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Ultrastructural Characteristics of Fresh and Frozen-Thawed Ovine Embryos Using Two Cryoprotectants¹

^a Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria A, Dpt. de Reproducción Animal y Conservación de Recursos Zoogenéticos, 28040 Madrid, Spain ^b Consejo Superior de Investigaciones Científicas, Centro de Investigaciones Biológicas, 28006 Madrid, Spain ^c Servicio de Investigación Agroalimentaria, Dpt. de Tecnología en Producción Animal, 50080 Zaragoza, Spain

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

 
Cryopreservation of sheep embryos with ethylene glycol as a protectant appears to be more effective than glycerol, particularly at the morula stage, as has been demonstrated on the basis of in vitro and in vivo development rates after thawing. In this study we compare the ultrastructure of fresh morulae, thawed morulae, and blastocysts cryopreserved with either ethylene glycol or glycerol at the electron microscopic level, to look for cellular damage that could be responsible for proven differences in embryo survival after transfer. Embryos cryopreserved with glycerol showed unequal degrees of conservation even among blastomeres within a single embryo. In morulae, inner blastomeres were completely damaged, whereas external ones appeared to be intact. Both morulae and blastocysts cryopreserved with ethylene glycol showed a higher uniformity in blastomere conservation than embryos with glycerol. The most remarkable features in this experimental group were the presence of desmosomes following tight junctions between blastomeres and the presence of many microvilli on the outer surface of external blastomeres. These characteristics are similar in fresh embryos of the control group. Our results show that ethylene glycol protects membrane and cytoplasmic structures of embryonic cells from cryoinjury much better than glycerol. In vivo survival of embryos confirmed the ultrastructural observations. A limited permeability of glycerol would explain the observed ultrastructural differences in blastomere integrity, which depends on blastomere location and the differences between morulae and blastocysts. We conclude that the low reproductive yield after cryopreservation using glycerol can be attributed to the lack of protection of inner cells.

early development, embryo, pregnancy, reproductive technology, trophoblast

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

 
Successful cryopreservation of embryos was first demonstrated almost three decades ago in mice [1, 2]. Since that time cryopreservation has been used for the storage of embryos from many species, including domestic animals and humans. Today, cryopreservation has become a key factor in embryo transfer technology with commercial and research purposes.

The first lambs produced from cryopreserved embryos were born in 1976 [3]. Once the procedure was established, research continued to increase embryo survival by using different cryoprotectants and freezing-thawing protocols [4–6].

Conventional slow-rate freezing of embryos has given alternatives in herd reproduction practices. However, pregnancy rates after cryopreservation, ranging from 50% to 60%, are lower than those of fresh embryo transfer [7]. In sheep, in the breeding program for the Rasa Aragonesa during 1999, the rate of live-born lambs per transferred embryos was 30% lower for cryopreserved embryos than it was for fresh embryos [8].

The loss of viability associated with freezing can be attributed to the type and concentration of cryoprotectant, the freezing protocol, the temperature at which embryos are transferred to liquid nitrogen, and the developmental stage of the embryo. In vitro and in vivo embryo survival are different between sheep embryos cryopreserved with either glycerol or ethylene glycol [4, 9], as well as between morulae and blastocysts [10].

Light microscopic evaluation of embryo viability after thawing appears to be inconclusive. Embryos appearing as morphologically good after thawing sometimes failed to produce pregnancies when transferred [10]. Therefore, the aim of this work was to perform a qualitative study of the fine structure of fresh and frozen-thawed embryos. Injuries or anomalies detected at this level, but not with conventional light microscopy, could account for the loss of viability found after transfer. Ultrastructure of fresh sheep embryos has been studied before [11, 12], but as far as we know, this is the first report showing an ultrastructural analysis of frozen-thawed sheep embryos.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

 
Embryo Production

In vivo-produced embryos (n = 153) were obtained from superovulated ewes in four different runs performed during the breeding season. Donors were synchronized with 40 mg of fluorogestone acetate intravaginal sponges (FGA Chronogest, Intervet International B.V., Boxmeer, The Netherlands) for 14 days. Superovulatory treatment consisted of six decreasing doses of FSH (Schering Plough Co., Kenilworth, NY). The administration protocol consisted of 4, 4, 3, 3, 2, and 2 mg at 12-h intervals, starting 48 h before sponge removal. Ewes were mated under control conditions with rams introduced at 36 and 48 h after sponge removal. Embryos were recovered on Day 6 or 7 after sponge removal by abdominal laparotomy under anesthesia with a mixture of rompun, atropin sulphate, and ketamine chlorhydrate. Dulbecco PBS supplemented with BSA (4 g L^-1) was used for embryo recovery and manipulation. Morphological scoring of embryos was performed according to previously established criteria [13], and only embryos considered as excellent or good were selected for freezing. Fresh embryos for the control group, consisting of eight blastocysts and six morulae, were directly processed for transmission electron microscopy.

Freezing

Embryos evaluated as excellent or good (n = 139) were randomly distributed between two cryoprotection protocols within 1 to 3 h after collection. Two cryoprotectants prepared in PBS plus 20% fetal calf serum (FCS) pH 7.2 were used: glycerol (at a final concentration of 1.4 M, n = 72) and ethylene glycol (at a final concentration of 1.5 M, n = 67). Equilibration with glycerol was performed in two steps (0.7 M for 20 min and 1.4 M for 20 min) and with ethylene glycol in three steps (0.5 M for 10 min, 1 M for 10 min, and 1.5 M for 20 min). Embryos were loaded in groups of two to four into 0.25-ml straws. A Lauda compact low-temperature thermostat RKP 20 (Lauda-Konigshofen, Germany) was used for slow-controlled freezing. The slow freezing system was essentially the same as that described by Willadsen et al. [3]: 1°C per min^-1 to -7°C; seeding, 0.3°C per min^-1 to -30°C, and 0.1°C per min^-1 to -35°C. Straws were then plunged into liquid nitrogen and stored for up to 11 mo.

Thawing was carried out by immersion in water at 32°C. Embryos were then transferred to a 0.25 M sucrose solution in PBS plus 20% FCS pH 7.2 for 10 min, and washed 3 times in PBS-FCS for 5 min.

Electron Microscopy

Fresh (n = 14) and frozen-thawed embryos (glycerol, 10 blastocysts and 4 morulae, n = 14; ethylene glycol, 9 blastocysts and 5 morulae, n = 14) were fixed in 2.5% glutaraldehyde solution in PBS for 2 h at 4°C, washed 3 times in cacodylate buffer for 30 min, and kept overnight at 4°C. They were postfixed in 2% osmium tetroxide for 1 h at 22°C, and washed 3 times in 0.05 M cacodylate buffer for 30 min. Dehydration was carried out through a graded series of ethanol. Embryos were individually flat-embedded in Spurr resin (ERL 4206; TAAB, Berkshire, England), and blocks were allowed to polymerize for at least 7 h at 70°C. Ultrathin sections of 50–70 µm were prepared using a Reichert-Jung Ultracut E ultramicrotome (Reichert A.G., Vienna, Austria) and collected onto formvar-coated 50-mesh copper grids. Sections were stained with uranyl acetate and lead citrate, and examined using a Phillips 300 electron microscope (Phillips, Eindhoven, The Netherlands).

Embryo Transfer

Frozen-thawed embryos of both cryoprotectant groups (glycerol, n = 58; ethylene glycol, n = 53), morphologically evaluated as "good" after thawing, were transferred into synchronized recipient ewes by laparotomy as previously described [14]. Transfers were performed at Day 7 or 8 after sponge removal depending on the stage of development of the embryos (morula or blastocyst). Two embryos per recipient were implanted, except in two cases in which only one per ewe was available. Data were statistically analyzed by using the chi-square test.

RESULTS

Fresh embryos were classified as excellent or good as shown with transmission electron microscopy as having undergone a good preservation protocol of the amorphous zona pellucida and blastomeres. Their cells showed a well-organized nucleus surrounded by a continuous double membrane in every cell of the inner cell mass (Fig. 1, A and B). More than one nucleolus, showing a reticulate ultrastructure, which is typical of activity with fibrillar centers, dense fibrillar component, and granular component, were observed in blastomeres (Fig. 1A).

View larger version (166K): [in this window] [in a new window]   FIG. 1. Electron microscopy of fresh blastocysts classified as viable embryos. A) A well-preserved inner cell mass cell (ICM) and a trophoblast cell (TP). Note the nuclear (Nu) and nucleolar (No) organizations that are typical of activity, and abundant mitochondria (mt), vacuoles (v), and lipid droplets (ld) in the cytoplasm. B) Higher magnification of the square in A, showing the well-fixed double membrane of the nuclear envelope (arrows) and a pore complex (arrowhead). C) Cytoplasm with abundant vesicles containing flock material (arrows) or membrane residues (big arrows) and mitochondria (mt). Bars = 1 µm

Two remarkable features were found in cytoplasmic components, a high number of vacuoles and the presence of abundant osmiophilic lipid droplets (Fig. 1A). Many vesicles varying in diameter, which appeared to be membrane-bound, were preferently observed in inner cells but also in outer cells, containing flocculent material or membrane residues in some cases (Fig. 1C). Mitochondria of variable shapes and organizations, from spherical to cylindrical, were abundant and showed developed cristae with double membranes (Fig. 2A). Endoplasmic reticulum cisternae and ribosomes were also observed. Microvilli were present in the outer surface of trophoblastic cells (Figs. 2B and 3A), whereas cytoplasmic protusions were formed on the sides that were in contact with adjacent blastomeres. Adhesion between blastomeres took place by means of tight junctions, followed by a high number of desmosomes with associated filaments (Figs. 2B and 3A).

View larger version (139K): [in this window] [in a new window]   FIG. 2. Fresh blastocysts. A) Mitochondria with different shapes and organizations in the blastomeres with abundant transverse cristae and associations with the endoplasmic reticulum (arrow). Golgi cisternae (thick arrow), and rough endoplasmic reticulum cisternae (rer). B) Two trophoblast cells (TC) with tight junctions at the contact zone (arrows) and microvilli in their outer surface (arrowheads). Residual material in the previtelline space (rm), zona pellucida (zp), blastocoel (b), and lipid droplet (ld). Bars = 1 µm

When fresh embryos morphologically scored as fair were analyzed, blastomeres with clear symptoms of autolysis were found. A high degree of disorganization and loss of intercellular junctions could be observed (Fig. 3B) as well as vacuoles with remains of cytoplasmic components and lipid droplets (Fig. 4A). Images corresponding to a high activity of exocytosis were often detected in low-viability embryos (Fig. 4B).

View larger version (154K): [in this window] [in a new window]   FIG. 3. Fresh blastocyst. A) Higher magnification of the contact zone between two trophoblast cells showing tight junctions (arrows) and a desmosome with associated filaments (arrowhead). B) Fresh embryo classified as fair showing a loss of intercellular junctions and abundant vacuoles (v). Bars = 1 µm.

View larger version (131K): [in this window] [in a new window]   FIG. 4. Typical cellular components of fresh embryos scored as "low viability." A) Higher magnification of a vacuole (v) containing residues of cytoplasmic components including a lipid droplet (ld). B) Exocytosis figures (ex) frequently observed in fair embryos. Bars = 1 µm

The first outstanding appreciation of transmission electron microscopy evaluation was that cryopreservation with glycerol gave uneven results, depending on several factors. As observed in Figure 5, A and B, only one embryo (1/14 = 7.1%) showed intact structures after thawing, whereas some others were completely destroyed (7/14 = 50%). Morphological evaluation with light microscopy gave the following scoring: 9 out of 14 (64.3%) degenerated embryos, 2 (14.3%) fair embryos, and 3 (21.4%) good embryos. The use of glycerol proved to be better for early blastocysts and blastocysts than for morulae, in both good and fair embryos (35.7%). Differences in the degrees of integrity after thawing were observed even between blastomeres from the same embryo. Some of them presented cytoplasm with numerous mitochondria, ribosomes, vesicles, microfilaments, and even microtubules (Fig. 6A), whereas others showed clear signs of alteration (low number of mitochondria, abundant vesicles with various types of contents, and empty areas; Fig. 6B). In some cells, symptoms of lysis in different cell components such as nuclear envelope disruptions, nuclear cavities, and broken mitochondria were observed (Fig. 7, A and B).

View larger version (119K): [in this window] [in a new window]   FIG. 5. Glycerol-cryopreserved embryos. A) A well-preserved blastocyst displaying a well-maintained ultrastructure of most of the blastomeres after thawing. B) Degenerated embryo presenting several breaks in many cells. Bars = 10 µm

View larger version (160K): [in this window] [in a new window]   FIG. 6. Glycerol-cryopreserved embryos. A) Portion of a well-cryopreserved cell. All intracellular components appear intact. Nucleus (Nu). Mitochondria showing connections with the rough endoplasmic reticulum (arrows). Microtubules with associated vesicles (small arrows). Endoplasmic reticulum cisternae associated with vacuoles (arrowhead). B) Portion of a blastomere with clear ultrastructural disruptions such as broken membranes (arrows), empty spaces in the cytoplasm (big arrows), disorganized Golgi complexes (GC), mitochondria (mt), and lipid droplet (ld). Bars = 1 µm

View larger version (149K): [in this window] [in a new window]   FIG. 7. Portions of cells from glycerol-cryopreserved embryos showing severe disruptions. A) Cell with the nuclear envelope disrupted in several points (arrowheads) showing alterations in the nucleus (Nu) and nucleolus (No). The nucleus and cytoplasm have extensive empty spaces (*). B) Mitochondria with altered membranes and cristae and evaginations of the internal contents (big arrow). C) Glycerol-cryopreserved morula displaying a better preservation of the outer blastomeres than of the inner ones. The core blastomeres show no visible organelles at this magnification. Bars in A and B = 1 µm. Bar in C = 10 µm

The most noticeable image obtained in this experimental group was from morulae that were evaluated by conventional light microscopy as morphologically good, as the one shown in Figure 7C. Two out of three studied morulae showed different degrees of preservation of blastomere ultrastructure depending on cell position within the embryo. External blastomeres observed at 400x presented a well-differentiated nucleus, electrondense granules in the cytoplasm, and close adherence of cells. However, internal blastomeres showed no distinguishable organelles or nucleus, and the cells exhibited a uniform texture. In addition, blastomeres located in the inner part of the morulae were loosely bound and presented incipient cell separation. These ultrastructural differences were not found in any other experimental group.

Evidence of lysis was also found on internal blastomeres of morulae treated with glycerol. As shown in Figure 8A, most of the components of the cytoplasm were degenerated. Some organelles were hard to identify, as was the case of swollen, large mitochondria and a reduced contrast due to the loss of their internal organization, which appeared intermingled, with others showing minor alterations and a normal size (Fig. 8B). Empty areas not limited by a membrane indicated alterations in the fine structure of the cytoplasm.

View larger version (153K): [in this window] [in a new window]   FIG. 8. A) Inner blastomere from the morula shown in Figure 7C showing a completely disrupted cell organization. Only the lipid droplets (ld) appear similar to those in control embryos. B) Higher magnification of the upper part of the cell in A showing the altered structure of mitochondria (mt), some of which appear swollen (smt) with very few cristae in a marginal position (small arrows). Many of the nonswollen mitochondria show empty areas in their interiors (arrowheads). Bars = 1 µm

Morphology of embryos cryopreserved with ethylene glycol revealed a better conservation than those treated with glycerol, although in this experimental group, we obtained embryos with extreme ultrastructural preservations, either with an optimal preservation in almost all cells, as observed in 8 out of 14 (57%) embryos in this group (Fig. 9A), or with all blastomeres disorganized or destroyed (6/14 = 42.9%), and in which most of the cytoplasmic organelles were difficult to identify (Fig. 9B).

View larger version (154K): [in this window] [in a new window]   FIG. 9. Ethylene glycol-cryopreserved embryos. A) Morula cell showing an excellent preservation of its components, as well as intercellular junctions (arrows) and desmosomes (arrowheads). Compare with Figure 1, which shows a control blastocyst. Nucleus (Nu), nucleolus (No), mitochondria (mt), lipid droplets (ld), vacuoles containing membrane residues (big arrows) or flock material (double arrows). B) Severely altered cell from a morula. It shows many empty spaces in the cytoplasm (*). The cell components are hard to identify, except for the lipid droplets (ld). Altered mitochondria (mt). Bars = 1 µm

In contrast with the glycerol-cryopreserved embryos, homogeneity in cells preserved with ethylene glycol was high. Blastomeres showed a similar degree of integrity as in fresh embryos, although mitochondria were less electron-dense and vacuoles with hyalin contents were observed. Lipid droplets and cytoplasmic vesicles remained intact. Mitochondria showed double membranes in the periphery and in cristae, and ribosomes were either dispersed in the cytosol or clustered in polysomes (Fig. 10A). Fine structure of the cytoplasm indicated no breaks in the cytoskeleton. An intact, double-nuclear membrane was also observed in the majority of blastomeres (Fig. 10B).The most remarkable feature was the presence of well-defined desmosomes (Fig. 11), which consisted of inner osmiophilic layers of plasma membranes of two adjacent blastomeres plus electrondense, associated microfilaments. The role of this type of structure is to strengthen cellular junctions between blastomeres. Outer blastomeres showed desmosomes on the internal side, whereas the remainder contained several microvilli. We considered that both desmosomes and microvilli are signs of good cell preservation.

View larger version (145K): [in this window] [in a new window]   FIG. 10. Details of the cell organization of embryos cryopreserved with ethylene glycol. A) Mitochondria with typical shapes and structures displaying well-organized cristae. Most are associated with cisternae of the rough endoplasmic reticulum (arrows). Golgi complexes with abundant vesicles (big arrows) and rough endoplasmic reticulum cisternae (rer) with polyribosomes (small arrows). B) Detail of a cell showing the well-preserved double membrane of the nuclear envelope (ne), and also a mitochondria associated with both the rough endoplasmic reticulum (arrowhead) and the nuclear envelope (double arrows). Abundant polyribosomes are also observed (flat arrowheads). Bar in A = 1 µm. Bar in B = 0.5 µm

View larger version (133K): [in this window] [in a new window]   FIG. 11. Cells from an embryo with intermediate-quality preservation. Even in these cases, the tight junctions (arrows) and desmosomes (arrowheads) are very resistant, showing a well-defined ultrastructure with associated bundles of filaments (fb), mitochondria (mt), Golgi complex (GC), vacuole (v), and rough endoplasmic reticulum cisternae (rer). Bar = 1 µm.

The in vivo survival after transfer of frozen-thawed embryos, cryopreserved either with ethylene glycol or glycerol, is shown in Table 1. After morphological evaluation, only good embryos were transferred. Embryos with clear damage were discarded. At this level of evaluation, blastocysts preserved with ethylene glycol showed significantly better integrity than those preserved with glycerol. This difference between cryoprotectants was not observed in morulae. However, the lambing rate was significantly higher for the group that received morulae cryopreserved with ethylene glycol than with glycerol. The overall embryo survival rate resulted in significant differences in favor of ethylene glycol only in morulae. On the other hand, in these experimental conditions, in vivo survival of blastocysts was not affected by the type of cryoprotectant, and most of the differences could be detected by morphological evaluation before transfer.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS DISCUSSION REFERENCES

 
Information about cellular damage produced during or after embryo cryopreservation is scarce, but it would provide useful, nonempirical information for understanding cellular sensitivities to cryopreservation [7]. In this paper we investigate differences in ultrastructure between frozen-thawed ovine embryos cryopreserved with either ethylene glycol or glycerol in comparison to the ultrastructure of fresh embryos as controls. Although the ultrastructural characteristics of fresh embryos have already been reported [11, 12], this is to our knowledge the first time that ovine frozen-thawed embryos have been evaluated at the electron microscopic level.

In vivo survival results corroborated previous studies reporting a better efficacy of ethylene glycol as a cryoprotectant in comparison to glycerol, particularly at the morula stage [10]. The fact that the lambing rate and survival were significantly higher for morulae preserved with ethylene glycol, despite eliminating the damaged ones, supports the idea that cellular alterations are not detectable by morphological evaluation, which causes a decrease in embryo viability.

The ultrastructural features observed in our nonfrozen morulae and blastocysts are essentially consistent with those reported by Calarco and McLaren [11] in the same system, but with some particular disparity. Those researchers described the most obvious difference in blastocyst stage, compared with earlier stages, the almost complete absence of vacuoles in the cells of the inner cell mass and trophoblast. In contrast to this observation, we found a high number of vacuoles in early blastocysts, blastocysts, and expanded blastocysts, even in the trophoblast and inner cell mass cells. Membrane-bound structures were also observed, which were interpreted as degradation bodies.

The observed ultrastructure of fresh embryos was similar to findings in other species at the same stage of development [15–17], such as the organization of the nucleus and nucleolus in blastomeres. In every type of embryonic cell, the nucleus was surrounded by a double membrane, and some of the blastomeres presented more than one nucleolus. Furthermore, the external surface of trophoblast cells showed many microvilli, whereas lateral sides of adjacent blastomeres were interdigitated, and tight junctions and desmosomes with associated microfilaments were present.

Cytoplasm of blastomeres and trophoblast cells was abundant, with isolated or clustered ribosomes, and with oval mitochondria containing well-developed cristae, although small spherical mitochondria in association with endoplasmic reticulum cisternae were also present in scarce amounts in both morulae and blastocysts. This type of immature mitochondrion has also recently been found in bovine compact morulae [18], although other authors have reported the presence of this type, but not further than the 8- to 16-cell stage [15]. A difference found in comparison with bovine embryos is the presence of abundant lipid droplets and vesicles, as already reported by other authors [11]. Embryos of fair quality showed vacuoles containing altered cytoplasmic components, a clear sign of degeneration, as the most remarkable feature.

Observations through transmission electron microscopy demonstrated that ethylene glycol is a better protector of embryonic structures than glycerol, which is consistent with the results of embryo survival after transfer. Morulae and blastocysts preserved with ethylene glycol showed a similar ultrastructural preservation than the control group.

There were differences in the extent of damage observed in frozen-thawed embryos not only between embryos, but also between cells of a single embryo, mainly with glycerol-frozen embryos. Well-preserved blastomeres kept an intact structure and maintained cellular unions. The slight changes observed in the fine structure of the cytoplasm, mainly empty areas with no contents and no membranes surrounding them, are likely due to crystallization, and are in agreement with previous descriptions made on different species and stages of development [19]. In glycerol-frozen embryos, large vacuoles and some disruptions in the nuclear membrane were found in blastomeres that appeared intact. Embryonic cells not surviving freezing frequently showed broken membranes and cellular disorganization, similar to those reported in other species when using dimethyl sulfoxide [15, 19].

The cytoskeleton is a network of microfilaments and microtubules that are responsible for cell structural organization and movement. Cryoprotectants are known to influence cytoskeleton dynamics, although their interactions with cellular structures of embryos are not consistent across species or stages of development [7, 20]. Therefore, the drastically disturbed cell organization that we observed, principally in morulae cryopreserved with glycerol, can be due to a disruption of microfilaments, microtubules, or both, in this cleavage stage of sheep embryo, as reported in other species [21].

Apart from the different alterations in cellular organelles, depending on the cryoprotectant used, the most striking result when comparing ethylene glycol and glycerol was related to the global structure of the embryo. Although highly permeable cryoprotectants avoid high degrees of dehydration of embryos during equilibration [22], in our results both cryoprotectants contributed to a good preservation of most embryonic cells of blastocysts; however, morulae showed remarkable differences between cryoprotectants. With ethylene glycol, the majority of cells were intact in surviving morulae, whereas morulae with alterations did not survive and were easily detected by conventional light microscopy evaluation. As evidence of this, we found no differences in lambing rates or embryo survival between morulae and blastocysts preserved with ethylene glycol. On the contrary, in morulae preserved with glycerol, while external blastomeres looked like intact cells, internal blastomeres appeared as a rather uniform mass, in which membranes, nuclei, or cellular organelles could not be detected. Morulae showing such dramatic alterations could hardly develop after transfer. These morphologically undetectable damaged embryos are, in our opinion, an explanation for the low yield obtained after transfer of morulae cryopreserved with glycerol.

The different damage induced by freezing in the blastomeres, according to their positions in the embryo, and the confirmation of this positional effect by electron microscopy in cells with otherwise identical compositions and ultrastructural organizations [11], suggest a lack of accessibility of glycerol to the inner cells, most probably due to a lower permeability of glycerol compared to ethylene glycol, as demonstrated by Szell and Shelton [23], instead of a low efficiency of the cryoprotectant in this species or stage of development of the embryo.

Our observations provide morphological and ultrastructural evidence for dramatic alterations produced in cells located in less-accessible areas. This damage involves basic cell organelles and components that are essential for proper cell functioning, and thus for cell viability. The extension of damaged cells would account for the low rates of in vitro development and prolificacy obtained in our studies using frozen sheep morulae that had been morphologically selected before transfer. Therefore, the survival of morulae cryopreserved by classic techniques could be improved by favoring cryoprotectant permeation and uniform distribution throughout embryonic cells.

	ACKNOWLEDGMENTS

 
We thank Dr. S.J. Dieleman for critical review of the manuscript.

	FOOTNOTES

 
First decision: 10 July 2001.

¹ This work was supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (project SC97-020-C2).

² Correspondence: M.J. Cocero, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Dpt. de Reproducción Animal y Conservación de Recursos Zoogenéticos, Carretera de Ca Coruña km.5.9, 28040 Madrid, Spain. FAX: 34 915490956; cocero{at}inia.es

Accepted: November 21, 2001.

Received: June 6, 2001.

	REFERENCES

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