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Biol Reprod. 2008 November; 79(5): 832–840.
Prepublished online 2008 July 16. doi:  10.1095/biolreprod.107.067371
PMCID: PMC2658816
NIHMSID: NIHMS101150

Efficient Term Development of Vitrified Ferret Embryos Using a Novel Pipette Chamber Technique1

Abstract

Development of an efficient cryopreservation technique for the domestic ferret is key for the long-term maintenance of valuable genetic specimens of this species and for the conservation of related endangered species. Unfortunately, current cryopreservation procedures, such as slow-rate freezing and vitrification with open pulled straws, are inefficient. In this report, we describe a pipette tip-based vitrification method that significantly improves the development of thawed ferret embryos following embryo transfer (ET). Ferret embryos at the morula (MR), compact morula (CM), and early blastocyst (EB) stages were vitrified using an Eppendorf microloader pipette tip as the chamber vessel. The rate of in vitro development was significantly (P < 0.05) higher among embryos vitrified at the CM (93.6%) and EB (100%) stages relative to those vitrified at the MR stages (58.7%). No significant developmental differences were observed when comparing CM and EB vitrified embryos with nonvitrified control CM (100%) and EB (100%) embryos. In addition, few differences in the ultrastructure of intracellular lipid droplets or in microfilament structure were observed between control embryos and embryos vitrified at any developmental stage. Vitrified-thawed CM/EB embryos cultured for 2 or 16 h before ET resulted in live birth rates of 71.3% and 77.4%, respectively. These rates were not significantly different from the control live birth rate (79.2%). However, culture for 32 h (25%) or 48 h (7.8%) after vitrification significantly reduced the rate of live births. These data indicate that the pipette chamber vitrification technique significantly improves the live birth rate of transferred ferret embryos relative to current state-of-the-art methods..

Keywords: assisted reproductive technology, cryopreservation, embryo, ferret, term development, vitrification

INTRODUCTION

The development of an efficient cryopreservation technique for early embryos is critical for the long-term maintenance of valuable genetic traits and for the development of effective assisted reproductive technologies. Key examples include transgenic animals that model human diseases and endangered species. The domestic ferret (Mustela putorius furo) is a member of the Mustelidae family and has been used extensively as an animal model in biomedical research involving virology, reproductive physiology, and endocrinology. The ferret has marked similarities to humans in its airway structure and lung cell biology, and it therefore has the potential to become a model of choice for the study of genetic lung diseases, including cystic fibrosis [13]. Indeed, cloning of ferrets from cystic fibrosis transmembrane conductance regulator gene-targeted fibroblasts has recently generated a cystic fibrosis model in the ferret [3]. This species is also considered an excellent model for the recovery and conservation of related endangered species, such as the black-footed ferret and the European mink [4].

Embryo cryopreservation was first accomplished in the mouse [5] and since that time, a variety of methods have been used successfully with cattle, goats, and sheep. The development of cryopreservation methods for mustelid embryos, on the other hand, has lagged behind that of these other species. Initial efforts toward cryopreservation of Mustelidae began with the stoat (Mustela erminea) in 1993 [6], and it was a full 10 years later before embryos from the domestic ferret were successfully cryopreserved [7]. In the latter case, embryos grown to the expanded blastocyst stage were preserved using a slow-rate freezing technique. In total, 93 frozen and thawed embryos were transferred into nine recipient females, with 10% producing live pups [7]. The same group later reported a 16% survival rate resulting from the use of embryos vitrified by an open pulled straw (OPS) method [8]. Thus, OPS-based vitrification improved embryo development relative to slow-rate freezing, yet the efficiency of the approach with respect to embryo transfer (ET) and the rate of live births was still quite low.

The low survival rate associated with OPS-vitrified ferret embryos may be due to the fact that the plastic straw used for vitrification in the OPS procedure is not reliably at the desired diameter (i.e., <0.8 mm) and also floats in liquid nitrogen rather than remaining submerged [9, 10]. Indeed, a larger, floating straw decreases the cooling rate during vitrification, and may thus compromise embryo viability. Alternative approaches to overcome these problems include microdrop methods [11] and the use of a glass micropipette (GMP) as a vessel for vitrification. The disadvantage of this latter method is that the GMPs are quite fragile, resulting in embryo loss [10]. However, use of GMPs improved survival rates of vitrified bovine blastocysts, a finding that may be attributed to increasing the rate of freezing due to the vessel's small size and loading volume [10].

Embryos from domestic animals [12] are rich in lipid droplets (LDs) relative to embryos from rodent or human species [13]. A high concentration of LDs may make the ferret embryo more prone to damage during freezing [14, 15]. Indeed, lipid-rich pig embryos are highly sensitive to freezing, experiencing severe ultrastructural damage to intracellular LDs at low temperatures [16]. However, removal of intracellular lipid from early cleavage stage pig embryos before freezing results in improved developmental competence following ET [17]. Taken together, these data suggest that freezing-induced damage to LDs in pig embryos may be irreversible and may ultimately contribute to compromised developmental competence [16]. In ferrets, however, little is known about the distribution of LDs in preimplantation embryos and whether or not freezing-induced ultrastructural damage occurs.

Vitrification-induced damage to microfilament (MF) structure is also well established in the developing embryo. Cryopreservation-induced MF damage is reversible in mouse and rabbit oocytes and embryos [18, 19], but in livestock such changes in MF structure may persist and impact survival following ET [14]. In pigs, for example, the degree of vitrification-induced MF damage appears to be inversely correlated with the extent of developmental competence [20]. Specifically, vitrified morula and early pig blastocysts display extensive MF damage and are developmentally incompetent using this method, whereas expanded and hatched blastocysts undergo variable MF damage and development. Microfilament structure and developmental competence can be improved in expanded and hatched vitrified pig blastocysts, however, by pretreating the embryos with the MF inhibitor cytochalasin B (CCB) to relax MF structure. However, no CCB-mediated improvement in either parameter occurs in pig embryos vitrified at the morula (MR) or early blastocyst (EB) stages [20]. Thus, it appears that vitrification-induced MF damage is irreversible in early-stage pig embryos using this technique and may contribute to their developmental incompetence following ET. Microfilament damage has not been assessed in vitrified ferret embryos.

The study described here was designed to test the hypothesis that reduced chamber size and loading volume will improve the developmental potential of vitrified ferret embryos following ET. These chamber features were captured using a commercially available Eppendorf microloader pipette tip. We also sought to determine the extent to which this pipette chamber vitrification technique affects the structure of LDs and MFs in developing ferret embryos.

MATERIALS AND METHODS

Chemicals and Animals

Chemicals were purchased from Sigma Chemical Co. (St. Louis, MO) and Invitrogen Co. (Grand Island, NY) unless otherwise noted. Ferrets were purchased from Marshall Farms (North Rose, NY). Sable and albino coat-color Jills (nullipara, 6–7 mo of age, weight 610–851 g) were in estrus when delivered. Breeder male ferrets (10–12 mo of age) were used for mating with Jills for embryo production, and vasectomized males were used to induce pseudopregnancy. Vasectomized males were confirmed as sterile at Marshall Farms by the lack of spermatozoa in ejaculates and the inability to reproduce following several mating attempts. All ferrets were housed in separate cages under controlled temperature (20°C–22°C) and a long day light cycle (16L:8D). The use of animals in this study was carried out according to a protocol approved by the University of Iowa Institutional Animal Care and Use Committee and it conformed to or exceeded National Institutes of Health standards.

Mating of Embryo Donors and Recipients

Jills with maximal vulval swelling were considered ready for mating. The embryo donor Jills were placed into the breeder male cage for 24 h, and the recipient Jills were mated with a vasectomized male during a 24-h period. The mating day is recorded as Day 0.

Embryo Recovery

Embryos were retrieved from mated donor Jills after the animals were weighed and euthanized at Days 4, 5.5, and 6 following the first mating to collect MR (cell number approximately 8–16 cells), compact morula (CM; cell number approximately 21–32 cells), and EB (cell number approximately 60 cells) embryos. It should be noted that occasionally, both CM- and EB-stage embryos were recovered at Day 6. The ovaries, oviducts, and uteri of donors were removed and washed with 0.9% (w/v) saline supplemented with 1% (v/v) penicillin-streptomycin (Invitrogen Co.) at 37°C–38.5°C. Uterine horns and oviducts were flushed with PBS (Dulbecco PBS supplemented with 0.1% [w/v] d-glucose, 36 mg/l pyruvate, and 0.4% [w/v] BSA) to release embryos into a Petri dish. Recovered embryos were evaluated under a stereomicroscope (40×) for their developmental stage and photographed.

Vitrification, Warming, and Culture of Embryos

The procedure for vitrification and thawing was conducted essentially as described by Piltti et al. [8] with one key modification. We chose to change the vitrification “chamber” in an attempt to improve the efficiency of ferret embryo survival and in vivo development following cryopreservation. To this end, we adapted a standard Eppendorf microloader pipette tip (Eppendorf, catalogue no. 5242–956.003) for use as the embryo chamber (Fig. 1A). This pipette tip provides a relatively constant inner diameter of 0.25 mm and a uniform wall thickness of 0.03 mm (Fig. 1B). We reasoned that these physical parameters would be highly suitable in light of both the cooling and warming rates associated with successful vitrification. Two ferret embryos were loaded into each pipette tip (Fig. 1C) and vitrified according to the procedure described below.

FIG. 1.
The pipette tip chamber technique for ferret embryo vitrification. A) Modified Eppendorf microloader tip used for embryo vitrification. B) Schematic transverse section of the tip illustrating the inner diameter (ID) and the thickness of the outer wall. ...

The pipette tip was cut at a diameter of 0.25 mm. Embryos of all stages were incubated at 37°C in PBS medium for 1–2 min, then in cryopreservation medium I (PBS, 20% fetal calf serum [FCS], 1.34 M ethylene glycol, and 1.05 M dimethyl sulfoxide [DMSO]) for 4 min, and finally in cryopreservation medium II (PBS, 20% FCS, 2.95 M ethylene glycol, 2.32 M DMSO, and 0.9 M sucrose). Two embryos then were aspirated together into an Eppendorf tip in 0.05 μl cryopreservation medium II. All cryoprotectants were used at 37°C. Within 30–40 sec, the tips were immersed in cryotubes filled with liquid nitrogen (LN2) and stored in LN2 for 2–40 days. Embryos were warmed by immersing the tips at a 30°–40° angle within 3 sec after removal from LN2 into 37°C PBS medium containing 0.3 M sucrose, and holding them in this position for 1 min. The embryos then were placed into PBS medium containing 0.15 M sucrose for 5 min. After an additional 5 min in PBS without sucrose, embryos were washed and cultured in 199 medium containing 10% FCS. The duration of in vitro culture was 2, 16, 32, or 48 h after thawing. The embryos were then washed, kept in PBS medium on a 38.5°C warm stage, and transferred into recipient Jills.

Evaluation of LDs in Ferret Embryos by Light Microscopy and by Transmission Electron Microscopy

Control embryos (without vitrification) and pipette chamber-vitrified thawed embryos were fixed in a 2.5% solution of glutaraldehyde in sodium cacodylate buffer (0.067 M, pH 7.2–7.4) and stored at 4°C until processing. After postfixation for 5 min in 2% (w/v) osmium tetroxide in cacodylate buffer, embryos were embedded in Agar. Semithin and continuous ultrathin sections of embryos were processed conventionally for transmission electron microscopy (TEM). Semithin sections analyzed by light microscopy were stained with toluidine blue. Ultrathin sections were cut and stained with uranyl acetate and lead citrate and examined by TEM (Philips 420 electron microscope) at 80 kV. Control and vitrified-thawed MR, CM, and EB ferret embryos were processed, and five to seven embryos in each group were analyzed.

Evaluation of MF Structure in Ferret Embryos

To visualize MF structure, embryos were fixed overnight in 4% paraformaldehyde, permeabilized by immersion in 0.1% Triton X-100 in PBS for 10 min, and then stained for 1 h at room temperature with a 15 μg/ml solution of Alexa Fluor 488-phalloidin (Molecular Probes Europe BV) in PBS to label the actin filaments. Labeled embryos were mounted on glass microscope slides with an antifade medium to retard photobleaching (Vectashield; Vector Laboratories, Burlingame, CA), sealed under a coverslip using nail polish, and stored in the dark at room temperature until they were analyzed with a Leica confocal laser-scanning microscope. The quality of microfilament structure (i.e., the actin cytoskeleton) was scored as previously described [21] with modifications. A grade I cytoskeleton was characterized by the precise restriction of actin staining to the cell borders. A grade II cytoskeleton was characterized by indistinct cell outlines and the presence of small actin clumps in the cytoplasm.

Transfer of Control and Vitrified Ferret Embryos into Recipient Jills

A single stock solution of saline containing 10 mg/ml each of ketamine HCl (Abbott Laboratories, Chicago, IL) and xylazine (Phoenix Pharmaceutical Inc., St. Joseph, MO) was prepared. The recipient ferrets were routinely anesthetized by i.p. injection of this solution to a final concentration of 20 mg/kg of ketamine and 20 mg/kg xylazine. If the depth of anesthesia was insufficient, an additional dose of the stock solution was administered, up to a total dose of 30 mg/kg of each drug. During the surgery, a 3- to 4-cm incision was made along the midline of the abdomen to expose the oviducts and uteri. Embryos were transferred into either of the bilateral uteri using a fine glass pipette. The ferret ET usually required 30–60 min. After the incision was sutured, the ferrets were returned to their cages and closely monitored until they recovered from anesthesia.

Statistical Analysis

Data were analyzed using arcsine transformation and compared by one-way and two-way analysis of variance (ANOVA) using Statistics Package for Social Science (SPSS) software. Differences with a P < 0.05 were considered significant.

RESULTS

In Vitro Development of Pipette Chamber-Vitrified Ferret Embryos

Ferret embryos at different stages of development were collected from Jills mated at different times. A total of 60 (97%) of 62 MR-stage embryos, 41 (98%) of 42 CM-stage embryos, and 30 (100%) of 30 EB-stage embryos were recovered following vitrification (Table 1). All recovered embryos were cultured in vitro to assess the rate of blastocyst formation. Morula embryos are shown before vitrification (Fig. 2A), after postvitrification thawing (Fig. 2B), and at the blastocyst stage following an additional 72 h of in vitro culture after thawing (Fig. 2C). CM are also shown before vitrification (Fig. 2D), after postvitrification thawing (Fig. 2E), and at the blastocyst stage following an additional 48 h of in vitro culture (Fig. 2F). Finally, EB embryos are shown before vitrification (Fig. 2G), after postvitrification thawing (Fig. 2H), and at the blastocyst stage following an additional 48 h of in vitro culture (Fig. 2I). Table 1 compares the rates of blastocyst development among nonvitrified (control) embryos following in vitro culture and among embryos vitrified and thawed at various developmental stages following in vitro culture. Five to seven replicate experiments were conducted for each group. In the case of embryos vitrified at the earliest stages (MR, 8–16 cells), 58.7% developed to the blastocyst stage, whereas significantly more (85%; P < 0.05) of their nonvitrified, control counterpart embryos developed to blastocysts under identical conditions. However, this difference appeared only when embryos were vitrified at these early developmental stages; embryos that were vitrified at the CM stage or the EB stage did not exhibit significant differences in the rates of blastocyst development relative to their control counterparts following in vitro culture (Table 1).

TABLE 1.
In vitro rate of blastocyst formation for control and vitrified ferret embryos at different developmental stages.
FIG. 2.
Ferret embryos before vitrification (Pre-vitrification) and after thawing (Post-vitrification thawing). Blastocysts obtained after in vitro culture of vitrified and thawed embryos (Post-vitrification culture). A) Previtrification MR embryos. B) Thawed ...

LD Distribution and Ultrastructure in Ferret Embryos Before Vitrification and after Pipette Chamber Vitrification and Thawing

A high concentration and heterogeneous distribution of intracellular lipid in the developing embryo has been associated with reduced survival following vitrification [16]. In addition, damage to the structure of LDs, mitochondria, and other intracellular organelles has been reported in porcine oocytes following cooling [22]. Given the high concentration of lipid within ferret embryos, we first chose to examine the distribution and structure of LDs before and after pipette chamber vitrification to assess the extent of cryo-induced damage associated with this new technique. Representative semithin (light microscopy) and ultrathin (TEM) sections from a control (fresh, without vitrification) MR, CM, and EB are shown in Figure 3. MR embryos were found to contain a high concentration of LDs that appear relatively homogeneous in both size and intracellular distribution (Fig. 3, A and B). However, development to the CM (Fig. 3, C and D) and EB (Fig. 3, E and F) stages showed a progressive heterogeneity in droplet size and distribution concomitant with cavitation and cell differentiation.

FIG. 3.
Distribution and ultrastructure of LDs in the developing ferret embryo. Representative semithin sections (A, C, E) and ultrathin sections (B, D, F) of an MR (A, B), CM (C, D), and EB (E, F). MR embryos contain a high concentration of LDs in the blastomeres ...

A comparative ultrastructural analysis of control and vitrified MR, CM, and EB embryos by TEM revealed few differences between the LDs at each developmental stage. Lipid droplets from a control (Fig. 4A) and a vitrified (Fig. 4B) CM embryo illustrate these differences and are shown in Figure 4. Before vitrification, each LD showed an electron-dense rim of polar lipid and protein (Fig. 4A, arrow) surrounding a less dense hydrophobic neutral lipid core [23]. Many droplets were also found in close association with mitochondria and smooth endoplasmic reticulum. Postvitrification LDs were nearly identical to control LDs, except that partial damage to the electron-dense rim was apparent in some cases (Fig. 4B, arrowheads). However, the association with mitochondria and smooth endoplasmic reticulum was maintained following freezing.

FIG. 4.
Ultrastructural changes in LDs following pipette chamber vitrification of ferret embryos. A) Previtrification, LDs were surrounded by an electron-dense rim (arrow) and frequently associated with smooth endoplasmic reticulum (*) and mitochondria (Mt). ...

Analysis of MF Structure in Ferret Embryos Following Vitrification and In Vitro Culture

An intact MF structure is essential for normal embryo development, and freeze/thaw procedures including vitrification can lead to cytoskelatal damage and poor embryo development following transfer into a recipient female. Therefore, assessing MF quality following vitrification and in vitro embryo culture may be one good measure of the in vivo developmental potential of vitrified embryos. Embryos with a sharp and distinct network of actin fluorescence surrounding each blastomere following vitrification, thawing, and in vitro culture were thus classified as grade I (Fig. 5A), whereas identically treated embryos displaying abnormal actin fluorescence—characterized by indistinct fluorescence throughout the embryo, including small clumps of cytoplasmic staining—were classified as grade II (Fig. 5B).

FIG. 5.
Microfilament structure in vitrified, thawed, and in vitro-cultured ferret blastocysts. Actin filaments were stained with Alexa Fluor 488-phalloidin (green). A) Grade I blastocyst showing a precise, sharp restriction of actin staining to the cell borders ...

Microfilament structure was assessed and classified, as described above, in nonvitrified control and vitrified blastocyst embryos following their in vitro culture (Fig. 6). The percentages of grade I control (72.7%, n = 11) and vitrified (77.6%, n = 17) blastocysts derived from MR embryos were very similar to one another, as were those derived from control and vitrified CM (83.3%, n = 6 vs. 75%, n = 16, respectively) and EB (75%, n = 8 vs. 80%, n = 10, respectively) embryos. Taken together, these data suggest that either MF structure was not adversely affected by this pipette chamber vitrification method or, alternatively, that any resultant MF damage was repaired during in vitro culture.

FIG. 6.
Classification of microfilament structure in vitrified and nonvitrified blastocysts. Proportion of grade I (gray) and grade II (black) blastocysts derived from vitrified (V) and nonvitrified control (C) morula (M), compact morula (CM), and early blastocyst ...

Development of Vitrified Ferret Embryos to Term

We next sought to determine the rate at which pipette chamber-vitrified ferret embryos develop to term following thawing and ET. Vitrified CM- and EB-stage embryos were used in these experiments because of their superior rate of in vitro development relative to vitrified MR-stage embryos (Table 1). Control embryos were transferred to recipient females immediately after collection without in vitro culture. Vitrified embryos were thawed and incubated in vitro for 2, 16, 32, or 48 h prior to ET. A total of 22 to 28 embryos in each group were transferred to three to four recipient females. Our rationale for varying the incubation time prior to ET was two-fold. First, we reasoned that it would allow for embryo acclimation following the potential stress associated with freeze/thawing, and second, it would enable us to determine the developmental potential of vitrified embryos after increasing periods of in vitro culture.

The developmental potential of control and vitrified embryos under this range of conditions is summarized in Table 2. These data illustrate three important points. First, the live birth rate in the control group (79.2%) was considerably higher than that previously reported (42%) [24], suggesting that our ET technique may be more efficient than earlier approaches (for review, see Amstislavsky et al. [25]). Second, there was no significant difference (P < 0.05) in live birth rates between the control group (79.2%) and the 2-h (71.3%) or 16-h (77.4%) culture interval groups. Third, a longer culture interval, that is, from 32 h (25%) to 48 h (7.8%), significantly (P < 0.05) reduced developmental potential relative to control and shorter thaw-to-transfer interval groups following ET.

TABLE 2.
Effects of in vitro culture on in vivo development of vitrified CM/EB ferret embryos.

Live ferret pups derived from vitrified embryos are shown in Figure 7. Sable pups are shown with their albino foster mother 5 days after birth (Fig. 7, A and C) and again at 5 wk of age (Fig. 7, B and D). Taken together, our data indicate that ferret ET is optimal when either CM- or EB-stage embryos are used for vitrification, and the time interval between thawing and transfer of these vitrified embryos is relatively short (i.e., 2–16 h).

FIG 7.
Live ferret pups born from vitrified, thawed, and in vitro-cultured Sable embryos transferred to recipient albino Jills. A, C) Five day-old pups with or without their foster mother. B, D) Pups after 5 wk with or without their foster mother.

DISCUSSION

In the process of cryopreservation by vitrification, the rate of cooling is critical to embryo survival. In earlier methods, a straw containing the embryo(s) was plunged into liquid nitrogen, and the cooling rate depended upon three factors: 1) the internal diameter of the straw; 2) the thickness of the straw wall; and 3) the related “holding” volume of medium surrounding the embryo(s). Subsequent development of the OPS technique resulted in a reduction of the straw's inner diameter and its holding volume, thereby increasing the survival rate of thawed porcine embryos from 18.5% to 29.0% [26]. OPS vitrification also increased the survival rate of transferred ferret embryos to 16% [8]. To increase survival rates in ferrets even further, we have developed a new approach that uses a standard microloader pipette tip manufactured by Eppendorf as the vitrification chamber (Fig. 1A). This pipette tip chamber differs from the traditional OPS plastic straw in that its inner diameter is narrower (i.e., 0.25 mm vs. 0.80 mm) and the vessel wall is thinner (0.03 mm vs. 0.07 mm; Fig. 1B). These differences in dimension translate into a smaller volume of holding buffer being required (i.e., ~0.05 μl vs. ~1–2 μl). Moreover, using this new technique we have efficiently vitrified ferret embryos (see below) without removing cytoplasmic lipids, a process often required when using the older vitrification techniques for lipid-rich pig embryos.

To test the performance of our pipette chamber vitrification technique, we first evaluated the rate of in vitro development of ferret embryos following cryopreservation and thawing (Fig. 2 and Table 1). We found that the majority of vitrified CM (93.6%) and EB (100%) embryos developed to the blastocyst or re-expansion stages following in vitro culture at rates similar to those for the development of nonvitrified control embryos at the same stages (100%). Thus, our data demonstrate that nearly all vitrified ferret embryos at the CM and EB stages are able to develop in vitro following cryopreservation by our pipette chamber technique. In contrast, previous vitrification methods achieved blastocyst re-expansion in only approximately one half (51%) of either MR- or blastocyst-stage embryos following warming and in vitro culture [8]. Our data also suggest that ferret embryos at the CM and EB stages are likely more resistant to the cooling damage associated with vitrification than those at the MR stages. These data from ferrets appear to be consistent with those obtained from pig, in which embryos at the prehatching stages are more resistant to the effects of freezing than earlier-stage embryos [27]. This difference is presumably due to a reduction in the size of LDs within the pig embryo at more advanced stages [27]. On the other hand, and somewhat surprisingly, expanded ferret blastocysts are not efficiently vitrified when compared with earlier MR- and EB-stage embryos [24]. However, this difference may relate to the large size of the expanded blastocyst and the resultant need to load a larger volume of medium during vitrification [24]; a larger volume of medium may reduce the rate of cooling and compromise embryo viability. Taken together, all available data indicate that CM- and EB-stage embryos are best for vitrification in the case of ferrets.

It is well known that cryopreservation can severely disrupt the cellular organization of developing embryos [16]. Intracellular LDs appear to be especially sensitive to reduced temperature, with organelle damage contributing to reduced developmental competence following vitrification. These effects appear to be magnified in embryos from lipid-rich species like pigs [17]. The concentration and structure of LDs have been characterized in pig embryos undergoing preimplantation development [12]. Ferret embryos are also lipid rich; however, to our knowledge no previous study has examined the concentration and structure of LDs during preimplantation development nor the potential vitrification-associated damage to these organelles in this species. Our analysis demonstrates that ferret MR-stage embryos contain an abundance of lipid droplets that are relatively homogeneous in both size and distribution but become more heterogeneous at later stages of development (Fig. 3). Ultrastructural analysis of LDs in these embryos following pipette chamber vitrification revealed limited damage to the electron-dense rim that appeared at all stages of development. However, no changes in the association of droplets with mitochondria or smooth endoplasmic reticulum were observed (Fig. 4). These data illustrate two important points. First, the vitrification-induced damage to the structure of LDs occurs regardless of the stage of preimplantation development, and therefore independently of associated changes in droplet size and intracellular distribution. Second, this damage, whether reversible or not, does not significantly impede the survival of pipette chamber-vitrified embryos following in vitro culture and ET (Table 2).

An intact cytoskeleton is essential for successful mitosis and cell division. When either process is irreversibly compromised, the death of individual cells, or even of the entire embryo, may result. Thus, a successful cryopreservation technique must preserve cytoskelatal integrity [27]. In our study, we observed no differences in the appearance of MF structure between vitrified thawed embryos and nonvitrified, control ferret embryos (Figs. 5 and and6).6). However, given that vitrified embryos were cultured for more than 24 h prior to actin staining, we are unable to determine whether pipette chamber vitrification induces minimal damage to MF structure or whether more extensive damage is efficiently repaired during extended in vitro culture. Whichever of these possibilities is correct, our data clearly demonstrate that any vitrification-induced damage to MF structure that does occur does not impede embryo survival following ET (Table 2).

Transfer of pipette chamber-vitrified CM/EB-stage ferret embryos after 2 or 16 h of in vitro culture resulted in live birth rates (71.3% and 77.4%, respectively) that were not statistically different from those observed with nonvitrified, control ferret embryos (79.2%; Table 2). This rate represents a more than 4-fold increase in the live birth rate relative to that achieved using the OPS vitrification method [8]. However, the live birth rate we achieved with our control embryos was higher than that previously reported in ferret (79.2% vs. 42% [24]), and therefore more efficient ET could contribute to the higher birth rates we observe with pipette chamber vitrification. Nevertheless, the live birth rate we achieved with this technique still represents ~94% of the control rate, whereas the earlier rate, using the OPS vitrification method, represents ~38% of control levels—a ~2.5-fold increase in the rate of live births using pipette chamber vitrification. Given that the concentrations of cryoprotectants used for vitrification in the current study were similar to those used in the earlier OPS study [8], we conclude that the higher rate of development to term is primarily the result of our pipette chamber technique. However, we cannot rule out the possibility that our use of PBS rather than synthetic oviductal fluid [8] may also have contributed to the improved rates we observed.

Notably, the live birth rate with vitrified ferret embryos declined significantly with increasing periods of postthawing in vitro culture. Only 25% of transferred embryos produced live offspring after 32 h of in vitro culture, with the rate dropping to 7.8% by 48 h (Table 2). These data are consistent with the results reported using OPS-vitrified ferret embryos [8]. The basis for the lack of developmental potential among late-stage ferret blastocysts is unknown; however, our data may provide some important clues. For example, our results show that embryo survival rates are not affected by asynchrony in the developmental stage of the donor embryos and the recipients (data not shown). In addition, we found that embryo expansion in vitro was compromised regardless of the vitrification method used (Sun et al., unpublished data). Thus, it may be that long-term in vitro culture itself is detrimental to embryo survival and that short postthawing incubation times should be maintained to maximize embryo survival.

Taken together, our data demonstrate that the pipette chamber-based method for vitrification results in a significant improvement in the birth rate of transferred ferret embryos relative to current state-of-the-art methodologies. Therefore, the novel technique described here should be useful for the long-term maintenance of valuable genetic specimens of Mustelidae, especially in relation to the domestic ferret as a biomedical research model for human diseases and the conservation of related endangered species.

Acknowledgments

The authors want to thank Dr. R. Scipioni Ball and her staff at Marshall Farms for their kind assistance with ferret care and reproduction. The authors also wish to thank the personnel at the University of Iowa Animal Facility for their efforts in maintenance of the ferret colony and Jian Shao with the Cell Morphology Core facility at University of Iowa for excellent technical assistance with TEM.

Footnotes

1Supported by the Cystic Fibrosis Foundation (ENGELH04G0 to J.F.E. and G.H.L.) and the National Institutes of Health (DK47967 to J.F.E. and HL61234 to Michael J. Welsh, P.I.).

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