93 PRODUCTION OF LIVE PIGLETS BY CRYOPRESERVATION OF IN VITRO-PRODUCED PORCINE ZYGOTES

Abstract

Successful cryopreservation of in vitro-produced porcine zygotes is reported in the present study. Follicular oocytes were collected from prepubertal gilts. They were matured (IVM), fertilized (IVF), and cultured (IVC) in vitro (Kikuchi et al. 2002 Biol. Reprod. 66, 1033–1041). Ten or 23 h after IVF, the oocytes were centrifuged at 10 000g at 37°C for 20 min to permit visualization of pronuclei. Zygotes with two or three pronuclei were selected under stereomicroscope and used for solid surface vitrification (SSV). Briefly, after equilibration in 4% ethylene glycol (EG) for 15 min, zygotes were washed in vitrification solution (35% EG, 5% polyvinyl pyrrolidone, and 0.3 m trehalose), and then dropped with about 2 µL vitrification solution onto the dry surface of aluminum foil floating on the surface of liquid nitrogen (LN₂). Microdroplets were transferred into cryotubes and stored in LN₂. During warming, vitrified droplets were transferred in warming solution (0.4 m trehalose) at 37°C for 1 min, and then consecutively transferred for 1-min periods into 0.2 m, 0.1 m, or 0.05 m trehalose solutions. Survival of vitrified/warmed zygotes was determined by their morphology. To assess their developmental competence, vitrified (SSV), cryoprotectant-treated (CT), and untreated (control) zygotes were cultured in vitro for 6 days. There was no difference in developmental competence between control and CT zygotes in terms of cleavage rates (88.1% and 86.1%, respectively), blastocyst rates (23.2% and 20.8%, respectively), and blastocyst cell numbers (38.0 ± 2.0 and 41.2 ± 1.7, respectively). The rate of live zygotes after SSV and warming was similar to that of the control (93.4% and 100%, respectively). Cleavage rates (71.7% and 86.3%, respectively) and blastocyst rates (15.8% and 24.5%, respectively) of SSV were significantly reduced after vitrification compared to control (P < 0.05, ANOVA). Blastocyst cell numbers of SSV and control embryos were similar (41.2 ± 3.4 and 41.6 ± 3.3, respectively). There was no difference in developmental ability between zygotes cryopreserved at an early (10 h after IVF) or late (23 h after IVF) pronuclear stage. When embryo culture medium was supplemented with 1 µm of the antioxidant glutathione, development of cryopreserved zygotes to the blastocyst stage did not differ significantly from that of the control zygotes (18.6% and 22.1%, respectively). To test their ability to develop to term, 150 vitrified zygotes were transferred into a recipient, resulting in pregnancy and the production of five live piglets. These data demonstrate that a high rate of porcine zygotes could be successfully cryopreserved at the pronuclear stage, preserving their full developmental competence.