Cryobiology of Rat Embryos I: Determination of Zygote Membrane Permeability Coefficients for Water and Cryoprotectants, Their Activation Energies, and the Development of Improved Cryopreservation Methods During the 1nd Semester 2012-2013 A Research Paper Presented to Mr. Luis Gallardo Mago In partial fulfillment Of the requirements in Nat. Sci. Subject

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Table of contents

Abstract
Materials and methods
Results
Discussion
References
Figures
Tables

Abstract

Cryobiology of Rat Embryos I: Determination of Zygote Membrane Permeability Coefficients for Water and Cryoprotectants, Their Activation Energies, and the Development of Improved Cryopreservation Methods.
Reinhold T. Pfaffa,b, Yuksel Agcaa,b, Jun Liua, Erik J. Woodsa, Augustine T. Peterb and John K. Critser2,,a,b a Cryobiology Research Institute, Herman B Wells Center for Pediatric Research, Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana 46202 Department of Veterinary Clinical Sciences, b School of Veterinary Medicine, Purdue University, West Lafayette, Indiana 47907
New rat models are being developed at an exponential rate, making improved methods to cryopreserve rat embryos extremely important. However, cryopreservation of rat embryos has proven to be difficult and expensive. In this study, a series of experiments was performed to characterize the fundamental cryobiology of rat fertilized 1-cell embryos (zygotes) and to investigate the effects of different cryoprotective agents (CPAs) and two different plunging temperatures (Tp) on post-thaw survival of embryos from three genetic backgrounds. In the initial experiments, information on the fundamental cryobiology of rat zygotes was determined, including 1) the hydraulic conductivity in the presence of CPAs (Lp), 2) the cryoprotectant permeability (PCPA), 3) the reflection coefficient (), and 4) the activation energies for these parameters. PCPA values were determined for the CPAs, ethylene glycol (EG), dimethyl sulfoxide (DMSO), and propylene glycol (PG). Using this information, a cryopreservation method was developed and the cryosurvival and fetal development of Sprague-Dawley zygotes cryopreserved in either EG, DMSO, or PG and plunged at either -30 or -80°C, were assessed. The highest fetal developmental rates were obtained using a Tp of -30°C and EG (61.2% ± 2.4%), which was not different (P > 0.05) from nonfrozen control zygotes (54.6% ± 3.0%).

Introduction

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) . 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 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.

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 , 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.

Materials and methods

Fundamental Cryobiology of Rat Embryos
Source of embryos. Rats, 8–10 wk of age from three lines were used in these experiments. One was an outbred stock (HSD: Sprague-Dawley, SD); and two that were originally derived from crossbreeding eight different inbred strains (ACI, BN, BUF, F344, M520, MR, WKY, and WN) and selected through 15 generations for either high alcohol drinking (HAD) tolerance or low alcohol drinking (LAD) tolerance .
The estrous cycles of the female rats were monitored by daily evaluation of vaginal cytology in late afternoon . Female rats in proestrus or early estrus were mated, and mating was verified on the following morning by the presence of a vaginal plug, sperm in vaginal smears, or both.
For collection of zygotes, rats were i.m. injected with 0.1 ml of a 10:2 (v/v) mixture of 100 mg/mL ketamine (Ketaset, Fort Dodge Labs., Fort Dodge, IA):100 mg/ml xylazine (Rompun, Bayer Agriculture Division, Shawnee Mission, KS) for sedation, then killed. The oviducts were excised and placed into a culture dish containing PBS (Life Technologies Inc., Grand Island, NY) supplemented with 1 mg/ml hyaluronidase (Sigma Chemical Co., St. Louis, MO) and 4 mg/ml BSA (Sigma). The extended, translucent oviductal ampulla was dissected to release the clutch of zygotes into the solution in a culture dish.
Zygotes were incubated with hyaluronidase for approximately 5 min to enable dissociation and removal of the cumulus cells. A stereodissecting microscope (Nikon SMZ-2T, Fryer Co. Inc., Huntley, IL) was used to evaluate zygotes for the presence of polar bodies and pronuclei to verify fertilization. The cumulus-free zygotes were washed by transferring them through three drops of hyaluronidase-free PBS before being placed into 40-µl culture drops of Hams F10 under oil in culture dishes, and placed in an incubator (humidified 5% CO2/95% N2) at 37°C. The zygotes were subjected to osmotic experiments within 0.25 to 5 h after collection.
Perfusion and image analysis. Rat zygotes were washed in PBS, transferred into a 10-µl drop of PBS in the perfusion dish, and then immobilized using a holding pipette as previously described by Gao et al.. Two ml of 1.5 M cryoprotectant medium (either EG, DMSO, or PG in PBS) were pre-equilibrated at the experimental temperatures, then added to the perfusion dish where the zygotes were perfused at either 25, 14, or 3°C. The osmotic responses were observed using 40x magnification on an inverted Nikon compound microscope (Diaphot, Nikon, Fryer Co. Inc.) and videotaped for image analysis. The videotaped images were digitized (Haupage Computer Works, Haupage, NY) to enable collection of volume and surface area data. These data were based on two diameter measurements (minimum and maximum diameter, which were taken at an angle of approximately 90°) on the two-dimensional image of the zygote. Assuming a spherical shape, the average of the two diameter measurements was used to calculate the volume (V) and surface area (A) of a zygote.

For embryos perfused at 25°C, diameter measurements were performed at 4-sec intervals during the cell shrinkage phase, and at 10-sec intervals during the initial minutes of the swelling phase, then at 20-sec intervals during the later part of the cell-swelling phase. For those zygotes perfused at 14°C, diameter measurements were obtained at 10-sec intervals during the shrinkage phase, and at 10- and 20-sec intervals during the swelling phase. For embryos perfused at 3°C, diameter measurements were obtained at 60-sec intervals during both shrinkage and swelling phases.
Determination of the permeability coefficient of CPAs. Kedem and Katchalsky formulated a theoretical model for the permeability of membranes based on the theory of irreversible thermodynamics. For a ternary solution consisting of a permeable solute (in these experiments either EG, DMSO, or PG), and an impermeable solute (NaCl), a pair of coupled nonlinear equations that describe the cell volume and the concentration of the permeating solute in the cell as functions of time are used:

where V is cell volume, A is the surface area of a zygote, M is osmolality, and (where m is the molal concentration). The superscripts "i" and "e" refer to intracellular or extracellular cell compartments, respectively. Lp, PCPA, and are the hydraulic conductivity, the permeability coefficient of the CPA, and the reflection coefficient, respectively. Temperature and the universal gas constant are indicated by T and R.
For the impermeable solute (NaCl), the intracellular osmolality of NaCl is given by:

where Vb is the osmotically inactive cell volume and Vs = (s is the partial molar volume of the CPA). The superscript "(o)" represents the values at t = 0.

During the kinetic experiments, zygotes were perfused with a 1.5 M concentration of either EG, DMSO, or PG in PBS. The extracellular CPA concentration (1.5 M) and NaCl osmolality () were assumed to be invariant with time. At t = 0, there was no CPA inside the cell, so (t = 0) = Viso, where Viso, the isosmotic volume, is determined at the beginning of the osmotic response experiments. These were the initial conditions for the first and second differential equations. The commercial software package, MLAB (Civilized Software, Inc., Bethesda, MD) was used to solve the differential equations using the Gear method. The Marquard-Levenberg method, as implemented in MLAB, was used to carry out the three-parameter fit to the experimental data and to estimate the values of Lp, PCPA, (PEG, PDMSO, and PPG), and . Similar calculations were repeated at three different temperatures (25, 14, and 3°C).
Determination of the activation energy for Lp and PCPA. The Arrhenius relationship was used to determine the activation energies for the parameters Lp(T), PCPA(T), and . The Arrhenius relationship assumes that the value of a parameter (Para) will change with temperature as:

where Para = Lp, or PCPA, or , and Ea(Lp, PCPA, ) is the activation energy for the associated parameter. The subscript "(o)" represents the values at a reference temperature To. Activation energy values, Ea(Lp, PCPA, ), can be determined as the product of the term of (-R) and the slope of the Arrhenius plot (ln[Para] vs. 1/T).
Statistical analysis. Embryos from individual donor animals were blocked by donor and randomly assigned to treatment groups. All data were analyzed using standard ANOVA approaches using the Statistical Analysis System . Tukeys multiple range tests were used to compare means among treatments .
Rat Embryo Cryopreservation and Transfer
The protocol used for embryo cryopreservation and thawing was developed using a theoretical model (see accompanying article by Liu et al. in this issue). Physical-chemical data developed for DMSO solution properties were used in conjunction with the permeability characteristics measured in this study because the most complete set of information exists for solutions of this CPA (i.e., critical concentrations/cooling and warming rates necessary for vitrification; phase diagram information for the DMSO/NaCl/H2O ternary system) .

Equilibration with cryoprotectant, cooling, and storage. After removal of cumulus cells and evaluation for normal fertilization (polar bodies and pronuclei), the zygotes were placed into 1.5 M CPA for 12–15 min. After equilibration, 20 zygotes per straw were loaded into the CPA-containing column within 0.25-ml plastic straws, and the straws were then heat-sealed. The proximal 60% of the straws contained a column of fluid consisting of 0.5 M sucrose in PBS, and the distal 40% of the straws contained three small columns consisting of permeable CPA in PBS solutions. All solution columns were separated by air bubbles to prevent mixing. The embryos were placed into the middle column of the three permeable CPA-containing columns using a disposable, pulled and polished micropipette (Fisher Scientific, Pittsburgh, PA) with an inner lumen diameter of approximately 180–200 µm. The straws were then placed into a programmable freezer (Planer, Model Kryo 10 Series II; Perkasie, PA), cooled to the seeding temperature of -7°C, and held at -7°C for 15 min. Five min after reaching the seeding temperature, seeding (ice crystallization in the extracellular space) was induced by pinching the straw containing the sucrose/PBS column with forceps, which had been dipped in LN2. Ten min after seeding, cooling was continued at a rate of 0.5°C/min until reaching the plunging temperature (-30°C or -80°C), at which time the straws were quickly removed from the freezing apparatus and immediately immersed in LN2 until thawed.
Thawing and zygote transfer into recipients. For embryo transfer, straws containing the zygotes were thawed by quickly retrieving the plastic straws from the LN2 dewar followed by immediate immersion and rapid thawing in a 37°C water bath for 20 sec. After drying the straws with tissue paper, the tips of the straws were cut and the contents of the straw were emptied into a Petri dish. The zygotes were maintained for approximately 5 min in the CPA-sucrose-PBS mixture that was expelled together with the CPA solution, and then transferred into an isotonic PBS solution.
Assessment of embryo post-thaw survival. After thawing and removal of the CPA, zygote morphology was evaluated using a stereodissecting microscope (Nikon SMZ-2T) to assess post-thaw survival rates. Only those zygotes that were found after unloading and that had an intact zona pellucida and intact and normal-appearing plasma membrane and cytoplasm were counted as viable and considered suitable for transfer into recipient rats. The post-thaw survival ratio was defined as the ratio of the number of intact and normal-appearing zygotes over the number of cryopreserved zygotes (20 per straw).
Embryo transfer. To obtain pseudopregnant embryo recipient rats, 8- to 10-wk-old female rats of the Long Evans (HSD: LE [LE]) stock were synchronized using the same protocol as for zygote production, and mated 1:1 with mature, vasectomized LE males. For verification of mating and hence pseudopregnancy, the female rats were removed the following day between 0800 and 0930 h and checked for the presence of a vaginal plug. In addition, vaginal cytology was evaluated for the presence of cells consistent with either predominant presence of cornified superficial cells (indicating metestrus), presence of a mixed population of predominantly cornified superficial cells and leukocytes (late metestrus/early postestrus), or predominant presence of leukocytes alone (indicating metestrus II). Synchronous embryo transfers were performed (i.e., a recipient's estrous cycle stage was timed to be synchronous with the embryo developmental stage) because this approach has been reported to provide good results .
For embryo transfer, the recipient rats were anesthetized by injecting 0.13 ml of the ketamine:xylazine mixture (described earlier) into the biceps femoris muscle. Approximately 5 min after the injection, the dorsal and lateral skin was clipped and the surgery site prepared with three alternate scrubs of Betadine and alcohol. The skin of the dorsum was sagittally incised (10–15 mm in length) on the midline at the level of the paralumbar fossa. The skin was undermined by blunt dissection. The skin incision was then rolled laterally to superimpose the paralumbar fossa. The muscle layers and the peritoneal wall were opened via incision and spread to create a window about 8–12 mm in length, which permitted retraction of the ovarian fat pad. The ovary was gently grasped with a tissue forceps and retracted/extracted until the ovary, ovarian bursa, and oviduct were exposed and visualized using a stereomicroscope (Stereozoom #5, Leica and Nicholas Illuminator; Fisher Scientific) at 10x–15x. A few drops (25 µl) of epinephrine (VEDCO, St. Joseph, MO; 1 mg/ml) were sprinkled over the bursa to reduce bleeding. The bursa was gently dissected using watchmaker forceps to gain access to the infundibulum. Embryos were loaded into a small column consisting of a few µl of PBS within a 150-µm diameter transfer pipette. The transfer pipette was inserted into the infundibulum, 6–10 zygotes discharged into the oviduct, and then the ovary was carefully replaced into the peritoneal cavity. After repeating the procedure on the contralateral side, the skin was closed using wound clips (Autoclip Applier and 9-mm clips; Fisher Scientific).
Fetal development rates after transfer into pseudopregnant recipients were determined by assessing in vivo development at Days 16–18 of gestation (Day 1 = morning after mating with a vasectomized male). Pseudopregnancy was verified by the presence of a vaginal plug and the presence of a population of cells in the vaginal cytology, which is consistent with metestrus [28]. Recipient animals were sedated and killed between Days 16 and 18. The abdomen of the recipient was incised and the fetuses counted. Only those fetuses showing normal, homogeneous/synchronous development as expected at the day of pregnancy were included. Those fetuses that were smaller in size or being absorbed were not counted.

Results

Fundamental Cryobiology of Rat Embryos

Rat zygote volume excursion during perfusion with the CPA-containing solution was videotaped to enable subsequent image analysis. These videotaped images depicted zygote volume excursions during perfusion after a change in the extracellular medium from isotonic PBS to a solution containing 1.5 M CPA (either EG, DMSO, or PG) in PBS. All zygotes initially decreased in volume due to water efflux in response to the initial change in the chemical potential of water between the intracellular and extracellular compartments. This was followed by a slow increase in volume as CPA entered the cell and water followed to maintain chemical equilibrium. At 25°C, zygotes regained their original volume and eventually reached approximately 110% of their original isosmotic volume. At 14°C, the volume-increase phase was slightly prolonged, and at 3°C, both the volume-decreasing and, even more pronounced, the volume-increasing phase were prolonged (Fig. 1).
Membrane permeability data for different rat lines and CPAs at different temperatures are summarized in Tables 1, 2, and 3. There were no differences among the three CPAs with respect to membrane permeability for Lp and PCPA (P > 0.05) at a given temperature.
Temperature significantly affected (P p and PCPA. Mean values of Lp and PCPA decreased as the temperature was decreased among all three CPAs and all three rat lines. Sigma values increased with lower temperature, although the effect of temperature was not as large as that on Lp and PCPA.
Hydraulic conductivity. The Lp values for 25, 14, and 3°C are shown in Tables 1, 2, and 3.. There were no significant differences among Lp mean values in the presence of the different CPAs (P > 0.05) at any of the three experimental temperatures (25, 14, and 3°C), or among the three rat lines (P > 0.05). However, Lp values were significantly different among temperatures (P
CPA permeability. The PCPA values for 25, 14, and 3°C are listed in Tables 1, 2, and 3.. There were no significant differences (P > 0.05) among PCPA values in presence of the different CPAs at any of the three experimental temperatures (25, 14, and 3°C). However at 25°C, there were significant differences among PCPA mean values for the different rat lines (P of EG. This line difference was not significant at 3 or 14°C for any CPA.
Sigma. The values for 25, 14, and 3°C are listed in Tables 1, 2, and 3.. There were no significant differences among mean values at 25, 14, or 3°C (P > 0.05) among the different CPAs or different rat lines. Sigma values were significantly different (P CPA/line combinations (HAD/DMSO, HAD/PG, HAD/EG, and LAD/PG), but not significantly different (P > 0.05) for all other cases.

Activation energies. The activation energy (Ea) values for Lp () and PCPA () are shown in Table 4. The CPA-dependent activation energies for the hydraulic conductivity () ranged from 12.52 kcal/mol in the presence of EG for the LAD line, to 15.68 kcal/mol in presence of EG for the SD zygotes. The activation energies for PCPA () ranged from 11.78 kcal/mol for EG for the SD stock to 20.58 kcal/mol for PG for the SD stock. The activation energies for were negative values and ranged from -5.53 kcal/mol for PG/HAD to -1.41 kcal/mol for PG for the SD stock.
Rat Embryo Cryopreservation and Transfer
Embryo survival after thawing. The overall post-thaw viability of SD zygotes ranged from 66% to 81% (Table 5) and there were no significant differences (P > 0.05) among the three CPAs or between the two plunging temperatures (Tp). The highest post-thaw survival rates (81% ± 5%) were obtained using EG with a Tp of -80°C. The lowest post-thaw survival rates (66.0% ± 4.7%) were obtained using EG with Tp of -30°C. These rates were not different (P > 0.05) from rates obtained with either DMSO (72.5% ± 3.7% and 73.5% ± 8.6%) or PG (73.0% ± 2.6% and 72.0% ± 7.7%) at a Tp of -30 or -80°C, respectively.
Embryo development in utero. Overall fetal development of SD zygotes (to Days 16–18) ranged from 8% to 61% (Table 5). Fresh, nonfrozen zygotes were used as a control and resulted in fetal development of 54.6% ± 3.0%. Average fetal development was significantly affected by both cryoprotectant type (P (P highest when EG was used as the CPA (vs. DMSO or PG) and when -30°C was used as the Tp (vs. -80°C). The highest fetal development rate (61.2% ± 2.4%) was achieved using a combination of EG as the CPA, and -30°C as the Tp. Importantly, there were no significant differences (P > 0.05) in fetal development between the control (nonfrozen) zygotes or those cryopreserved using either EG or DMSO (averaged across plunging temperatures).

Discussion
Fundamental Cryobiology of Rat Embryos
Rat embryo cryopreservation is becoming increasingly important as a result of advances in genetic technologies that permit the rapid creation of new genetically engineered rat models for studying health and disease, and their effect on functional genomics. The zygote stage is predominately used as the developmental stage for production of these genetically altered animals. The increasing numbers of rat models, combined with the need to reduce the cost of animal housing, requirements for simple and efficient shipment, as well as the need to protect these animal models against catastrophic loss have combined to create a crucial need for effective and efficient cryopreservation of rat embryos. This study initiated the evaluation of the membrane permeability of the rat zygote from three different lines of rats for three different cryoprotectants. To the best of our knowledge, this is the first report describing the membrane permeability characteristics of rat embryos.
Hydraulic conductivity and its activation energy. The hydraulic conductivity (Lp) results obtained in this study are similar to those described for mouse zygotes (0.43 µm/min per atmosphere at 20°C in hypertonic saline and 0.81 µm/min per atmosphere at 24°C in the presence of 1.5 M DMSO ). These results are also similar to Lp values previously determined for metaphase II mouse oocytes (0.44 µm/min per atmosphere at 20°C in hypertonic saline and 0.4 and 0.07 µm/min per atmosphere at 20 and 3°C in 1.5 M DMSO ) as well as metaphase II bovine oocytes (0.57 and 1.16 µm/min per atmosphere at 20 and 3°C in 1.5 DMSO).
Measuring the hydraulic conductivity at different temperatures enabled the determination of the associated activation energies (). The () mean values ranged from 12.52 kcal/mol to 15.68 kcal/mol. The values are similar to those values reported for mouse zygotes (13 kcal/mol in hypertonic saline ), which are slightly smaller than those reported for metaphase II mouse oocytes (16.3 kcal/mol in DMSO ), and substantially larger than those reported for metaphase II hamster oocytes (7.9 kcal/mol in hyperosmotic saline ).
CPA permeability and its activation energy. Very little data are available regarding the cryoprotectant permeability of mammalian zygotes, which would allow direct comparison across species. Jackowski et al. determined the glycerol permeability (PGLY) of unfertilized and fertilized mouse ova at approximately 20, 12, and 3°C. They obtained values of 0.01 x 10-3, 0.002 x 10-3, and 0.0005 x 10-3 cm/min for unfertilized mouse ova; and 0.034 x 10-3, 0.013 x 10-3, and 0.0047 x 10-3 cm/min for fertilized ova, respectively, using a photographic method to determine the osmotic changes over time and calculating the labeled glycerol uptake. The mean PCPA values for EG, DMSO, and PG in this study are approximately two orders of magnitude higher when compared with the data from Jackowski's study . This may be the result of a general higher membrane permeability for DMSO, EG, and PG. The PCPA values from this study are similar to the values reported for metaphase II mouse oocytes: PDMSO of 1.44 x 10-3 cm/min at 20°C ; PDMSO of 1.07 x 10-3 and 0.078 x 10-3 cm/min at 20 and 3°C ; and metaphase II bovine oocytes: PDMSO of 2.6 x 10-3 and 0.3 x 10-3 cm/min at 20 and 4°C .

Measuring the CPA permeability (PCPA) at different temperatures enabled the determination of the associated activation energies for PCPA (). The mean value was highest for PG and lowest for EG. These mean values are similar to those reported for mouse zygotes (18.7 kcal/mol in glycerol ), but lower than those reported for metaphase II mouse oocytes (28.4 kcal/mol in glycerol and 23.2 kcal/mol in DMSO ).
Sigma and its activation energy. The selectivity of the membrane for water and the properties of the cryoprotectant compound used determine the value, which is constrained between zero and one; the larger the sigma value, the more selective the membrane is to a given solute. In this experiment, the pooled mean values ranged from 0.65 at 25°C to 0.91 at 3°C.
Measuring sigma at different temperatures enabled the determination of the associated activation energies (Ea) for (). The values in this study were significantly temperature-dependent and increased with a decrease in temperature. This suggests that the membrane becomes more selective for solutes at decreased temperatures, which may be due to phase transitions of plasma membrane components. The same phenomenon was previously reported and has been discussed in detail for mouse oocytes by Agca et al. .
Development of Improved Rat Embryo Cryopreservation Methods
Using the information gained from these experiments, which were designed to characterize the fundamental cryobiology of the rat zygote, a practical cryopreservation method was established on the basis of a theory (see Liu et al., this issue). The membrane permeability coefficients and their activation energies can be used in calculations to predict an optimal "two-step" cryopreservation protocol. The two-step or "interrupted slow freezing" (ISF) method is a common approach currently used to cryopreserve many different cell and tissue types.
This procedure consists of 1) a slow cooling step, in which the sample is cooled from the seeding temperature to an intermediate (or "plunging") temperature at a relatively slow cooling rate (B1); and 2) a nonequilibrium cooling step that occurs when a sample is plunged into liquid nitrogen (LN2). The strategy behind this method involves slow cooling to concentrate the intracellular solution high enough to reach a critical concentration (Cc) for either intracellular vitrification or formation of small innocuous ice crystals during the high cooling rates (B2) that are associated with plunging relatively small samples directly into LN2 .
The overall mean values (across CPAs and lines) of Lp and PCPA were used in the following calculations because the membrane permeability data characterized in this study indicated little variation in regard to plasma membrane permeability among the CPAs and lines. In order to obtain the most robust estimate of the Ea for the subsequent calculations, four Ea values were chosen for use: the minimum and maximum values of and over all Eas estimated for the different CPAs and lines. Four combinations of these Eas ( and ; and ; and ; and and ) were used in the calculations. It was determined that the cooling rate (B1) range is highly dependent on values and very insensitive to values. This makes it possible to adapt a common protocol for all investigated rat lines because the values are more consistent among them than the values.

Briefly, to achieve optimization of initial CPA concentrations ([CPA]0) (0.1 to 2.5 molal) and cooling rates (B1) (0.1 to 2.5°C/min), the ranges were categorized into 25 intervals for subsequent numerical calculation. For each combination of B1 and [CPA]0, the intracellular solute concentration ([S]i) and the degree of supercooling were calculated at each temperature point from the seeding temperature to -80°C. Next, the B1 and [CPA]0 combinations that would not be predicted to allow [S]i to exceed the critical concentration at any temperature, or that would be predicted to result in a high probability of IIF (as indicated by Mazur's three requirements for IIF ) were then excluded. For the remaining B1 and [CPA]0 combinations, the Tp values at which the [S]i would reach the required [CPA]c were determined (Liu et al., this issue). Hence, the optimal ranges of initial CPA concentrations, cooling rates, and plunging temperatures from a vast array of potential cryopreservation protocols were determined.
The theoretical calculations indicated that many B1 and [CPA]0 combinations could be used to cryopreserve rat zygotes with equal success. A relatively conservative protocol was chosen from these ranges to be used in the freezing experiments. Specifically, an initial CPA concentration ([CPA]0) of 1.5 M, an initial cooling rate (B1) of 0.5°C/min to a plunging temperature (Tp) of -30°C, followed by plunging into LN2 (Fig. 2) was chosen. Because this range was determined using the extreme values of and , the resulting protocol would be expected to be robust and satisfactory for a variety of different rat lines.
Embryo survival after cryopreservation. The post-thaw survival determined by morphological evaluation was similar among treatment groups. A fraction of the embryos (19%–34%) was lost, or severely damaged, lysed (or both) after thawing. These were discarded.
Embryo development in utero. The results of the experiments to address the fundamental cryobiology of rat embryos indicated rather small differences in the permeability characteristics. Based upon this observation, each of the three CPAs would have been expected to provide similar levels of protection during cryopreservation. However, although the mean PCPA values for the three CPAs used in this study were very similar, the results of the embryo transfer experiments indicated very different effects of the different CPAs. The different outcomes observed in regard to in vivo development of the SD zygotes after thawing and transfer between CPAs are likely the result of differing sensitivity of rat zygotes to toxic effects of the cryoprotectants (i.e., true biochemical toxicity vs. osmotic damage).
The different outcomes in regard to plunging temperatures represent a more complex situation. Because of the problems associated with obtaining a large number of rat embryos (described earlier), it was not possible to conduct a complete factorial experiment to examine all of the component steps in the cryopreservation process. Therefore, while two Tp treatments were investigated, a single warming rate was used. It is important to note that for many cell types, there is a well-characterized "cooling rate by warming rate interaction". For example Critser et al. previously reported for mouse embryos that slow cooling to low plunging temperatures requires slow rather than rapid warming to achieve high survival rates. Therefore, it is important to emphasize that in this study, although a significantly lower fetal developmental rate was obtained with the -80°C Tp treatments; if additional (i.e., slower) warming rates had been used in combination with a Tp of -80°C, it is very possible that those results would have been similar to those observed here with a Tp of -30°C and a relatively rapid warming rate.
The data described here represent, to the best of our knowledge, the first attempt to develop an understanding of the fundamental cryobiology of rat embryos. These data indicate that, at least among the lines investigated here, genotypic differences among rats do not result in large differences in the basic cryobiological properties of the zygote stage embryo. However, important differences in these parameters may become apparent among other rat genotypes. they may occur at later developmental stages, or both. The cryopreservation protocol developed here can serve to provide high survival of cryopreserved rat zygotes. Future experiments will focus on investigating the nature of the observed CPA toxicity to rat embryos as well as the characterization of the fundamental cryobiology of later developmental stage rat embryos.

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Figures
FIG. 1. Predicted volume changes of rat zygotes in 1.5 DMSO vs. time at three temperatures (3, 14, and 25°C). Volume changes were calculated using the estimated permeability parameters obtained by fitting the theoretical curve to the experimental data

FIG. 2. The combination of cooling rates (B1) and initial CPA concentrations ([CPA]0) that can be used to cryopreserve rat zygotes. Four combinations of Eas ( and ; and ; and ; and and ) were used in calculations. The maximum zone (triangles) and the minimum zone of (solid circles) B1 and [CPA]0 combinations are plotted in A. Plunging temperatures (Tp) for these B1 and [CPA]0 combinations are plotted in B. The B1 and [CPA]0 combinations within the minimum zone can be used for different rat lines and CPAs

Source: Biology of Reproduction 63, 1294-1302 (2000). © 2000 Society for the Study of Reproduction, Inc.
Tables
TABLE 1. Estimates (mean ± SEM) of Lp, PCPA, and {} for EG among different rat lines at 3, 14, and 25°C.*

TABLE 2. Estimates (mean ± SEM) of Lp, PCPA, and {} for DMSO among different rat lines at 3, 14, and 25°C.*

TABLE 3. Estimates (mean ± SEM) of Lp, PCPA, and {} for PG among different rat lines at 3, 14, and 25°C.*

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TABLE 4. Activation energies (Ea) for Lp, PCPA, and {} for different CPAs and rat lines.*

TABLE 5. Post-thaw embryo survival and fetal development of Sprague-Dawley rat zygotes