101 CRYOPRESERVATION OF PORCINE EMBRYOS DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

Abstract

In the production of cloned pigs, a large number of nuclear transfer (NT) embryos generally need to be transferred into a single surrogate. Thus, attempts to conduct embryo transfer can be frustrating when either a synchronized surrogate is not available, or enough NT embryos are not produced. This problem would be solved if one could cryopreserve the porcine nuclear transfer embryos. Cryopreservation of porcine embryos has been successful only for in vivo-derived embryos. In vitro-derived porcine embryos are sensitive to chilling, and this sensitivity has been attributed to the lipid droplets in the cytoplasm. In previous reports, the viability of cyropreserved embryos was improved by removal of lipid drops from the cytoplasm. Therefore we designed a procedure to cryopreserve cloned blastocysts by a combination of the open pulled straw (OPS) vitrification method with removal of lipid drops from the oocyte. In vitro-matured MII oocytes were enucleated, and centrifuged (10 000 rpm, 5 min) to polarize the lipid droplets. This was followed by removal of the polarized lipid droplets and transfer of a donor fetal-derived fibroblast cell into the perivitelline space by micromanipulation. After electrical activation and fusion, the NT embryos were cultured in PZM-3 medium with 4 mg/mL BSA. Day 5 and Day 6 blastocysts (manipulation day was Day 0) were vitrified by equilibration with 25 mM HEPES-buffered TCM-199 containing 10% ethylene glycol, 10% DMSO, and 20% fetal calf serum for 2 min, followed by exposure to 20% ethylene glycol and 20% DMSO. Embryos were loaded into an OPS straw and immediately plunged into liquid nitrogen. The process from exposure of embryos to vitrification solution to plunging was 25–30 s. Embryos were thawed by immersing the end of the OPS straw in 0.3 M sucrose in which embryos were kept for 5 min, and then in 0.2 M sucrose for 5 min. Some embryos were cultured in PZM-3 for 12 h to determine the percentage and cell number of re-expanded blastocysts. The others were transferred to the uterus of a surrogate gilt within 3 h of thawing. Lipid removal appeared to have no harmful effect on embryo development and cell number of the blastocysts. Interestingly, a higher blastocyst percentage (28.8%, 178/619) was obtained with NT embryos from which the lipid had been removed as compared to normal NT (19.6%, 44/225; P < 0.01). The cell number (31.2 ± 7.7) of re-expanded blastocysts in the delipation group was comparable with normal NT blastocysts (33.6 ± 14.1, P = 0.33). The survival rate of blastocysts after freezing and thawing was enhanced after delipation (delipation group: 66.7%, 14/21; normal NT group: 21.9%, 9/42; P < 0.01). Two hundred and fourteen delipatized NT blastocysts were transferred to four surrogates after freezing and thawing. Three of the surrogates showed a delayed estrus cycle and one is still pregnant as confirmed by ultrasound scanning. We show that the combination of the OPS vitrification method with removal of lipid drops of oocyte cytoplasm might be an efficient way to cryopreserve porcine NT blastocysts. Funding for this project was from the NIH HL51670 and RR018877 and Food for the 21st Century.