Theriogenology 65 (2006) 228235

www.journals.elsevierhealth.com/periodicals/the

Modifying oocytes and embryos to

improve their cryopreservation
George E. Seidel Jr.*
Animal Reproduction and Biotechnology Laboratory, ARBL Building, Foothills Campus,
Colorado State University, Fort Collins, CO 80523-1683, USA

Abstract
Numerous studies indicate that in vitro-produced bovine embryos do not survive cryopreservation
as well as those produced in vivo. Furthermore, embryos cultured in vitro in the absence of blood
serum are more cryotolerant than embryos cultured in media containing serum. Although in vivoproduced embryos are more cryotolerant, there appear to be breed differences. Most if not all of these
observations are correlated with cytoplasmic lipid content of embryos; more and larger lipid droplets
are associated with reduced cryotolerance. This review concerns strategies for modifying oocytes and
embryos to increase cryosurvival. Reduction of cytoplasmic lipid content of embryos with phenazine
ethosulfate (PES), a compound that oxidizes NADPH, even improved cryotolerance of bovine
embryos cultured in the absence of serum. Whether cytoplasmic lipid content per se or associated
changes in lipid composition of cell membranes are responsible for differences in cryotolerance is
unknown. Increasing cholesterol content of membranes of sperm and oocytes via cholesterol-loaded
cyclodextrin also appears to improve cryotolerance. While lipids have been emphasized and appear to
be important, non-lipid aspects of cell composition also likely affect cryotolerance, and might be
modified to improve cryotolerance. Additional research on mechanisms of variation in cryotolerance
will be applicable to circumvent cryo-intolerance attributable to variation associated with the
individual animal, breed, species, cell type, and factors such as nutrition and season of the year.
# 2005 Published by Elsevier Inc.
Keywords: Cryopreservation; Vitrification; Lipids; Oocyte; Embryo

* Tel.: +1 970 491 5287; fax: +1 970 491 3557.

E-mail address: gseidel@colostate.edu.
0093-691X/$ see front matter # 2005 Published by Elsevier Inc.
doi:10.1016/j.theriogenology.2005.09.025

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1. Introduction
A number of procedures have been developed to cryopreserve bovine embryos collected
from donors 68 days after estrus with quite good success; in most cases, with reasonable
management, pregnancy rates of thawed embryos are 8090% of those with
uncryopreseved controls [1]. Similarly successful cryopreservation occurs with in vivoproduced embryos at similar stages from other species of ruminants. However, in vitroproduced ruminant embryos are much more difficult to cryopreserve successfully, and in
vivo-produced embryos from some breeds survive cryopreservation less successfully than
those of other breeds [2].
Bovine oocytes are particularly difficult to cryopreserve successfully, and result in low
rates of blastocyst production after thawing, in vitro fertilization and culture of resulting
embryos [3,4]. This may be in part due to the large size of oocytes, with consequent low
surface to volume ratios, making it more difficult for water and cryoprotectants to move
across cell plasma membranes [5].
The most common approach to deal with the lower success rates of cryopreservation of
certain kinds of cells is to modify cryopreservation procedures [5], for example, by varying
concentration and types of cryoprotectants, studying different times and temperatures of
procedures, or using additives, such as sugars or surfactants. While this approach to
optimizing procedures usually results in improvements, they often are quite limited. An
alternative approach, modifying the cells themselves to make them more cryopreservable
is the subject of this review. Information from a variety of sources and cells types that bear
on this approach will be examined, including some recent work on modifying oocytes and
embryos.
Cells undergo considerable physical stress during cryopreservation procedures; some
kinds of cells tolerate these stresses better than others [5], and modifications to cell
membranes can result in changing tolerance of such stress. One major stress is osmotic in
nature; cells and cellular compartments undergo huge changes in volume due to
movements of water and intracellular cryoprotectants [5]. Thus, cells with more flexible
membranes will usually have less membrane damage than those with more rigid
membranes. Also, there will be less osmotic stress if permeabilities of cell membranes to
water and cryoprotectants are high. Such cellular properties are highly dependent on
membrane composition.
The major components of cell membranes are phospholipids, cholesterol, other lipids and
proteins. Except for the proteins, these components can be manipulated fairly readily in a
variety of ways including nutrition of donor animals and composition of culture media. One
other relevant property of cell membranes is that for normal function, they need to be in a
relatively fluid, sol state rather than a gel state. Certain regions of some cell membranes readily
change fluidity to gel at decreased temperatures [6]. Often the changes that occur in this phase
transition are not reversible, and upon warming, the cellular components are not reassembled
correctly [7]. It would seem to be best to eliminate the fluid to gel transition completely during
cooling; other strategies include having the transition occur at lower temperatures or to speed
the transition between these states with rapid cooling rates, to limit the opportunity for
damaging changes to occur. As with osmotic properties, the tendency for phase changes in
membranes during cooling is highly dependent on membrane composition [8].

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One clue as to how cells might be changed specifically is the relationship between the
ratio of cholesterol to phospholipids in cell membranes, and the success of
cryopreservation of sperm from species to species [9]. Low ratios, such as those found
in boars and stallions are associated with less successful cryopreservation, while high ratios
in men and bulls result in more successful cryopreservation of sperm. Whether genetic
differences in freezability of sperm from male to male within mammalian species, or breed
differences in freezability of blastocysts have a similar basis is unknown [2], although such
a relationship has been clearly demonstrated in poultry sperm [10]. Other differences in
lipid content or lipid composition of the cells and/or cell membranes also are likely to be
important. Studies demonstrating more successful cryopreservation after microsurgical
delipidation of porcine embryos [11] provide clear evidence of the benefit of reducing
cytoplasmic lipid content.
There also are several studies on changing the cryotolerance of cells by manipulating the
diet of the donor animal [12,13], or culturing the cells for a variable period (from minutes to
days) in media that change freezability of cells. This latter approach is particularly attractive
because one can control the process to improve cryopreservation success.
Changing the chemical composition of cells and cell membranes can be accomplished
acutely by modifying the composition of cryopreservation media by adding egg yolk and
extracellular cryoprotectants [13]. Additives, such as egg yolk, sugars, or serum albumin are
beneficial for cryoprotection [14], and the mechanisms for these benefits have been
elucidated in some cases [15]; however, it is not even entirely clear how most cryoprotectants,
intracellular or extracellular, exert their beneficial effects. While the study of additives to
cryopreservation media has been extremely important and productive, this review will
concern modification of cells prior to the process of cryopreservation. I will concentrate on
oocytes and embryos, but will include information on other cell types when instructive.

2. Thought-provoking observations
Anecdotal observations frequently provide a basis for formulating hypotheses to test,
particularly when large effects are observed. For example, dismal success rates for
cryopreserving porcine embryos at certain stages characterized by high lipid content
prompted development of various approaches to circumvent the high lipid content, such as
delipidation or delaying cryopreservation until the expanded blastocyst stage [11,16].
Jersey embryos result in lower pregnancy rates after cryopreservation than Holstein
embryos [2] and, this observation is correlated with the higher milk fat content in Jerseys.
In vitro-produced embryos are more difficult to cryopreserve than in vivo-produced
embryos [1720], which again may be correlated with their higher lipid content.
A second, more compelling type of evidence as to why some cells are poorly cryotolerant
comes from direct physical or chemical measurements that are correlative with
cryotolerance. For example, Pfaff et al. [21] determined that permeabilities of equine
blastocysts to cryoprotectants decreased markedly as they developed to diameters >250 mm,
a size at which they become more difficult to cryopreserve efficaciously. Likewise, there was
a correlation between a seasonal change in lipid composition of oocytes from Holstein cows

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and success of embryonic development [6]. Lipid composition, not just the amount of lipid,
also differs between in vivo- and in vitro-produced bovine embryos [22].
A third kind of information is derived from testing hypotheses more directly, by
experiments with different treatments to oocytes or embryos followed by their
cryopreservation. This type of evidence will form the topic of the bulk of this review.

3. Information from experiments with sperm

Recent studies in which addition of selected phospholipids [23] as well as cholesterol
[24] to sperm cell membranes improved freezability have been encouraging. While added
cholesterol decreases membrane fluidity at ambient temperatures, added cholesterol
increases membrane fluidity at lower temperatures [8]. Cholesterol appears to accomplish
this effect by modifying how membrane phospholipids interact with each other, making
membranes less contracted at lower temperatures. Added cholesterol also lowers the phase
transition temperatures of the membranes, or eliminates the phase transition altogether in
some circumstances [8].
Purdy and Graham [24] demonstrated this beneficial effect of added cholesterol with
bovine sperm and Moore et al. [25] confirmed the benefit for stallion sperm. In these studies
cholesterol was loaded into methyl-b-cyclodextrin, a hydrophilic molecule with a
hydrophobic center. Incubation of cells with this material results in a transfer of cholesterol
to cell membranes, and improved cryotolerance of these cells [24,25].

4. Oocytes
Horvath and Seidel [4] added cholesterol to bovine oocytes in an attempt to increase
success rates for vitrification. They found that it is quite important to have the correct
conditions for transferring cholesterol to cell membranes as was also true for sperm [24].
Removing serum albumin (fatty acid-free) from the medium during transfer of cholesterol
to oocytes improved success rates, likely because cholesterol was preferentially transferred
to the fatty acid-free albumin if present. Horvath and Seidel [4] demonstrated a modest
improvement of cleavage rates and development to the 8-cell stage of cholesterol-enhanced
bovine oocytes relative to controls after they were subsequently thawed and fertilized in
vitro (55% versus 41% 8-cell embryos; P < 0.05).
Zeron et al. [26] also studied modification of bovine oocyte lipids by incorporating
liposomes containing egg-phosphatidylcholine into oocyte cell membranes. This treatment
reduced the chilling injury from cooling to 0 8C so that more blastocysts were produced
upon IVF and culture post-warming.

5. In vitro-produced embryos
Culture of embryos in vitro results in fundamentally different embryos from those
produced in vivo, particularly for ruminants [27]. In general, the longer the in vitro culture,

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the more deviation occurs relative to embryos recovered in vivo; this phenomenon appears
to be exacerbated when in vitro oocyte maturation and in vitro fertilization procedures are
added on to embryo culture procedures [19].
One of the clearest demonstrations that different culture conditions can result in
embryos with different properties was the observation that ovine embryos cultured in
medium containing blood serum resulted in lambs with higher birth weights than for
embryos produced in medium with serum albumin [28]. It has also been confirmed several
times that culture in the absence of serum results in less lipid accumulation in embryos
[28,29]. A further finding is that embryos with less lipid, produced without blood serum in
the medium, also cryopreserve more successfully than the lipid-laden embryos produced in
the presence of serum [20,19]. While the success rates of cryopreservation of embryos
appear to be highly correlated with cytoplasmic lipid content, the specific mechanism is
unknown and may only be indirectly related to cytoplasmic lipids. Success rates of
cryopreservation, nevertheless, are becoming a measure of normalcy of bovine embryos in
studies of culture procedures [20,27].
While removing serum from culture media clearly decreases cytoplasmic lipid
accumulation compared to controls, embryos produced with fatty acid-free bovine serum
albumin or recombinant human serum albumin still have more cytoplasmic lipid than in
vivo controls [30]. We have conducted a series of experiments to lower cytoplasmic lipid
even further [30] (Barcelo-Fimbres and Seidel, unpublished), and have found that this can
be accomplished with phenazine ethosulfate (PES), a strong electron acceptor that oxidizes
NADPH readily. This lipid-lowering effect likely occurs because NADPH is required for
synthesizing numerous lipids, particularly long-chain fatty acids.
We have recently demonstrated (Barcelo-Fimbres and Seidel, unpublished) that bovine
blastocysts produced by culture with PES survived conventional cryopreservation better
than controls (P < 0.05; 94, 85 and 61% survival, N = 120 per group for PES, no additive,
or 10% fetal calf serum, respectively). Conclusions were the same for vitrification of
blastocysts for the same treatments: 90, 84 and 59% survival for respective groups (N = 120
blastocysts per group). Thus, not only is it possible to improve the success of
cryopreservation of in vitro-produced embryos by eliminating blood serum from culture
medium, but the embryos also can be manipulated metabolically to improve success of
cryopreservation even further. The embryos cultured in the presence of PES, although more
similar to in vivo-produced embryos than controls, still are more lipid-laden than the in
vivo-produced embryos [30], which correlates with the higher cryotolerance of in vivoproduced embryos.

6. Future studies
In most cases, modifying cryopreservation methods to fit the cells to be cryopreserved is
likely to be preferable to modifying cells to fit procedures for cryopreservation.
Nevertheless, there are opportunities to do the latter, and such modifications (for example,
removing serum from media) may also result in improved success of oocyte maturation and
culture of embryos and perhaps even in healthier offspring [28]. Various studies with
reproductive and non-reproductive cells provide numerous ideas for further study. Adding

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more cholesterol to in vitro-produced embryos via cyclodextrin may be worth pursuing.

Horvath and Seidel [4] showed that the zona pellucida of oocytes did not prevent transfer of
cholesterol from occurring with this procedure. Another area worth investigating is
stabilizing cell membranes with trehalose, a compound that plants use naturally to increase
cryotolerance. One issue is how to transfer the compound to the cytoplasm of the cell,
where it normally functions. Satpathy et al. [31] developed a method for loading red blood
cells with trehalose for freeze-drying, and a variation might work to move trehalose into
oocytes and embryonic cells, or trehalose might be injected into individual oocytes [32]. It
is unclear whether trehalose would be harmful after thawing, and if so whether the
molecule could be removed from cells readily. Kim et al. [33] used another approach by
modifying red blood cells with phosphoenolpyruvate to decrease membrane fragility.
Similar research with oocytes and embryos is likely to result in improved procedures for
their cryopreservation in addition to the considerable improvements already realized.

Acknowledgements
Several students and colleagues assisted with preparation of this review. Related
research was supported in part by National Research Initiative Grant No. 2003-3520313705 from the USDA CSREES and by the Colorado State University Experiment Station
via USDA Regional Project W-171.

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