10 min). Vitrified and nonvitrified embryos were cultured for 40 h, and their development into blastocysts was evaluated. The in vitro development of vitrified embryos to the blastocyst stage was compared with that of the nonvitrified controls on Day 6. In the embryo-transfer experiments, blastocysts at either Day 5 or Day 6 from both the vitrification and nonvitrification groups were transferred to 3 recipient gilts per group (25–32 blastocysts/gilt), and their development through farrowing was compared. To test long-term preservation, some of the vitrified morulae were kept in liquid nitrogen for 43 days, and their development to Day 30 fetuses was evaluated after transfer to an additional recipient. The differences in proportional data between the 2 groups were analyzed with the χ2-test. Of the 393 putative zygotes obtained by IVM/IVF, 169 (43.0%) developed into morulae. In vitro development of the vitrified morulae to blastocysts (66/85, 77.6%) was comparable with that of the nonvitrified morulae (67/84, 79.8%, not significant: NS). The embryo-transfer experiments resulted in pregnancy in all 6 of the recipients. The production efficiency of piglets (piglets/embryos transferred) was 17/88 (19.3%) for the vitrification group and 27/88 (27.7%, NS) for the nonvitrification group. Approximately 50% of the offspring in both groups were transgenic. Long-term cryopreservation using the HFV method resulted in similar piglet production efficiency (7 piglets produced out of 32 embryos transferred). This study demonstrated for the first time that the HFV method effectively cryopreserves IVM/IVF-derived transgenic pig embryos. Supported by the JST CREST program.