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Regular Article |
a Department of Animal Science, University of Connecticut, U-4040, Storrs, Connecticut 06269
ABSTRACT
Successful cryopreservation of mammalian oocytes would provide a steady source of materials for nuclear transfer and in vitro embryo production. Our goal was to develop an effective vitrification protocol to cryopreserve bovine oocytes for research and practice of parthenogenetic activation, in vitro fertilization, and nuclear transfer. Bovine oocytes matured in vitro were placed in 4% ethylene glycol (EG) in TCM 199 plus 20% fetal bovine serum (FBS) at 39°C for 12–15 min, and then transferred to a vitrification solution (35% EG, 5% polyvinyl-pyrrolidone, 0.4 M trehalose in TCM 199 and 20% FBS). Oocytes were vitrified in microdrops on a precooled (-150°C) metal surface (solid-surface vitrification). The vitrified microdrops were stored in liquid nitrogen and were either immediately thawed or were thawed after storage for 2–3 wk. Surviving oocytes were subjected to 1) parthenogenetic activation, 2) in vitro fertilization, or 3) nuclear transfer with cultured adult fibroblast cells. Treated oocytes were cultured in KSOM containing BSA or FBS for 9 to 10 days. Embryo development rates were recorded daily and morphologically high-quality blastocysts were cryopreserved for nuclear transfer-derived embryos at Day 7 or Day 8 of culture. Immediate survival of vitrified/thawed oocytes varied between 77% and 86%. Cleavage and blastocyst development rates of vitrified oocytes following in vitro fertilization or activation were lower than those of the controls. For nuclear transfer, however, vitrified oocytes supported embryonic development as equally well as fresh oocytes.
1 This research is supported in part by a grant from Connecticut Innovation, Inc. The manuscript is a scientific contribution (1396) of the Storrs Agricultural Experiment Station at the University of Connecticut. A.D. was supported in part by OTKA grants T023698 and T030698.
2 Correspondence: Xiangzhong (Jerry) Yang, Department of Animal Science, University of Connecticut, 3636 Horsebarn Rd. Ext. U-4040, Storrs, CT 06269-4040. FAX: 860 486 4375; xiangzhong.yang{at}uconn.edu
3 Current address: Roslin Institute, Roslin, Midlothian EH25 9PS, United Kingdom.
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