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Vitrification of the oocytes and embryos of
domestic animals
Gabor Vajta
´
)Centre for Early Human DeÕelopment,
Monash Institute of Reproduction and DeÕelopment, Monash UniÕersity, LeÕel 3, 27-31 Wright Street, Clayton, VIC 3168, Australia
Abstract
After the first successful application of vitrification for embryo cryopreservation 15 years ago, a rapid application of the method in domestic animal embryology was presumed. However,
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although the advantages of vitrification simplicity, cost efficiency, speed of the procedure were widely acknowledged, its use has been mainly restricted to experimental studies. For commercial embryo transfer purposes, the traditional slow-rate or equilibrium freezing has been used. This review attempts to explain the reasons for this phenomenon and discusses the theoretical and practical differences between the two technologies as well as their commercial prospects. Recent developments that improve the efficiency of vitrification and applications to other reproductive technologies are also summarized. These advances may result in considerable advantage and could lead to widespread application of vitrification in certain areas of domestic animal embryology. q2000 Elsevier Science B.V. All rights reserved.
Keywords: Cryopreservation; Vitrification; Oocytes; Embryos
1. Vitrification vs. equilibrium freezing
Ž The physical definition of vitrification is the solidification of a solution glass
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formation at low temperatures without ice crystal formation. The phenomenon can be regarded as an extreme increase of viscosity and requires either rapid cooling rates Žaccording to theoretical calculations, at a cooling rate of approximately 1078Crs even
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pure water vitrifies; Rall, 1987 or the use of cryoprotectant solutions, which depress ice crystal formation and increase viscosity at low temperatures. Until recently, the highest
)Tel.:q61-3-9594-7340; fax:q61-3-9594-7311.
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E-mail address: gabor.vajta@med.monash.edu.au G. Vajta .
0378-4320r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved.
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cooling rate of common vitrification procedures was limited to that which could be achieved by plunging of a sealed 0.25 ml insemination straw directly into liquid
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nitrogen, i.e. approximately 25008Crmin Palasz and Mapletoft, 1996 . On the other hand, to avoid fractures of the zona pellucida and embryos in sealed straws, even the
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achievable cooling and warming rates cannot be fully utilized Rall and Meyer, 1989; .
Kasai, 1997; Kasai et al., 1996 . These cooling rates require the use of approximately 5 to 7 M concentration of cryoprotectants, which is several orders higher than that needed
Ž
for traditional equilibrium freezing approximately 1 to 2 M; Rall, 1987; Massip et al., .
1989 . Additional factors, such as a small volume of solution or a considerable increase
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of the hydrostatic pressure, may also facilitate vitrification Fahy et al., 1984 , although the latter has few practical consequences in reproductive biology.
Controversially, some degree of vitrification occurs in any method resulting in successful cryopreservation, even in equilibrium freezing, as a consequence of the concentration of the solutions in and around the embryos or oocytes caused by the
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gradual formation of ice Rall et al., 1984 . To avoid confusion, the term of vitrification in cryobiology and embryology refers to methods where the whole solution containing the biological sample vitrifies completely.
The strategy of vitrification is basically different from that of freezing by slow cooling. A slow rate of cooling attempts to maintain a delicate balance between the various factors, which may result in damage, such as ice crystal formation, osmotic injury, toxic effect of cryoprotectants, concentrated intracellular electrolytes, chilling injury, zona and embryo fracture, and alterations of intracellular organelles, cytoskeleton
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and cell-to-cell contacts Massip et al., 1995; Dobrinsky, 1996; Kasai, 1996; Kasai et al., .
1996; Martino et al., 1996a; Saha et al., 1996 , whereas vitrification totally eliminates ice crystal formation.
A negative consequence of this strategy is the increased probability of nearly all forms of injury except for those caused by ice crystal formation. Different approaches are used to minimize toxic, osmotic and other injuries; application of less toxic
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chemicals, combination of two or three including at least one permeable cryoprotec-tants, stepwise addition andror exposure of cells to precooled concentrated solutions ŽFahy et al., 1984; Rall, 1987; Rall and Fahy, 1985; Massip et al., 1987, 1989; Kasai et
.
al., 1990; Smorag and Gajda, 1994; Palasz and Mapletoft, 1996 . In the past 15 years, at least 20 different combinations of cryoprotectants have been published for vitrification of mammalian oocytes and embryos. The number of variations regarding the concentra-tions, incubation times and other conditions is almost infinite.
On the other hand, the radical strategy of vitrification has resulted in some positive consequences apart from the total elimination of ice crystal formation. The increased cooling rate decreases chilling injury, i.e. damage of the intracellular lipid droplets, lipid-containing membranes and the cytoskeleton, passing rapidly through the dangerous
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q158toy58C zone Dobrinsky, 1996; Martino et al., 1996a,b; Isachenko et al., 1998; .
Zeron et al., 1999 . Moreover, vitrification does not require expensive coolers or special skill and can be performed very quickly.
Most of the publications comparing traditional freezing and vitrification of transfer-able stage domestic animal embryos report either equal in vitro or in vivo survival rates
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Dinnyes et al., 1995; Hasler et al., 1995; Reinders et al., 1995; Agca et al., 1996, 1998a;
´
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Kuwayama et al., 1996; Lane et al., 1998; O’Kearney Flynn et al., 1998 . However, to date, vitrification has not been widely accepted by embryo transfer practitioners, and there are no signs of a major change in the foreseeable future. In analyzing the reasons, the following factors appear to be important.
Ž .1 Traditional freezing has resulted in acceptable pregnancy and calving rates in cattle, sheep and goats. The usual 10% gap between the results after transfer of fresh vs.
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frozen embryos is difficult to reduce Niemann, 1991; Palasz and Mapletoft, 1996 . On the other hand, for porcine embryo cryopreservation, despite of some promising results in vitro, vitrification so far has not offered a commercially viable solution.
Ž .2 The extreme diversity of the vitrification methods and the lack of a standard and proved technology discourage practitioners. Moreover, most of the comparative results available are based on transfer of in vitro produced embryos, where handicapped embryo quality might also influence the outcome.
Ž .3 The practical advantages of vitrification are relative. The time required for vitrification may be as short as 3 min, but this is for each straw that must be cooled individually. Calculating the time for 15–25 embryos to be cryopreserved together, the difference is minimal and the work may be more difficult using vitrification. The vitrification procedure seems to be simpler than equilibrium freezing but in practice similar steps are required for both processes and the complicated part of the equilibrium freezing process is done automatically. Compared to the other expenses, the price of an embryo freezer is not really the decisive factor for an embryo transfer practitioner. On the other hand, there is little interest for equipment-producing companies in spreading a technology such as vitrification, which can be performed using a simple foam box.
The present and possible future role of vitrification in domestic animal embryology is therefore not to replace traditional freezing, but to offer solutions for special areas where the other methods have failed to produce satisfactory results.
2. Recent developments
Although the potential beneficial effects of further increased cooling and warming Ž
rates reduction of cryoprotectant concentration and toxicity, further decrease of chilling .
injury were widely known, until recently, few efforts were made to achieve this advantage for practical purposes. The traditional tools, i.e. cryovials and insemination straws, are far from ideal for this purpose and their widespread application for vitrification restricted the options available. However, the development of some alterna-tive ways of cooling and warming started nearly a decade ago and, more recently, has created some new opportunities. Nearly all the new rapid cooling techniques are based on direct contact between the cryoprotectant solution and the liquid nitrogen.
The simplest way to establish this contact is the direct dropping of embryos into
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liquid nitrogen. This was first suggested by Landa and Tepla 1990 for mouse embryos, Ž
then successfully used also for bovine embryos, zygotes and oocytes Riha et al., 1991; .
Yang and Leibo, 1999; Papis et al., 1999 . However, the size required for the droplet is
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this problem. Based on the results achieved in vitrification of Drosophila embryos ŽSteponkus et al., 1990; Mazur et al., 1992 , bovine oocytes were placed on the surface.
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of electron microscope grids before submerging into the liquid nitrogen Martino et al., .
1996b; Arav and Zeron, 1997 . The estimated cooling rate was 11 000 to 14 000 and 24 0008Crmin in liquid nitrogen or in slush of liquid nitrogen, respectively. In the Open
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Pulled Straw OPS technology, the carrier is a narrow plastic tube and the approximate
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volume of 1ml cryoprotectant solution is loaded into the open end Vajta et al., 1997a . The cooling rate in this method is approximately 20 0008Crmin. As the method is relatively simple and can be based on the traditional tools of embryo and semen cryopreservation, it was rapidly applied for different purposes. Moreover, in-straw dilution is also possible and high survival, pregnancy and calving rates were achieved with in vitro produced, OPS vitrified bovine blastocysts after direct embryo transfer ŽLewis et al., 1999; Vajta et al., 1999; Lewis, unpublished ..
These new high-speed vitrification methods decrease dramatically the chilling injury,
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permit the use of less concentrated i.e. less toxic solutions, and shorten the time of exposure with the final cryoprotectant both before cooling and after warming.
Addition-Ž .
ally, the small volume of the solution prevents heterogeneous ice formation Rall, 1987 . Consequently, a considerable advance in the cryopreservation of certain mammalian ova and embryos has been achieved. These results have generated a search for new methods
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for further increasing the cooling rates, with remarkable success Hamawaki et al., 1999; .
Lane et al., 1999 .
However, almost all these techniques are based on the direct contact of the liquid nitrogen and the cryoprotectant medium containing the oocytes or embryos, which may
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be a source of contamination Tedder et al., 1995; Fountain et al., 1997 . These dangers may be eliminated by using sterile liquid nitrogen for cooling, then wrapping the cooled
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embryos in a hermetic container before storage Vajta et al., 1998b; Lane et al., 1999 , but this procedure might be too complicated for everyday application. A possible solution to the problem is the minimum volume cooling method of Hamawaki et al. Ž1999 , where the embryos are loaded in an extremely low volume onto the wall of a. 0.25-ml insemination straw, which is sealed before cooling.
Another possibility is to use even more narrow and thin-walled straws than the OPS and to heat-seal them as used for normal straws. According to our recent observations based on serially diluted cryoprotectant solutions, the open end is not required if the inner diameter and the wall thickness are approximately 400 and 40mm. This is half the size of the OPS straws and a quarter of the standard 0.25-mm straw. The initial survival of embryos using this tool was not different from the results achieved by the OPS method. This simple modification of the size may result in a practical tool and method combining the safety of the sealed straw with the rapid cooling rate of the OPS technology.
3. Results and perspectives
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approach the values of the non-cryopreserved controls 15%, 25% and 30%; Martino et .
al., 1996b; Vajta et al., 1998a; Papis et al., 1999, respectively and their transfer resulted
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in live offspring Vajta et al., 1998a . The commercial application of oocyte vitrification has become a reality and a similar advance is also expected in other domestic animal species.
Another important area of vitrification concerns embryo cloning. Vitrification of pre-compaction stage bovine embryos did not decrease their further developmental
Ž ability, and the blastomeres were successfully used as donors for nuclear transfer Booth
.
et al., 1998; Peura et al., 1999 . Although the blastocyst rates were still compromised, recipient cytoplasts were also successfully cryopreserved and full-term development was
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achieved Booth et al., 1999 . This application may have considerable impact on the rapidly increasing cloning research and industry.
Until recently, vitrification was regarded as a possible way to resolve the problems of cryopreservation of in vitro produced bovine embryos. However, it has been revealed
Ž that the success of cryopreservation is profoundly affected by culture conditions Leibo and Loskutoff, 1993; Voelkel et al., 1992; Tervit et al., 1994; Massip et al., 1995;
.
Twagiramungu et al., 1997 . Recent developments in the culture methods have consider-ably improved the embryo quality, therefore the traditional equilibrium freezing method
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can also be successfully applied for this purpose Holm et al., 1999a; Galli, personal .
communication . On the other hand, vitrification may still have some advantage for Ž cryopreservation of ‘double-handicapped’, i.e. in vitro cultured and manipulated
bio-. Ž .
psied, cloned embryos Booth et al., 1997; Vajta et al., 1997b; Agca et al., 1998b . The appearance of new vitrification methods has generated a new wave of research to resolve the problem of cryopreservation of embryos from pigs, as well as from other mammalian species, predominantly those that are endangered. In spite of some excellent survival results in vitro and the birth of some piglets, the expected breakthrough
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permitting large scale porcine embryo transfer has not been achieved for long Kuwayama Ž .. et al., 1997; Vajta et al., 1997c; Kobayashi et al., 1998; Holm et al., in press b . Very recently, however, offspring were produced after OPS vitrification of porcine embryos
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in two laboratories Nottle, personal communication; Berthelot, personal communica-.
tion . A major challenge for researchers is to improve and stabilize these results and to establish a universal standardized vitrification method, which can be successfully applied for cryopreservation of oocytes and embryos of different species and different developmental stages.
Acknowledgements
The author wishes to thank H. Callesen, R.H.F. Hunter, J. Shaw, and A.O. Trounson for the useful suggestions and critical reading of the manuscript.
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