fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

Fetal development of mouse oocytes and zygotescryopreserved in a nonconventional freezing mediumq

James J. Stachecki,* Jacques Cohen, Tim Schimmel,and Steen M. Willadsen

Institute for Reproductive Medicine and Science of Saint Barnabas Medical Center, 101 Old Short Hills Road, Suite 501,

West Orange, NJ 07052, USA

Received 31 July 2001; accepted 2 January 2002

Abstract

This study (1) analyzed fetal development of mouse embryos after oocyte cryopreservation in CJ2, a

choline-based medium, (2) examined the effect of culture duration in vitro on subsequent fetal develop-

ment, and (3) compared survival and fetal development of zygotes frozen in embryo transfer freeze medium

(ETFM; sodium-based medium) or CJ2. Unfertilized oocytes and zygotes were cryopreserved using a slow-

cooling protocol. After thawing, oocytes were inseminated after drilling a hole in their zona, cultured in

vitro either to the two-cell or blastocyst stage, and transferred to the oviducts or uterine horns of recipient

mice. In parallel experiments, frozen–thawed zygotes were similarly cultured and transferred. Implantation

rates for transferred embryos were high (range 66–88%), regardless of whether they had been frozen as

oocytes or zygotes and whether they had been transferred to the oviduct or uterus. However, fetal de-

velopment was significantly higher when two-cell embryos were transferred. With blastocyst transfer,

control embryos implanted and produced a greater proportion of fetuses than did oocytes frozen in CJ2,

whereas transfer at the two-cell stage resulted in similar proportions of implantation sites and fetuses.

Blastocyst transfer of zygotes cryopreserved in ETFM or CJ2 produced similar fetal development rates

(23.6% vs 20.0%), but when frozen–thawed zygotes were transferred at the two-cell stage the fetal devel-

opment rates were higher in the ETFM group (53.3%) than in the CJ2 group (32.0%). A high proportion

(46.7%) of oocytes frozen in CJ2 in a nonprogrammable freezer and plunged at )20 �C developed into liveoffspring. This study shows that in the mouse (1) oocytes frozen in CJ2 can develop into viable fetuses, (2)

prolonging culture in vitro has a detrimental effect on embryo transfer outcome, and (3) CJ2 offers no

advantage for zygote cryopreservation. � 2002 Elsevier Science (USA). All rights reserved.

Keywords: Cryopreservation; Oocytes; Zygotes; Choline chloride; Mouse; Uterus; Oviduct; Embryo transfer

In previous reports we have described how the

results of cryopreserving unfertilized mouse eggs

according to a conventional slow-freezing proto-

col are substantially improved by the use of CJ2, a

choline-based medium, instead of a conventional

sodium-based medium, ETFM (Embryo Transfer

Freeze Medium; Gibco BRL, Gaithersburg, MD;

[24–26]). In the present study we examined (1)

fetal development of embryos produced from

oocytes cryopreserved in CJ2, (2) the effect of

Cryobiology 44 (2002) 5–13

www.academicpress.com

qThis work was funded by institutional sources.* Corresponding author.

0011-2240/02/$ - see front matter � 2002 Elsevier Science (USA). All rights reserved.PII: S0011 -2240 (02)00007 -X

culture duration in vitro on subsequent develop-

ment in vivo, and (3) the usefulness of CJ2 for

zygote freezing.

Our previously published results were ex-

pressed in terms of survival after thawing, fer-

tilization, and development to the blastocyst

stage in vitro. Ultimately, however, it is the

ability of embryos produced from cryopreserved

oocytes to develop into normal offspring that is

the best measure of success. Because we are

working toward developing a medium for storing

oocytes from a range of species, including hu-

mans, and because CJ2 is very different from any

other medium previously used, in being virtually

sodium-free, it is imperative that fetal develop-

ment be assessed and documented. Furthermore,

there have been less than a dozen studies that

have reported fetal development from cryopre-

served mouse oocytes [1,2,5,23]. The highest fetal

development rates obtained with slowly cooled

oocytes were 31% for two-cell oviductal transfers

and 23% for blastocyst uterine transfers [5] and a

rate of 42% for two-cell oviductal transfers [2],

although Bos-Mikich et al. [1] obtained 41% with

oviductal transfers of two-cell embryos produced

from vitrified oocytes. Of particular note is the

study by Schroeder et al. [23], in which a live pup

rate of 26% was obtained after oviductal trans-

fer. More recent studies describe oocyte cryo-

preservation success only in terms of the

blastulation rate after fertilization and culture in

vitro (for review see [10]). Relatively early in the

course of our experiments, a small trial estab-

lished that viable pups could develop from

blastocysts produced from oocytes cryopreserved

in CJ2. However, the success rate was lower than

expected from the high rates of development in

vitro (data not shown), similar to the findings of

Lane and Gardner [15] when they transferred

slowly cooled oocytes to recipients at the

blastocyst stage.

Choline and other organic osmolytes including

betaines are known to have osmoprotective and

cryoprotective effects on liposomes, erythrocytes,

plants, and mouse embryos [16,17,27]. The osmo-

lyte glycinebetaine has been found in animals,

bacteria, fungi, algae, and many drought- and

salt-tolerant plants [22]. Some of these com-

pounds protect enzymes and membranes from

cold [14,18,28], salt [7,9], and freezing damage

[8,32]. It is possible that choline has similar effects

on the membrane integrity of oocytes and em-

bryos. In support of this hypothesis, Toner et al.

[27] has shown that during cooling, zygote cell

membranes are more tolerant to hyperosmotic

stress from a mixture of sodium and choline ions

than from sodium ions alone.

In the present study we examined fetal devel-

opment to days 10–13 of gestation and briefly

examined development to term to obtain a more

clear indication of oocyte viability after cryopre-

servation in CJ2. Finally, we investigated whether

mouse embryo cryopreservation could be im-

proved by the use of CJ2, rather than ETFM, a

common sodium-based medium for freezing

mouse embryos.

Materials and methods

Collection and cryopreservation of oocytes and

zygotes

For all experiments, C57BL/6�BALB/c F1mice (The Jackson Laboratory, Bar Harbor, ME,

USA) were used [24]. Freshly ovulated metaphase

II mouse oocytes (collected 13 h after HCG) and

zygotes (collected 17 h after HCG) were treated

according to the methods described by Stachecki

et al. [24]. Oocytes and zygotes were frozen in ei-

ther CJ2 supplemented with 10% fetal bovine se-

rum or ETFM with 1.5 M 1,2-propanediol

(PrOH) and 0.1 M sucrose as cryoprotectants and

frozen using a slow-cooling protocol [24]. After

storage for at least 3 days, the straws were thawed

by exposing them to air at room temperature for

30 s followed by immersion in a 30 �C water bathfor an additional 10 s (30/10; [26]). After thawing,

the cryoprotectants were removed in five steps at

5-min intervals at 23 �C as described by Stacheckiet al. [24]: freezing medium supplemented with (I)

0.2 M sucrose and 1.0 M PrOH, (II) 0.2 M sucrose

and 0.5 M PrOH, (III) 0.2 M sucrose, (IV) 0.1 M

sucrose, and (V) freezing medium alone. Oocytes

and zygotes frozen in CJ2 were held for an addi-

tional 5 min in mCZB [3,11] for reequilibration

with a sodium-based medium and then incubated

on a slide warmer at 37 �C for 5 min. Embryosfrozen in ETFM were not moved to mCZB me-

dium, but incubated in ETFM on a slide warmer

at 37 �C for 5 min prior to being placed intoculture. The zonae of frozen–thawed oocytes was

opened during step IV (freezing medium with

0.1 M sucrose) of the thawing procedure by using

a Fertilase laser [24]. Spermatozoa were aspirated

from the epididymides of 12- to 16-week-old

C57BL/6�BALB/c F1 mice into Ham’s F10 me-dium (Sigma Chemical, St. Louis, MO, USA),

6 J.J. Stachecki et al. / Cryobiology 44 (2002) 5–13

supplemented with 6 mg/ml bovine serum albumin

(Fraction V; Sigma) and allowed to capacitate for

1.5–2 h before insemination. Directly after cryo-

protectant removal, the oocytes were transferred

to Ham’s F10 containing approximately 5� 105motile sperm for 5–9 h. Fertilized oocytes were

cultured in S1 (a low-glucose, 0.50 mM, preim-

plantation embryo culture medium containing

nonessential amino acids; Scandinavian IVF Sci-

ence, Gothenburg, Sweden) for 2 days before be-

ing transferred to S2 (a high-glucose, 3.15 mM,

preimplantation embryo culture medium con-

taining essential and nonessential amino acids;

Scandinavian IVF Science). Zygotes were cultured

in KSOM (a conventional mouse embryo culture

medium without amino acids and with 0.20 mM

glucose; Specialty Media, Lavallette, NJ, USA),

for 2 days before being transferred to a fresh drop

of KSOM. In earlier experiments we determined

that the use of sequential media (S1/S2) was nec-

essary to maximize the production of blastocysts

from frozen–thawed oocytes [26], whereas culture

in KSOM (our standard mouse embryo culture

medium) was adequate for obtaining a high rate

of blastocyst formation from frozen–thawed zy-

gotes. Nonfrozen zygotes served as controls and

were cultured in KSOM either to the two-cell or

to the early blastocyst stage.

Embryo transfers

Two-cell embryos that developed from frozen–

thawed oocytes or zygotes were transferred to the

oviducts of Day 1 (day of copulation plug)

pseudopregnant CD1 mice (The Jackson Labo-

ratory) along with nonfrozen control two-cell

embryos collected at the zygote stage [2,5]. Re-

cipients were anesthetized with tribromoethanol

(Avertin; 2.5% solution; 0.02 ml/g body weight).

Frozen oocyte- and frozen zygote-derived ex-

panding blastocysts that appeared to be mor-

phologically normal and had a prominent

blastocoel, along with in vitro cultured, nonfrozen

control blastocysts, were transferred to the uterine

horns of Day 3 recipient mice [5]. Six to 10 em-

bryos were transplanted per recipient. Control

embryos were transferred to one oviduct or uter-

ine horn, while an equivalent number of embryos

from cryopreserved oocytes or zygotes were

transferred to the con-tralateral oviduct or uterine

horn of the same recipient. Only the number of

embryos transferred to recipients that became

pregnant are shown in the tables. The recipient

mice were sacrificed 10–13 days after embryo

transfer, and the numbers of implantation sites

and fetuses were recorded.

Cryopreservation in a nonprogrammable freezer

To test the ability of oocytes cryopreserved in

CJ2 to develop into live-born fetuses and to fur-

ther demonstrate that oocytes can withstand the

trauma of cryopreservation, mouse eggs were

frozen in a nonprogrammable freezer using a liq-

uid nitrogen plunge temperature of )20 �C. Theoocytes were exposed to cryoprotectants as de-

scribed above, sealed into straws, and transferred

into a 500-ml beaker filled with methanol at room

temperature (23 �C). The beaker was subsequentlyplaced in the freezer compartment of a conven-

tional refrigerator/freezer (approximately )24 �C).To monitor temperature changes, a thermocouple

was placed inside a control straw containing the

same freezing solution as the straws that con-

tained the oocytes. All straws were seeded at )7 �Cand plunged into LN2 after cooling to )20 �C.The average cooling rate down to )7.1 �C (theseeding temperature) was )1.76 �C/min. The av-erage rate of cooling following seeding down to

the plunge temperature of )20.3 �C was )0.36 �C/min. Based upon the results of pilot experiments

involving plunging oocytes at )20 �C (data notshown), the straws were thawed by holding them

in room temperature air for 10 s and then sub-

merging them into 30 �C water for 10 s. Aftercryoprotectant removal, insemination, and culture

overnight, the resulting two-cell embryos were

transferred to the oviducts (five embryos per tube)

of pseudopregnant recipients as described above.

Live-born young were counted and examined vi-

sually for the presence of gross morphological

abnormalities.

Statistical analysis

Within experiments, implantation and fetal

development rates in control and cryopreserved

oocytes/embryo groups were analyzed using a v2

test with Yates’ correction for small sample sizes.

Results

The main result of the present study was the

demonstration that fully viable embryos can be

produced from unfertilized oocytes frozen in CJ2.

The experiments also show that extended culture

J.J. Stachecki et al. / Cryobiology 44 (2002) 5–13 7

in vitro reduced the viability of embryos produced

from frozen oocytes and zygotes.

Following oviductal transfers, implantation

rates were marginally different for control em-

bryos and embryos from frozen oocytes

(P ¼ 0:040; 62.3 and 73.0%, respectively; Table 1).After uterine transfer, embryos from the same two

groups had similar implantation rates (P ¼ 0:085;75.5 and 62.0%, respectively; Table 2). There was

a significant reduction (P < 0:001; 35.3% lower) infetal development when embryos from cryopre-

served oocytes were cultured in vitro to the

blastocyst stage before being transferred rather

than being transferred at the two-cell stage to re-

cipient mice (12.3% vs 47.6%, respectively). By

contrast, fetal development rates were similar for

the control and the cryopreserved groups when

oviductal transfers were used (P ¼ 0:180; 40.0 and47.6%, respectively; Table 1). The overall fetal

development rate for all oocytes frozen in CJ2 and

transferred at the two-cell stage was 40.2%

(0:844� 0:476). These data show that oocytesfrozen in CJ2 can develop into viable fetuses at

rates similar to those of control embryos.

The overall survival postcryoprotectant re-

moval (93.7 and 93.5%, respectively) and the de-

velopment rate to the two-cell stage (80.8 and

86.0%, respectively) were similar whether zygotes

were frozen in ETFM or in CJ2 (Tables 3 and 4).

Implantation rates were also similar (P > 0:05Þfor zygotes frozen in either ETFM or CJ2 and

control embryos, regardless of whether the em-

bryos were transferred to the oviduct (66.7, 66.0,

and 67.4%) or uterus (80.0, 88.3, and 87.0%, re-

spectively; Tables 3 and 4). By contrast, fetal de-

velopment rates after uterine transfer were

significantly lower for embryos cryopreserved in

ETFM (P < 0:001; 23.6%) or in CJ2 (P < 0:001;20%) than for nonfrozen control embryos (59.1%;

Table 4). Fetal development rates for embryos

transferred to the oviduct were similar for those

zygotes cryopreserved with ETFM (53.3%) and

the control embryos (54.7%) and slightly lower for

embryos frozen with CJ2 (P ¼ 0:043; 32.0%).Oocytes frozen in the nonprogrammable free-

zer and plunged at )20 �C did not survive as wellor develop as well as oocytes frozen in a BioCool

programmable freezer and plunged at )33 �C,

Table 1

Oviductal transfer results of two-cell embryos cryopreserved in CJ2 as unfertilized oocytes

Nonfrozen control Cryopreserved

Number of oocytes intact ND 833 (97.5)

Postcryoprotectant removal (%)

Two-cells (%) ND 721 (84.4)

Transferred (to mice that became pregnant) 175 185

Implanted (%) 109 (62.3) 135 (73.0)

Fetuses (%) 70 (40.0) 88 (47.6)

Note. ND, not determined. Data represent 22 straws frozen. There were 39 mice that received embryos. Of these, 33

mice were pregnant. Five control embryos were transferred to one oviduct and five embryos from cryopreserved oocytes

were transferred to the contralateral oviduct of the same recipient. There was 1 mouse that received only cryopreserved

oocytes.

Table 2

Uterine transfer results of blastocysts cryopreserved in CJ2 as unfertilized oocytes


Number of oocytes intact ND 1212 (89.1)

Postcryoprotectant removal (%)

Two-cells (%) ND 943 (69.3)

Blastocysts (%) ND 594 (43.7)

Transferred (to mice that became pregnant) 163 163

Implanted (%) 123 (75.5) 101 (62.0)

Fetuses (%) 77 (47.2) 20 (12.3)


mice were pregnant. Two to five control embryos were transferred to one uterine horn, while an equivalent number of

embryos from cryopreserved oocytes were transferred to the contralateral horn of the same recipient.


although the difference was not statistically sig-

nificant (71.4% vs 81.9% and 67.1% vs 76.3%,

respectively; Table 5). However, all three recipient

mice that received embryos developing from oo-

cytes frozen in the nonprogrammable freezer be-

came pregnant and delivered a total of 14 live,

morphologically normal young at full term. One

of three recipients in the other group (BioCool

programmable freezer) became pregnant and de-

livered 8 live young. Regardless of the reduced

rate of survival and development in vitro exhib-

ited by the oocytes cryopreserved in the nonpro-

grammable freezer, the fact that nearly half of the

transferred embryos developed into normal young

reconfirms that high rates of fetal development are

possible, following cryopreservation in CJ2.

Table 4

Uterine transfer results of blastocysts cryopreserved as zygotes


ETFM CJ2

Number of zygotes ND 490 490

Intact postcryoprotectant removal (%) ND 454 (92.6) 465 (94.9)

Two-cells (%) ND 393 (80.25) 423 (86.3)

Morulae (%) ND 309 (63.1) 330 (67.3)

Transferred (to mice that became pregnant) 115 55 60

Implanted (%) 100 (87.0) 44 (80.0) 53 (88.3)

Fetuses (%) 68 (59.1) 13 (23.6) 12 (20.0)


mice were pregnant. Five control embryos were transferred to one uterine horn and five embryos from cryopreserved

embryos were transferred to the contralateral horn of the same recipient.

Table 3

Oviductal transfer results of two-cell embryos cryopreserved as zygotes


ETFM CJ2

Number of zygotes ND 145 145

Intact postcryoprotectant removal (%) ND 141 (97.2) 129 (88.9)

Two-cells (%) ND 120 (82.7) 123 (84.8)

Transferred (to mice that became pregnant) 95 45 50

Implanted (%) 64 (67.4) 30 (66.7) 33 (66.0)

Fetuses (%) 52 (54.7) 24 (53.3) 16 (32.0)

Note. ND, not determined. Data represent six straws frozen. There were 19 mice that received embryos. Of these, 19

mice were pregnant. Five control embryos were transferred to one oviduct and five embryos from cryopreserved embryos

were transferred to the contralateral oviduct of the same recipient.

Table 5

Oviductal transfer results of two-cell embryos cryopreserved in CJ2 as unfertilized oocytes

Freezera BioCool, programmable Nonprogrammable

Plunge temperature (�C) )33 )20Thaw rate (s in air/s in 30 �C water) 30/10 10/10

Number of oocytes (experiments) 72 (2) 98 (2)

Intact postcryoprotectant removal (%) 59 (81.9) 70 (71.4)

Two-cells (%) 45 (62.5) 47 (48.0)

Transferred (recipients) 30 (3) 30 (3)

Pregnant recipients 1 3

Pups male:female (%) 5:3 (26.7) 6:8 (46.7)

Note. Five embryos were transferred to each oviduct. Recipients had only cryopreserved material transferred.aOocytes were frozen in either a BioCool III (methanol) programmable freezer or in a beaker of methanol placed in

the freezer compartment of a nonprogrammable refrigerator/freezer.


Discussion

In previous reports we have examined the main

factors governing survival of mouse oocytes dur-

ing cryopreservation with CJ2 as the freezing me-

dium [24–26]. The freezing method used in the

present experiments is relatively simple compared

to other published methods [2,5,10] and in our

hands consistently yields oocyte survival rates in

the 90% range, i.e., equal to or higher than any so

far reported in the literature [2,23, for reviews see

5,10]. By ‘‘survival’’ it is meant that the oocyte has

an intact cell membrane and looks normal when

viewed under a microscope at powers up to 400�.However, survival thus defined does not imply full

viability. Therefore this study was undertaken to

examine the ability of these eggs to develop in vivo.

Experience leads to the expectation that in the

course of cryopreservation the oocyte’s viability

will be reduced, although not necessarily to an

extent that makes it incapable of becoming fer-

tilized and developing into a viable conceptus. It is

worth bearing in mind that at ovulation the

mammalian egg possesses developmental poten-

tial in excess of what is strictly necessary for

launching a single viable embryo. Although the

precise nature of the damage caused by cryopre-

servation remains to be determined, cryopreser-

vation severely taxes the reserves of the oocyte. In

this perspective, all the procedural steps from

thawing onward are best viewed as one long res-

cue mission, the success of which depends on the

integrated optimal use of a number of techniques

and procedures that form no part of the actual

freezing and thawing of the oocytes. These sup-

portive techniques include in vitro fertilization, in

vitro culture, and embryo transplantation, all of

which, being themselves imperfect, are sources of

variation. Others have also reported within-labo-

ratory variations in fetal development rates [1].

Expertise and organization with regard to the

supportive techniques are therefore essential to

maximize the outcome of oocyte cryopreserva-

tion. In the present study, ensuring that suitable

recipient mice would be available presented par-

ticular logistical problems, since the mice could

not be kept at the laboratory under close super-

vision, but had to be obtained on a daily basis

from a facility about 25 miles away. This ar-

rangement was by no means ideal.

The overall rate of normal fetal development

achieved after transfer of two-cell embryos from

cryopreserved oocytes in this study is at the same

level as the best reported in the literature [1,2,5,6].

This was achieved despite the fact that we cannot

be considered particularly experienced in oviduc-

tal embryo transfer in the mouse. More experi-

enced operators, with more conveniently located

mouse colonies, may achieve at least marginally

better results with the same freezing method.

However, our results are somewhat better than

those of Schroeder et al. [23]. In their study, over

1000 mouse oocytes were frozen using a standard

slow-cooling protocol and live pups were pro-

duced at a rate of 26% following two-cell transfer

to the oviduct.

Significant progress has been made in oocyte

vitrification and live offspring have been produced

from vitrifiedmouse oocytes. Early studies byKola

et al. [12], Wood et al. [30], and Nakagata [19–21]

report fetal development rates of between 5 and

36%. In addition, Kono et al. [13] demonstrated

oocyte viability after vitrification by producing 36

live pups (51% of those transferred; 25% per frozen

oocyte). In a more recent study Lane and Gardner

[15] compared the use of nylon loop vitrification to

slow-cooling using CJ1 medium (our original

choline-based freezing medium). They report some

of the highest survival, fertilization, in vitro de-

velopment, and fetal development rates (52% of the

embryos transferred; 37.9% of oocytes frozen) for

frozen mouse oocytes in the literature, using the

vitrification process. Their initial survival results

with CJ1 were similar to those in our original

publication describing the use of this medium [25],

but it is unclear why their fertilization and devel-

opment rates were significantly lower. It is also not

clear why they chose to use CJ1 instead of CJ2

(a modified choline-based medium) which we have

shown to work substantially better [24,26]. How-

ever, their fetal development rate of 11% for slowly

cooled oocytes in CJ1, 41% for their nonfrozen

controls, and 52% for their best group, the nylon

loop-vitrified oocytes, are similar to the results

presented here for slow-cooling oocytes in CJ2 and

transfer to the uterus (12.3%), nonfrozen controls

(47.2%), and our best group, slowly cooled oocytes

transferred to the oviduct (47.6%). Our data sug-

gest that their fetal rates could have been even

higher, especially for the CJ1 frozen oocytes, if the

resulting embryos were transferred to the oviduct

at the two-cell stage.

In the present experiments, delaying the

transfer of embryos from cryopreserved oocytes

(and zygotes) until they had reached the early

blastocyst stage severely reduced the rate of nor-

mal fetal development, while such an effect was

not evident in the control groups. A similar effect


of prolonged culture in vitro was previously ex-

perienced and discussed by George et al. [5]. Since

these are the only two reports involving uterine

transfers of embryos from slowly cooled mouse

oocytes, there seems to be a consensus that it is

preferable to transfer embryos from cryopreserved

oocytes at the two- to four-cell stage (see also [1]

and for a review [5]). In previous studies, we found

that in vitro culture after thawing and insemina-

tion of frozen oocytes is detrimental to blastocyst

development and that this can be significantly

improved by using a sequential (S1/S2) culture

media system [24–26]. Even though blastocyst

development is improved using sequential media,

the significant postimplantation loss (87.7%

[100) 12.3%]) observed when such blastocystswere transferred shows that the embryos, al-

though judged to be morphologically normal by

microscopic inspection, were in fact compromised

and that in vitro culture conditions were poor

compared to an in vivo environment [15]. Al-

though there is an apparent discrepancy between

the use of S1/S2 for the culture of cryopreserved

oocytes and the use of KSOM for control em-

bryos, this study was not aimed at optimizing

culture medium, but rather concerned (1) the

ability and rate of oocytes cryopreserved in CJ2 to

develop into viable fetuses in vivo and (2) the

differences, if any, in fetal development rates in

zygotes cryopreserved in CJ2 and ETFM. The

control embryos simply served as a reference

point by which comparisons could be made.

The ability of mouse oocytes to survive cryo-

preservation was far greater than expected, espe-

cially in the light of the literature on survival and

development with plunge temperatures higher

than )30 �C [4,29,31]. Although more oocytesdied during thawing and failed to become fertil-

ized after being cooled in a nonprogrammable

freezer and plunged at )20 �C (a breakpoint 13 �Cwarmer than those eggs cooled in the BioCool

freezer), as opposed to being cooled in a pro-

grammable freezer and plunged at )33 �C, themajority survived and became fertilized, and

nearly half of all two-cell embryos transferred

developed to term. Potentially detrimental factors

such as intracellular ice formation would have had

a much greater chance of occurring with plunging

into LN2 from )20 �C than from )33 �C, due tothe level of cellular dehydration achieved at the

respective temperatures. However, this was not

the case, indicating that lethal intracellular ice

formation was negligible even after cooling to

only )20 �C followed by submersion into LN2.

Although there have been numerous reports of

live births from embryos and oocytes plunged

from )30 �C after slow cooling, this is the firststudy to report live births from oocytes plunged

into LN2 from )20 �C. Indeed, it is still commonpractice to use plunge temperatures below )33 �C.Karlsson et al. [10] examined oocyte survival and

development with plunge temperatures ranging

from )30 to )150 �C, with )80 �C yielding max-imal recovery of oocytes. The present experiments

suggest that there are factors that lead to oocyte

demise during cryopreservation other than the

stresses induced by plunging from the relatively

high temperature of )20 �C. One such factorwould appear to be the dominance of sodium ions

in conventional cryopreservation media, but there

are most likely others.

The overall rate of fetal development obtained

when two-cell embryos from zygotes frozen in

ETFM were transferred to the oviduct was only

slightly higher than that obtained with embryos

from cryopreserved oocytes, and when comparing

the two results, it should be kept in mind that the

fertilization rate in vivo is not uniformly 100%.

However, while the oocyte group had a higher

implantation rate, fewer of the embryos that im-

planted gave rise to a fetus in this group than in

the zygote (ETFM) group (65.2% vs 80.0%); the

fetal development rate of implanted embryos from

zygotes frozen in CJ2 was lower than both

(48.5%). Thus, cryopreservation of oocytes ap-

pears to be more damaging to the cells’ ability to

form a fetus than cryopreservation of zygotes. We

are not able to adequately explain why CJ2 is

more effective as a freezing medium for oocytes

than ETFM, but it is interesting to note that no

significant advantage was observed regarding zy-

gotes with the protocol used in this study.

Acknowledgments

We thank Giles Tomkin for his critical review

of the manuscript, Anna Blasczyk for help in

preparing the mice used for these experiments,

and Dr. Fred Zander for providing the use of the

Fertilase system.

References

[1] A. Bos-Mikich, M.J. Wood, C.J. Candy, D.G.

Whittingham, Cytogenetical analysis and develop-

mental potential of vitrified mouse oocytes, Biol.

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fetal development of mouse oocytes and zygotes cryopreserved in a nonconventional freezing medium

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