Since the first report of successful mouse embryo cryopreservation , cryopreservation of many mammalian embryos has become a relatively routine and efficient technology. Embryo cryopreservation has become a widespread and common procedure for the long-term storage of mouse strains; however, rat embryos are less frequently cryopreserved, and limited data are available on successful methods for their cryopreservation.
Most previous reports of rat embryo cryopreservation have been based upon the use of standard cryopreservation protocols that were previously developed for embryo cryopreservation of other species (e.g., cattle or mouse) [2–7]. However, a few previous reports have investigated protocols that were specifically developed for rat embryos.
Among these were Utsumi et al.  using various polyols as cryoprotective agents (CPAs) and cooling the embryos within plastic straws in liquid nitrogen (LN2) vapor. In that study, the efficacy of various polyols as cryoprotectants (1–7 OH groups per molecule, and molecular weights between 32 and 212) in rat embryo cryopreservation was evaluated.
More recent reports have addressed rat embryo cryopreservation using vitrification methods [9–13] for embryos at the 1-cell, 2-cell, and morula stages. Most of these experiments represent only a few genetic backgrounds of rats, of which the Wistar rat was predominant. To date, for the rat, it is not known whether there are genetic differences in membrane permeability or embryo survival rates following cryopreservation, as has been shown for the mouse .
Methods for superovulation of immature rats have been described using FSH  or using eCG . Unlike the mouse model, however, these superovulation protocols do not reliably result in large numbers of oocytes per donor. Superovulation attempts often result in large numbers of unfertilized oocytes or retarded embryos . Therefore, a major issue contributing to the difficulty in cryopreserving rat embryos is related to the generally poor superovulatory response of rats and the difficulty in obtaining embryos.
The problem of obtaining rat embryos is exacerbated when attempts are made to cryopreserve embryos at later developmental stages because naturally mated, nonsuperovulated, postpubertal rats are commonly used as embryo donors in these cases. For example, Isachenko et al.  obtained an average of about 5.4 embryos (7–12 blastomeres per embryo on Day 4) using naturally cycling adult Wistar rats. In this regard, it is more efficient to use an earlier developmental stage, such as the zygote, which can be readily recovered from the oviductal ampulla, instead of the oviducts and uteri.
The value and implications of understanding the membrane permeability coefficients of embryos with respect to solution-effects injury and onset and extent of intracellular ice formation (IIF) as a function of cryoprotectant concentration and cooling rate were extensively described by Mazur [16, 17].
Solution-effects injury refers to the detrimental effects of long exposure of cells to high intracellular solute concentration, which is primarily caused by slow cooling, whereas IIF is usually a result of rapid cooling. Osmotic responses of cells during cooling are in large part dependent upon their membrane permeability coefficients, and even more on the associated activation energies (because of their exponential relationship with the temperature). These intrinsic properties of cells determine how the cells will respond to the series of steps involved in the cryopreservation process.
Knowledge of the underlying cryobiological properties of a given cell type allows the determination of specific optimal components of the overall cryopreservation process. In this regard, optimal CPA type and concentration, cooling rate, plunging temperature, and warming rate combinations can be determined. A central premise of cryobiology is to use a cooling rate that is high enough to minimize solution effects injury but low enough to prevent IIF.
The determination of the permeability of cells to water and cryoprotectant, together with the availability of osmotic tolerance limits, enables the design of optimal procedures to load CPA into cells and remove CPA from cells. Osmotic tolerance limits have been established for a variety of cell types, including human, mouse, and boar spermatozoa [18–20], bovine oocytes , murine fertilized ova , murine and bovine embryos , pancreatic islets , and human umbilical cord blood hematopoietic progenitor cells . The determination of the activation energies enables theoretical calculation of intracellular state (such as cell water volume, solute concentration, and supercooling) during cooling. This information, together with critical concentration and IFF, make it theoretically possible to design a cryopreservation protocol.
The objective of this study was to first initiate characterization of the fundamental cryobiological parameters of rat zygotes for three selected lines of rats, including 1) the hydraulic conductivity (Lp), cryoprotectant permeability coefficient (PCPA), and ; and 2) the activation energies of these parameters; and second, to evaluate the effects of three different CPAs and two plunging temperatures on rat embryo cryosurvival and subsequent in vivo development.