In vitro and in vivo survival of frozen‐thawed bovine oocytes after IVF, nuclear transfer, and parthenogenetic activation

Abstract

Cryopreservation of bovine oocytes would be beneficial both for nuclear transfer and for preservation efforts. The overall objective of this study was to evaluate the viability as well as the cryodamage to the nucleus vs. cytoplasm of bovine oocytes following freezing-thawing of oocytes at immature (GV) and matured (MII) stages using in vitro fertilization (IVF), parthenogenetic activation, or nuclear transfer assays. Oocytes were collected from slaughterhouse ovaries. Oocytes at the GV, MII, or MII but enucleated (MIIe) stages were cryopreserved in 5% (v/v) ethylene glycol; 6% (v/v) 1,2-propanediol; and 0.1-M sucrose in PBS supplemented with 20% (v/v) fetal bovine serum. Frozen-thawed oocytes were subjected to IVF, parthenogenetic activation, or nuclear transfer assays. Significantly fewer GV oocytes survived (i.e., remained morphologically intact during freezing-thawing) than did MII oocytes (47% vs. 84%). Subsequent development of the surviving frozen-thawed GV and MII oocytes was not different (58% and 60% cleavage development; 7% and 12% blastocyst development at Day 9, respectively, P > 0.05). Parthenogenetic activation of frozen-thawed oocytes resulted in significantly lower rates of blastocyst development for the GV than the MII oocyte groups (1% vs. 14%). Nuclear transfer with cytoplasts derived from frozen-thawed GV, MII, MIIe, and fresh-MII control oocytes resulted in 5%, 16%, 14%, and 17% blastocyst development, respectively. However, results of preliminary embryo transfer trials showed that fewer pregnancies were produced from cloned embryos derived from frozen oocytes or cytoplasts (9%, n = 11 embryos) than from fresh ones (19%, n = 21 embryos). Transfer of embryos derived by IVF from cryopreserved GV and MII oocytes also resulted in term development of calves. Our results showed that both GV and MII oocytes could survive freezing and were capable of developing into offspring following IVF or nuclear transfer. However, blastocyst development of frozen-thawed oocytes remains poorer than that of fresh oocytes, and our nuclear transfer assay suggests that this poorer development was likely caused by cryodamage to the oocyte cytoplasm as well as to the nucleus.

Short-Term Preservation of Mouse Oocytes at 5.DEG.C..

“…For long-term storage of genetic resources, the cryopreservation of eggs has been established as a useful technique for laboratory animals [2,3,12,15] and domestic animals [7,9,10]. For short-term preservation, such as during long-distance transport, eggs do not always need to be frozen.…”

Closed system for bovine oocyte vitrification

“…Nevertheless, as Vajta et al (2009) write, the survival of vitrified oocyte is determined by a number of factors and the effect on the overall spindle condition should not be overestimated when compiling the protocol. This fact is also supported by results from MII stage oocyte cryopreservation quite comparable to GV stage (in which the spindle is not yet assembled) oocyte cryopreservation results (Fuku et al 1992;Kubota et al 1998;Zhou et al 2010). Our data confirmed that because the survival rate and following development of fertilized oocytes in preheated vitrification solution were equal to results reached at room temperature conditions.…”

Protective effects of the cumulus-corona radiata complex during vitrification of horse oocytes

“…Cryopreserved oocytes can, in theory, be used to produce offspring via assisted reproductive techniques such as in vitro embryo production (Fuku et al 1992, Vajta et al 1998 or oocyte transfer (Maclellan et al 2002), and can also serve as host cytoplasts for cloning by nuclear transfer (Kubota et al 1998, Dinnyes et al 2000, Atabay et al 2004. However, while offspring have been produced using frozen-thawed oocytes in various species (e.g.…”