Effect of freezing rate and exposure time to cryoprotectant on the development of mouse pronuclear stage embryos

M.A. Nowshari1,3 and G. Brem2

1 Interuniversitäres Forschungsinstitut für Agrarbiotechnolgie, Tulln and 2 Ludwig Boltzmann Institute für immuno-, zyto- und molekulargenetische Forschung, Vienna, Austria


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
BACKGROUND: The effects of exposure time (20 versus 45 s) to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) and freezing rates (1200–10 300°C/min) during rapid freezing of mouse pronuclear stage embryos on survival and development to blastocysts were investigated. Different freezing rates were achieved by directly plunging the straws (rapid freezing) and open pulled straws (OPS) in liquid nitrogen (OPS freezing) and by plunging the straws (super rapid) and OPS (super OPS) in a super cooled liquid nitrogen chamber (at –212°C) before storage in liquid nitrogen. METHODS: Morphologically intact mouse zygotes (n = 891) pre-equilibrated in 1.5 mol/l ethylene glycol for 5 min were either loaded in 0.25 ml straws containing cryoprotectant or loaded in OPS with 2 µl cryoprotectant. After 20 or 45 s of loading the straws or mixing in cryoprotectant and loading in OPS, they were plunged either directly in to liquid nitrogen or were plunged first in to liquid nitrogen in a super cooled chamber and then stored in liquid nitrogen. Zygotes were thawed and intact embryos cultured in vitro. RESULTS: The rate of survival was higher (91%, P < 0.01) when zygotes were frozen with rapid freezing compared with super rapid, OPS and super OPS freezing rates with an exposure time of 20 s (70, 65, and 76% respectively). When zygotes were exposed to cryoprotectant for 45 s and frozen with rapid freezing rates, the survival was higher (86%, P < 0.01) compared with those frozen with OPS (62%) but was not different from those frozen with super rapid and super OPS freezing rates (81 and 75%). A higher rate of survival was observed when zygotes were exposed to cryoprotectant for 45 s and frozen with super OPS than with OPS freezing (75 versus 62%; P < 0.05). The rate of cleavage and development of intact zygotes to blastocysts was not different among the different groups. CONCLUSION: Exposure of zygotes to a high concentration of cryoprotectant (7.0 mol/l ethylene glycol with 0.5 mol/l sucrose) for 20 or 45 s did not influence their survival and development and increasing the freezing rate from 1200–10 300°C/min was of no advantage when using a rapid freezing procedure for freezing mouse pronuclear stage embryos.

Key words: cryopreservation/embryo/mice/rapid freezing/vitrification


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Cryopreservation of human embryos has been introduced into clinical IVF in order to preserve supernumerary embryos for a later transfer. Human embryos at different developmental stages have been frozen with variable success rates (Mandelbaum et al., 1998Go, for review). The pronuclear stage appears to be the optimal stage for cryopreservation (Veeck et al., 1993Go; Damario et al., 1999Go). The unicellular form and lack of spindle apparatus may account for its high post-thaw survival and implantation potential. Using this stage for freezing, there are no ambiguities about whether embryos survive thawing because subsequent embryo cleavage essentially proves cellular integrity. In addition, in some European countries, the freezing of cleaved stage embryos is illegal, thus limiting the choice to freezing of either unfertilized oocytes or pronuclear stage embryos. Slow cooling procedures have been most widely used for freezing of human pronuclear stage embryos with variable results (Mohr et al., 1985Go; Ashwood-Smith and Simons, 1986Go; Siebzehnrübl et al., 1986Go; Testart et al., 1987Go; Veeck et al., 1993Go; Van den Abbeel et al., 1997Go; Al-Hasani et al., 1999Go; Damario et al., 1999Go). Freezing human pronuclear embryos using a rapid procedure such as reported by Trounson et al. (Trounson et al., 1987Go) has been reported by a few IVF groups also with variable results (Gordts et al., 1990Go; Feichtinger et al., 1991Go). Slow cooling procedures have the disadvantage in that they are time consuming and require accurately controlled expensive freezing units, making them unsuitable for use where cost and time is a consideration. Different freezing protocols that are faster and cheaper and achieve higher survival and development rates after freezing and thawing than do conventional freezing procedures have been reported for mice pronuclear stage embryos (Van der Auwera et al., 1990Go; Shaw et al., 1991Go; Bernart et al., 1994Go; Nowshari et al., 1995Go; Van der Elst et al., 1995Go). Some factors, like type of cryoprotectant, its concentration and the thawing procedure, have been shown to influence the survival and development in vitro and in vivo using rapid freezing procedures (Nowshari et al., 1994Go, 1995Go; Nowshari and Brem, 1998Go, 2000aGo).

Exposure time to the high concentration of cryoprotectant(s) during the rapid freezing procedure and vitrification has been proved to be one of the main factors influencing the survival of embryos and oocytes (Scheffen et al., 1986Go; Vanderzwalmen et al., 1988Go; Ishimori et al., 1992Go; Shaw et al., 1992Go; Nowshari et al., 1994Go). However, there are practical limitations in reducing the exposure time beyond a certain minimum, because once embryos/oocytes are exposed to high concentrations of cryoprotectant, time is required to load them in to straws and to seal the straws before plunging them in liquid nitrogen. Another factor which has been less studied and which may influence the survival of embryos/oocytes during rapid freezing or vitrification is the rate of freezing. A freezing rate of about 1200°C/min can be achieved by directly plunging straws into liquid nitrogen. Recently, different approaches have been suggested and applied to increase the freezing rate of embryos/oocytes, however, nearly all the cooling techniques are based on direct contact between the cryoprotectant solution and the liquid nitrogen. The simplest way to establish this contact is to immerse embryos directly into liquid nitrogen (Landa and Tepla, 1990Go). This procedure developed for mouse embryos was then successfully used for bovine embryos, zygotes and oocytes (Riha et al., 1991Go; Yang and Leibo, 1999Go; Papis et al., 1999Go). A similar effect was achieved by using a cryoloop for freezing of mouse and human blastocysts (Lane et al., 1999Go). The surface of an electron microscope grid (Martino et al., 1996Go) or a metal slab (Dinnyés et al., 2000Go) have both been used for placing the bovine oocytes/embryos before submerging them into liquid nitrogen. Using an electron microscope grid, the estimated cooling rate was 11 000–14 000 and 24 000°C/min in liquid nitrogen or in the slush of liquid nitrogen respectively (Arav et al., 2000Go). Open pulled straw (OPS) technology (Vajta et al., 1998aGo) involves the use of a narrow plastic tube and an approximate volume of 1–2 µl cryoprotectant solution, loaded into the open end of the straw and directly plunged into liquid nitrogen. A device (Vit-Master®; Mini Tübe, Landeshut, Germany) with which the temperature of liquid nitrogen can be decreased down to –212°C by creating a low atmospheric pressure (Arav et al., 2000Go) has been introduced. Using this device the freezing rate of straws from + 20°C to –10°C was increased from 1200–4000°C and, when using 2 µl of cryoprotectant and an OPS, the freezing rate increased from 5300 to 10 300°C/min.

In this study we examined the effect of decreasing the exposure time (from 45–20 s) of pronucleate embryos to a high concentration of cryoprotectant and increasing the freezing rate from 1200°C to about 10 300°C by using two different freezing vessels, i.e. straws and OPS, and plunging them either directly in to liquid nitrogen or after immersion in Vit-Master® (–212°C) before final plunging in liquid nitrogen. The mouse was used as a model for development and testing of freezing methods to assess the effect of factors which may influence the viability of human embryos in a cryopreservation procedure, though the freezing sensitivities of different species cannot be expected to be identical. The rapid procedure used here has previously been shown by us to be suitable for cryopreservation of pronuclear stage mouse embryos (Nowshari et al., 1995Go). Intact and biopsied mouse embryos at different stages frozen with this freezing procedure have resulted in the birth of live animals (Nowshari et al., 1995Go; Nowsahri and Brem, 1998 Nowsahri and Brem, 2000a,b).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Embryo collection
Female virgin F1 mice (C57 BLxCBA, 28–42 days old) were superovulated with 7.5 IU i.p. eCG (Folligon®; Intervet, Vienna, Austria) and HCG (Ekluton®; Intervet) administered ~48 h apart. The injections were given between 1400 and 1500 h and lights were on between 0800–2000 h. At the time of HCG injection, the females mice were individually caged with males and checked for copulation plugs the next morning. Mice were killed by cervical dislocation 23–24 h after HCG injection, ovaries removed and cumulus complexes collected in phosphate buffered saline (PBS; Gibco BRL, Life Technologies, Vienna, Austria) with 10% fetal calf serum (FCS). Cumulus cells were removed by incubation in hyaluronidase (150 IU/ml, Sigma, Vienna, Austria) in PBS and then washing three times in PBS medium. Morphologically abnormal embryos were discarded at this stage, and the denuded intact pronuclear stage embryos from all mice were pooled together. Embryos were randomly divided into eight groups and frozen, using either straws or OPS after an exposure time of 20 and 45 s to cryoprotectant and different freezing rates. Each experimental condition had five to six replications.

Freezing
Freezing solutions of 1.5 mol/l ethylene glycol (Sigma) with 0.25 mol/l sucrose (Sigma) and 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose and thawing solution of 0.5 mol/l sucrose were made with PBS supplemented with 10% heat-inactivated FCS. All manipulations were performed in 35 mm plastic Petri dishes (Nunc, Denmark).

Embryos were frozen using a rapid freezing procedure described earlier (Nowshari and Brem, 1998Go). However, in this experiment either normal 0.25 ml straws (IMV, L'Aigle, France) or OPS (Vajta et al., 1998aGo), a narrow plastic tube (i.d. 0.8 mm) manually pulled out from a heat softened straw, were used.

Freezing and thawing protocol
For freezing embryos, using a 0.25ml straw or OPS, embryos were pre-equilibrated at room temperature (22–23°C) in 1.5 mol/l ethylene glycol with 0.25 mol/l sucrose in medium PBS for 5 min.

Freezing and thawing of straws
Pre-equilibrated embryos were directly loaded with a mouth controlled glass pipette into the middle of a 0.25 ml straw containing 40 µl of 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose (Figure 1Go). The straws were capped (no heat sealing) and after 20 or 45 s either dipped slowly and vertically into liquid nitrogen (rapid freezing) or immersed first in a super-cooled (–212°C) liquid nitrogen chamber (Vit-Master®) and then transferred to liquid nitrogen for further storage (super rapid freezing). Embryos were stored in liquid nitrogen for periods not less than 24 h.



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Figure 1. Schematic representation of the straw loaded with embryos and cryoprotectant for rapid freezing.

 
The straws with frozen embryos were thawed in a water bath at 25°C. The dilution of the cryoprotectant was achieved by transfer of embryos to 0.5 mol/l sucrose for 5 min. Embryos were washed three times in medium PBS. Morphologically intact embryos were washed in medium M16 (Whittingham, 1971Go) with 4 mg/ml BSA (Crystalline®; ICN, Meckenheim, Germany) and transferred to 50 µl of this medium for further culture. The droplets of M16 were maintained in an incubator at 37°C in an atmosphere of 5% CO2 in air. Eighteen hours later two cells were counted and uncleaved ova removed; 65 h after assessing the 2-cell stage the number of blastocysts (Hogan et al., 1986Go) was recorded.

Freezing and thawing of OPS
After pre-equilibration in 1.5 mol/l ethylene glycol with 0.25 mol/l sucrose for 5 min, 5–10 embryos in 2 µl of medium were transferred to a drop of 40 µl of 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose and mixed together. Embryos were immediately taken out with a mouth controlled pipette with 2 µl of medium and pushed out to make a droplet on the surface of a Petri dish. Embryos were then loaded into an OPS by simply touching the droplet thus using the capillary effect of the straw. The straws were then either plunged directly into liquid nitrogen (OPS freezing) or were first transferred to the super cooled liquid nitrogen chamber (Vit-Master®) maintained at –212°C (super OPS) and then plunged into liquid nitrogen. The straws were later transferred to a liquid nitrogen container for further storage.

The OPS were thawed by leaving the straws in air for 5 s and then releasing the embryos into a drop of 0.5 mol/ l sucrose as described by Vajta et al. (1998a). Embryos were left in this medium for 5 min and then washed and cultured as described for those frozen and thawed in straws.

Statistical analysis
Differences in rates of survival and development between treatment groups were tested for significance with {chi}2 test. The level of significance was set at 5%.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The rate of survival, cleavage and development of pronuclear stage embryos to blastocysts, frozen in straws with an exposure time of 20 versus 45 s and with different freezing rates (rapid freezing versus super rapid) is shown in Figure 2Go. The rate of survival of embryos exposed to cryoprotectant for 20 s and frozen with rapid freezing rates was higher (P < 0.01) compared with those frozen with super rapid freezing rates. However, there was no difference in the rate of development of embryos among the different groups exposed to 20 or 45 s and cooled with different freezing rates. When using OPS and embryos exposed to cryoprotectant for 45 s, a higher survival (P < 0.05) was recorded with super OPS compared with OPS freezing rates (Figure 3Go). However, there was no difference when embryos were exposed to 20 s and frozen with OPS or super OPS freezing rates. There was no difference in the rate of cleavage and development of embryos among different groups when OPS were used to freeze the embryos (OPS freezing and super OPS). While comparing the survival and development of embryos frozen in straws with those frozen in OPS, the best survival and development of embryos to blastocysts was achieved using straws with an exposure time of 20 s to the cryoprotectant and a freezing rate of about 1200°C/min (rapid freezing). This rate of survival was higher (P < 0.01) compared with those frozen with OPS and super OPS freezing rates. When zygotes were exposed to cryoprotectant for 45 s, the survival was higher (P < 0.01) with rapid freezing rates compared with those frozen with OPS freezing. The rate of cleavage and development of intact zygotes to blastocysts was not different among the different groups.



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Figure 2. Survival and in-vitro development of pronuclear stage mouse embryos frozen in straws with 7.0 mol/l ethylene glycol with0.5 mol/l sucrose and exposure time of 20 or 45 s and direct plunging in liquid nitrogen (rapid) or immersion in super cooled chamberat –212°C (super rapid) followed by plunging in liquid nitrogen. The value labels represent the number of embryos.

 


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Figure 3. Survival and in-vitro development of pronuclear stage mouse embryos frozen in open pulled straws (OPS) with 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose and exposure time of 20 or 45 s and direct plunging in liquid nitrogen (OPS) or immersion in super cooled chamber at –212°C (super OPS) followed by plunging in liquid nitrogen. The value labels represent the number of embryos.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
In this study, we have tested the effects of exposure time of zygotes to the high concentration of cryoprotectant and the freezing rate during rapid freezing of pronuclear stage mouse embryos. During rapid freezing or vitrification of embryos/oocytes, since a very high concentration of cryoprotectant is used, it is preferable to expose the embryos/oocytes to the cryoprotectant for a very short period to avoid its toxic effects. It has been demonstrated previously that time of exposure to high concentrations of cryoprotectant can affect the survival and development of embryos and oocytes (Scheffen et al., 1986Go; Vanderzwalmen et al., 1988Go; Ishimori et al., 1992Go; Shaw et al., 1992Go; Nowshari et al., 1994Go; Van der Elst et al., 1995Go). However, there are limitations in reducing further the time of exposure, since some time is obviously required to load the embryos into straws/vials and seal them properly before final immersion in liquid nitrogen. In our experience, time required to load the embryos into a straw and heat seal it takes between 30 and 40 s. This practical necessity was the reason behind selecting an exposure time of 45 s for freezing of different stages of embryos and oocytes using our rapid freezing procedure (Nowshari et al., 1994Go, 1995Go; Nowshari and Brem, 1998Go, 2000aGo, 2000bGo). Since we intended to reduce the time of exposure to the cryoprotectant in this experiment, we did not heat seal the plastic straws; instead, we fixed a cap on the open end of the straw and the other end was plugged with cotton. Plugging a straw with a cap takes just a few seconds, so the time of exposure to the cryoprotectant could be drastically reduced. However, one of the disadvantages of using a cap to plug the straw is the occasional seepage of liquid nitrogen into the straws. Thus, sometimes straws explode and lose their caps, which may result in the loss of embryos. In the present experiment, three straws lost their caps during thawing but none of the embryos was lost. Reducing the time of exposure from 45 to 20 s in 7.0 mol/l ethylene glycol with 0.5 mol/l sucrose does not seem to influence the survival of zygotes when using a rapid freezing protocol. We have previously shown (Nowshari et al., 1994Go) that exposure of mouse oocytes to high concentration of cryoprotectants can be deleterious for the survival and development of oocytes when exposed for 2.5 min compared with 45 s. However, the present study shows that further reduction in the exposure time from 45 to 20 s may not have any advantage in terms of survival and development of pronuclear stage embryos. Reducing the time of exposure from 20 to 15 s during vitrification of mouse oocytes has been previously reported (Shaw et al., 1992Go). These results are not comparable to the present experiment because a different freezing protocol, cryoprotectants and strain of mice were used in that experiment.

In this experiment we have also shown that increasing the freezing rate of mouse embryos from 1200°C/min, achieved by direct plunging of straws in to liquid nitrogen, to higher freezing rates may not be of any advantage, at least while using the cryoprotectant and freezing protocol used in this study. Freezing by loading the embryos in a minimal amount of freezing medium in an OPS, thus increasing the freezing rate from about 1200–5300°C/min, or by plunging the straws or OPS first in super cooled liquid nitrogen (–212°C), thus increasing the freezing rate to about 4000 and 10 300°C/min, may also not have a positive influence on the survival and development of embryos. A higher survival of embryos, exposed for 20 s and frozen at 1200°C/min compared with those frozen at higher rates (4000–10 500°C/min), indicates that just increasing the freezing rate may be at times rather disadvantageous. To obtain higher survival and development of embryos and oocytes after freezing and thawing, in addition to the rate of freezing, other factors like exposure time, type and concentration of cryoprotectant and freezing sensitivity of oocytes/embryos ought to be taken into consideration. The use of OPS for vitrification of embryos and oocytes in different species has been reported to improve their survival and development (Vajta, 2000Go, a review). However, there are a few drawbacks in using this freezing procedure. One of the main disadvantages is the possibility of contamination of the biological material from contaminated liquid nitrogen and vice versa, thus compromising the sanitary status of the embryos. Recently, embryos loaded in OPS exposed to contaminated liquid nitrogen with bovine viral diarrhoea virus (BVDV) and bovine herpesvirus-1 (BHV) were tested positive for viral association (Bielanski et al., 2000Go). It has also been acknowledged that many viral and bacterial pathogenic agents may adhere to the intact zona pellucida and that most infectious viral and bacterial agents easily survive in cryoprotectants and liquid nitrogen (Melnick, 1965Go; Wallis and Melnick, 1968Go; Fountain et al., 1997Go). As alternative preventive steps against contamination, measures such as liquid nitrogen filtration and the application of accessory protective storage containers have been proposed (Vajta et al., 1998bGo). However, the practicability of such measures are questionable and the method can then no longer be considered a simple method.

The rapid freezing procedure used in the present experiment has been shown by us previously to be very efficient for freezing different stages of mouse embryos and oocytes (Nowshari et al., 1994Go, 1995Go; Nowshari and Brem, 1998Go, 2000aGo). In the present experiment only in-vitro development of embryos was investigated; however, we have previously reported the in-vivo development of pronuclear stage embryos collected at a similar stage (Nowshari et al., 1995Go), embryos frozen once or twice (Nowshari and Brem, 1998Go) and those frozen in solutions supplemented with chemically defined macromolecules (e.g. PVA) using this freezing procedure (Nowshari and Brem, 2000cGo).

In summary, a high proportion of pronuclear stage embryos survive freezing and retain their development capacity after using the simple rapid freezing protocols with a high concentration of ethylene glycol with sucrose. We have shown that reducing the time of exposure from 45 to 20 s does not significantly affect the survival and development of embryos. Further, we have shown that increasing the freezing rate either by direct exposure of embryos to liquid nitrogen in an OPS or by immersion in super-cooled liquid nitrogen may not be of any advantage, when using this freezing protocol for cryopreservation of mouse pronuclear stage embryos. These findings need to be given consideration while developing faster freezing procedures for embryos and oocytes of human as well as animal origin.


    Acknowledgements
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The authors thank Dr Thomas Kolbe for technical help and Sandra Schönkhammer for animal maintenance. We thank M/S Mini-Tüb, Landeshut, Germany for providing us with Vit-Master® for the experiment.


    Notes
 
3 To whom correspondence should be addressed at: Agrobiogen GmbH., Biotechnologie-Larezhausen, Thalmannsdorf 25,86567 Hilgertshausen, Germany. E-mail: mnowshari{at}hotmail.com Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
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Submitted on March 16, 2001; accepted on August 2, 2001.