CSIRO PUBLISHING

www.publish.csiro.au/journals/rfd Reproduction, Fertility and Development, 2007, 19, 719–731

Novel gamete storage

A. DinnyesA,C , J. LiuA and T. L. NedambaleB

A BiotalentumLtd, Aulich L. 26, Godollo 2100, Hungary.
BAnimal Production Institute, Indigenous Genotype Physiology and Biotechnology
Development, Private Bag X2, Irene 0062, South Africa.
C Corresponding author. Email: biotalentum@yahoo.com

Abstract. The aim of this review is to outline recent advances in gamete storage that are beneficial for rescuing endangered
species or for the breeding of companion animals. Much more information is available on the technical resolutions and
practical applications of sperm cryopreservation in various species than of female gametes, reproductive tissues or organs.
Mammalian sperm cryopreservation often works relatively efficiently; however, the ability of female gametes to be
cryopreserved and still be viable for fertilisation is also essential for rescuing endangered species. For a proper evaluation
of gamete cryopreservation possibilities in a given species, it is essential to understand the basic mechanism affecting the
survival of cryopreserved cells, the technical and physical limitations, the available techniques and the new avenues to
resolve the specific problems in that species. This paper is aimed to provide some help for this process. The limited length
of this paper resulted in the omission of information on many important areas, including most data on teleosts, amphibian
and insect cryopreservation.

Current status of knowledge
The roots of modern applied gamete storage date back more
than 50 years to the time when an efficient cryopreservation
technique for preserving spermatozoa with glycerol was acci-
dentally discovered (Polge et al. 1949). As a result, serious
development began and sperm cryopreservation has become
an important method in agriculture and medicine and also
an option for rescuing endangered species. To some extent
gamete storage can provide insurance for preserving the exist-
ing genetic diversity within endangered species. These species
survive in nature but often in fragmented habitats, or live in
geographically disparate zoos and they are also susceptible to
inbreeding depression, environmental catastrophes, epidemics,
and even to drastic shifts in social and political structures (Wildt
2000; Wildt et al. 2001; Pukazhenthi et al. 2006a). However,
there have been many encouraging results following artificial
insemination (AI) of frozen–thawed semen at zoological parks
around the world (Monfort et al. 1993). For example, AI using
frozen–thawed spermatozoa has been used with success in the
cheetah, leopard cat and ocelot (Howard 1999). The use of AI
with thawed spermatozoa also would assist genetic manage-
ment through the distribution of genes among geographically
dispersed breeding centres and zoos. Availability of frozen sper-
matozoa would permit international transport of genetic material
without compromising political and legal restrictions. Effective
sperm cryopreservation could salvage precious genetic material
from wild animals that are captured in the field for various rea-
sons. Application of assisted reproduction technologies (ART)
using frozen–thawed gametes of rare and endangered species
will become increasingly important in conservation biology
programs (Wildt 2000).
Polge et al. (1949) successfully cryopreserved bull sperma-
tozoa by introducing glycerol as a cryoprotective agent (CPA)
into freezing medium for the first time. Later many other
non-electrolytic permeating CPA, such as dimethyl sulfoxide,
ethylene glycol and propylene glycol, were introduced for other
cell types (such as embryos and oocytes) in various species
(Mazur 1970).
Although there have been some recent exciting studies involv-
ing freeze drying and vitrification (see later section) of sper-
matozoa as a means of conserving this genetic material, slow
equilibrium freezing is still the most broadly practiced sperm
cryopreservation method. It consists of the following series of
non-physiological steps:
(1) adding a permeating CPA (e.g. glycerol, ethylene glycol,
dimethyl sulfoxide, propylene glycol) or non-permeating
CPA (e.g. sucrose, glucose, fructose, sorbitol, trehalose or
raffinose) to the cells or tissues before cooling;
(2) seeding the samples a few degrees below the freezing
point of the cell suspension and cooling the cells to a
low temperature at rates calculated to minimise intracel-
lular ice formation, then storage at below glass transition
temperatures (in the range of −130 to −196◦ C);
(3) thawing the cells; and
(4) removing the CPA from the cells and rehydrating them
before utilisation.
In general, we may expect coupled flows of water and CPA
when CPA are added, during freezing, thawing, and when CPA
are removed from cells. During the cryopreservation process,
cells are subjected to a series of anisosmotic conditions. Cells
are initially subjected to a hyperosmotic environment during the

© CSIRO 2007 10.1071/RD07035 1031-3613/07/060719

720 Reproduction, Fertility and Development
addition of the CPA, which is usually at a concentration of around
1.0 M or less for sperm cells. They shrink as water is driven out of
the cells by osmosis then return to their isosmotic volumes as the
permeating CPA enters the cells and water follows to maintain
chemical potential equilibrium across cell membranes. During
freezing the cells dehydrate and shrink as the water in the cell
suspension gradually becomes frozen below the freezing point
of the more and more concentrated solution. The cells remain
shrunken during storage, then return to their isosmotic volume
again upon thawing. Finally, the cells are subjected to potentially
lethal swelling upon CPA dilution and removal. These shrinkage
and swelling events are capable of causing damage or even cell
death (Mazur and Schneider 1986).
During the cooling process, if cells are cooled too rapidly,
water does not exit the cells fast enough to maintain equilibrium.
The cells become increasingly supercooled and eventually freeze
intracellularly (Mazur 1990). In most cases, intracellular ice for-
mation (IIF) is lethal for cells (Muldrew and McGann 1994).
If cooling is relatively slow, the cells will lose water rapidly
enough to concentrate the intracellular solutes sufficiently to
eliminate supercooling. Although rates low enough to prevent
internal freezing injury are necessary for high survival, freezing
at slow rates over a long duration can cause ‘solution effects’
injury resulting from the extreme concentration of extracellu-
lar and intracellular solutes that may decrease survival as well
(Critser et al. 2002), probably due to the effects of the solutes on
the cellular membrane or through osmotic dehydration. There-
fore, a cooling rate either ‘too high’ or ‘too low’ can kill cells,
although the mechanisms underlying cell damage are different.
Typically, cells that survive cryopreservation respond to cooling
rates in an inverted ‘U’-shaped pattern, hence there is a cool-
ing rate for maximum survival. This rate, however, can vary
greatly depending on the cell type (Mazur 1963). Current sperm
cryopreservation protocols commonly utilise cooling rates of
10–100◦ C min−1 .
A cell that has survived cooling to low subzero temperatures
still faces challenges during warming and thawing, which can
exert effects on survival comparable to those of cooling (Mazur
et al. 1984). These effects depend on whether the prior rate of
cooling has induced intracellular freezing or cell dehydration. In
the former case, if the cells are not destroyed through lysis from
IIF, they may end up with an accumulation of smaller, thermody-
namically unstable ice crystals that may subsequently undergo
recrystallisation, forming bigger ice crystals that rupture the cell
membrane thus leading to fatal damage. Rapid thawing can res-
cue many cells, possibly by preventing the harmful growth of
small intracellular ice crystals by recrystallisation.
The most important characteristics of CPA solutions are that
they are, for the most part, readily able to permeate spermato-
zoa and are relatively non-toxic in concentrations of 1 M or less.
The primary basis for their protective action is that they lower
the concentration of electrolytes during freezing and therefore
decrease the extent of osmotic shrinkage at a given temperature.
The extent of protection depends primarily on the molar ratio
of the CPA to endogenous solutes inside and outside the cells,
and the general protective mechanism of action is colligative.
If cells are cooled slowly enough to avoid intracellular freez-
ing through dehydration, the response to warming rate is highly
A. Dinnyes et al.
variable among cell types. In some cells warming rate makes
little or no difference, but in others, rapid or slow warming is
required, and is often dependent on the rate of cooling (Mazur
1984).
Mazur developed a theoretical model to study how cells lose
intracellular water during the cryopreservation process at a spe-
cific cooling rate (Mazur 1963). Over the years others have added
to this model to develop more refined methods of mathematically
predicting optimal conditions for cell cryopreservation process-
ing, and a convergence of theoretical and empirical approaches
has begun. With recent progress in understanding mechanisms of
injury to cells during cryopreservation, physical modelling using
mathematical formulations has been developed to simulate a
cell’s response to environmental change during the cryopreserva-
tion process and to predict optimal cryopreservation conditions
(Toner et al. 1990; Pitt et al. 1992; Karlsson et al. 1994; Liu et al.
2000). More advanced knowledge on the biophysical character-
istics of mammalian spermatozoa have been used to optimise
cryopreservation protocols (Gao et al. 1995; Gilmore et al.
1997).
Recently, new cryopreservation methods have emerged such
as freeze drying and vitrification.
Freeze-dried spermatozoa
Cryopreservation of spermatozoa, oocytes and embryos, as well
as somatic cells or cell lines for cloning by nuclear transfer, are all
options for the long-term storage of unique genotypes and endan-
gered species. Sperm cryopreservation and storage currently
requires liquid nitrogen or ultra-low refrigeration-based meth-
ods for long- or short-term storage, with routine maintenance
and extensive space requirements.
Besides the current methods available for cryopreservation
of spermatozoa by equilibrium freezing (see previous section),
recently, freeze drying has also been used to preserve mammalian
spermatozoa, serving as an alternate method of cryopreserva-
tion (Wakayama and Yanagimachi 1998; Kusakabe et al. 2001;
Kaneko et al. 2003a, 2003b; Ward et al. 2003; Liu et al.
2004; Poleo et al. 2005). Because freeze-dried spermatozoa can
be stored at an ambient or refrigerated temperature, the costs
required for the maintenance and shipping of spermatozoa would
be greatly reduced.
Freeze drying is a procedure designed for easy preserva-
tion and transportation of biological materials (such as viruses,
bacteria, yeasts and fungi), pharmaceuticals and other delicate,
solvent-impregnated materials. It is achieved through a process
in which frozen material is dried by the sublimation of ice.
The freeze drying procedures for spermatozoa described ear-
lier allow storage of the samples in sealed ampoules at 4◦ C or
room temperature (Kusakabe et al. 2001; Kaneko et al. 2003b;
Ward et al. 2003; Kaneko and Nakagata 2005).
The drying of cells usually leads to damage of cellular
membranes and proteins, which often results in cell death.
The removal of intracellular water causes changes in molecu-
lar interactions. When cell membranes are dehydrated, the water
molecules that help to maintain the spacing between the polar
phospholipid head groups are removed. The result is that the lipid
fatty acid chains and polar head groups come closer to each other
Novel gamete storage
and the membrane collapses; an increase in the membrane phase
transition temperature (Tm) occurs as the membrane changes
from a biologically active fluid phase to a gel phase (Crowe
et al. 1992, 1997). Subsequently, as water is added back during
rehydration, the membrane undergoes another phase transition,
resulting in leakage of soluble cell contents through each mem-
brane (Leslie et al. 1994; Crowe et al. 1997). In the conventional
sense, freeze-dried spermatozoa are dead. They are non-motile
and unable to fertilise either in vivo or in vitro, but they are able
to produce live offspring when injected into oocytes by the pro-
cedure of intracytoplasmic sperm injection (ICSI). Apparently,
the procedure for this level of freeze drying does not seem to
affect sperm nuclear DNA integrity.
Recently, a significant effort has been directed at preserving
spermatozoa in the dry state. In 1992, Katayose and colleagues
demonstrated that hamster and human spermatozoa can be used
following storage in a dehydrated state for 12 months at 4◦ C
(Katayose et al. 1992). They used the ICSI procedure and
injected the reconstituted freeze-dried spermatozoa into hamster
oocytes, and then examined the formation of pronuclei. Later,
the same group reported 85 and 90% pronuclear formation for
hamsters and humans, respectively (Hoshi et al. 1994). Live mice
have been produced by ICSI (Kimura andYanagimachi 1995). To
date, embryonic development after ICSI with freeze-dried sperm
heads has been reported in large domestic animals, including cat-
tle (Keskintepe et al. 2002) and pigs (Kwon et al. 2004), and live
offspring in mice (Wakayama and Yanagimachi 1998; Kusakabe
et al. 2001; Kaneko et al. 2003a, 2003b; Ward et al. 2003), rab-
bits (Liu et al. 2004), rat (Hirabayashi et al. 2005) and fish (Poleo
et al. 2005) have been produced.
Vitrification as an alternative to conventional freezing
Cryopreservation without ice formation (vitrification) could be
beneficial in comparison to freezing methods because it does not
need any expensive equipment and takes only a few seconds for
cooling and warming. Vitrification is considered an attractive
alternative to conventional freezing and has been used success-
fully for the cryopreservation of spermatozoa, embryos, oocytes,
stem cells and organs.
Vitrification is a process of converting a material into a glass-
like amorphous solid that is free of any crystalline structure,
achieved by rapid cooling and mixing with high concentrations
of CPA. In general, the rate of cooling or warming and the con-
centration of cryoprotectants required to achieve vitrification are
inversely related. In other words, the faster cooling and warm-
ing is undertaken, the lower the critical solute concentration
necessary to obtain ice-free vitrification.
In 1985, Rall and Fahy successfully vitrified embryos using
high CPA concentrations and a relatively low speed of cooling
and warming (Rall and Fahy 1985). This breakthrough opened
the avenue for the practical use of vitrification. A typical vit-
rification requires a high concentration of cryoprotectants in
the medium (30–50% compared with 5–10% for slow freezing)
at a cooling rate easily achievable for conventional labora-
tory conditions and set ups. Such high CPA concentrations
can be damaging to cells, causing both biochemical alterations
and lethal osmotic injury (Fahy et al. 1984). Some of the
Reproduction, Fertility and Development 721
deleterious effects of exposure to CPA can be avoided by opti-
mising regimens for their addition and removal (Gao et al. 1995;
Katkov et al. 1998), as described for human and animal sperma-
tozoa. To minimise osmotic and toxic effects associated with
concentrated CPA, many researchers are exploring vitrification
methods that would require lower CPA concentrations. Toxic-
ity can be reduced by combining two CPA or adding precooled
solutions. However, it is possible to lower the critical perme-
able CPA concentration by adding non-permeable CPA (Fahy
et al. 1984; Fahy 1986a, 1986b). Additives such as disaccharides
(e.g. sucrose or trehalose) or high molecular weight molecules
(e.g. Ficoll, poly vinyl alcohol or poly vinyl pyrrolidone) can
significantly reduce the amount of permeable CPA required.
However, these methods also have their limitations. Regardless
of the mechanism of damage, in the majority of species, sperma-
tozoa cannot tolerate the high concentrations of cryoprotectants
used conventionally in vitrification.
The obvious alternative is thus to increase cooling and warm-
ing rates to meet the requirement of vitrification. To achieve
even higher cooling rates, the volume of vitrification solution
should be minimised by using specially designed cell suspension
containers such as open-pulled straws (OPS) (Vajta et al. 1997,
1998), electron microscope copper grids (Martino et al. 1996),
cryoloops (Lane et al. 1999), cryotops (Kuwayama and Kato
2000), gel-loading tips (Tominaga and Hamada 2001) and other
similar devices, or even without any containers in minimum-size
drops (Arav and Zeron 1997), in microdrops (Papis et al. 2000)
or by solid-surface vitrification (SSV) (Dinnyés et al. 2000).
These methods have been used to achieve better results for the
vitrification of embryos in species that are particularly suscep-
tible to cryodamage and for human and animal oocytes (Chen
et al. 2000; Yoon et al. 2003).
Although the application of vitrification started with embryos
and oocytes, recently Nawroth et al. (2002) carried out a chal-
lenging experiment with sperm vitrification. They used a copper
loop of 0.5-cm diameter, with 20 µL liquid attached. They esti-
mated the thickness of the liquid film to be as small as 0.7 mm
and the speed of cooling as high as 720 000 K min−1 . Using 10%
serum albumin in their standard sperm holding medium, they
believed that it was possible to vitrify human spermatozoa in the
absence of cryoprotectants, and they succeeded. They vitrified
swim-up-prepared spermatozoa and compared their results with
the conventional glycerol freezing method. Immediately after
thawing, swim-up-prepared spermatozoa frozen conventionally
with cryoprotectants showed 38% motility compared with 49%
after vitrification without cryoprotectants. The numbers of mor-
phologically normal spermatozoa recovered after conventionally
frozen swim-up-prepared spermatozoa with cryoprotectant was
not different from the vitrified ones (27 v. 26%, respectively)
(Nawroth et al. 2002; Isachenko 2003).
Two basic variables of success or failure of vitrification are
the size of the cells and the relative concentration of soluble
macromolecules within the cells. First, the shape and size of
the sperm head could define the cryosensitivity of the cell.
Comparative studies (Nauk 1991) in mammals (boar, bull, ram,
rabbit, cat, dog, horse and human) have shown a negative corre-
lation between the size of the sperm head and cryostability. The
results show that the human spermatozoon is the smallest with
722 Reproduction, Fertility and Development
Table 1. Comparison of the three approaches for cryopreservation of oocytes
CPA, cryoprotective agent; NA, not applicable
Parameters
Freezing
Oocyte storage container Straw
CPA concentration 1.3–1.5 M
Time in CPA (concentration NA
for equilibration)
Time in CPA (concentration 15–20 min
for cooling)
Time required for cooling 90–120 min
Ice crystal formation Yes
Osmotic injury Low risk
Toxic injury Low risk
Chilling injury High risk
Warming rate Low to moderate
Cost High
Commercial applications Extensive
the highest cryostability. Second, spermatozoa naturally con-
tain high concentrations of proteins, which helps vitrification.
Usually, sperm cells have a very high osmotically inactive vol-
ume (∼60%), hence osmotically inactive water is also higher in
spermatozoa and is bound to several macromolecular structures
such as DNA, histones and hyaluronidase. Consequently, this
would enhance both the viscosity and glass transition tempera-
ture (Tg) of the intracellular cytosol for spermatozoa, whereas
for embryos the probability of lethal ice formation during cool-
ing and warming would be several orders of magnitude higher.
Extensive compartmentalisation of intracellular compounds may
also contribute to the successful survival of spermatozoa. It is
this combination of the unique characteristics of spermatozoa
and a high speed of cooling and warming that makes it possible
to successfully vitrify human spermatozoa without CPA.
Cryopreservation methods for oocytes
Successful cryopreservation of female gametes is a viable tool
for preserving the female genome and to assist in rescuing endan-
gered species. Subsequently, development of cryopreservation
techniques for preserving gametes of wild animals relies greatly
on the results obtained in livestock and laboratory species. How-
ever, they are not always suitable models for conserving wild
species. To date, research has demonstrated remarkable differ-
ences among species. It is clear from the studies that optimal
cryopreservation methods tend to be species specific, due largely
to variations in gamete size, permeability and sensitivity to
cryoprotectant and cooling or warming rates.
Cryopreservation of oocytes is one of the greatest challenges
facing ART today. However, survival rates of cryopreserved
oocytes are lower, with poor developmental competence com-
pared with their fresh counterparts in hamster (Barnett et al.
1996), rabbit (Carroll et al. 1989), pig (Rojas et al. 2004; Somfai
et al. 2006; Wu et al. 2006; Gupta et al. 2007), mouse (Rall and
Fahy 1985; Glenister et al. 1987), cow (Vajta et al. 1998; Dinnyés
et al. 2000; Lane and Gardner 2001; Schmidt et al. 2004), human
A. Dinnyes et al.
Cryopreservation techniques
In-straw vitrification Ultrarapid vitrification
Straw Cryovial, straws, special
devices or no container
5.5–7.5 M 3.5–5.5 M
2–5 min 2–5 min
≥1 min ≥10 s
2–3 min <0.1 s
No No
High risk High risk
High risk High risk
Low risk Low risk
Moderate to high High
Low Low
Limited Limited
(Gordts et al. 1990; Kuleshova et al. 1999; Chung et al. 2000)
and equine (Tharasanit et al. 2006).
The development of female gamete cryopreservation tech-
niques in dogs and cats is important both for these companion
animal species and for the conservation of related endangered
species. Research is still in progress in the study of dog oocyte
maturation, which is still at a much lower rate than most domestic
species. Problems are due to dissimilarities of the reproductive
physiology of the dog compared with other species and the lack
of precise information concerning the oviductal environment,
in which oocyte maturation, fertilisation and early embryonic
development take place (Yamada et al. 1993). In cat, success-
ful in vitro embryo production has been achieved with oocytes
matured in vitro, and kittens were born after transfer (Luvoni
2006). Although sperm cells have been frozen successfully in
dogs and cats, cryopreservation of oocytes in dogs remains elu-
sive (Luvoni 2000). Cat oocyte cryopreservation has had some
success (Comizzoli et al. 2004). More research on cryostorage
of female gametes in dogs and cats is needed, together with refin-
ing techniques for oocytes maturation and fertilisation in vitro
to allow for establishment of a cryogene bank.
Recent efforts have been devoted to improve the survival of
frozen or vitrified oocytes by developing cryopreservation tech-
niques. Currently, there are three strategies for female gamete
storage: freezing, traditional vitrification (vitrification in straw)
and ultra-rapid vitrification (Table 1). Slow freezing has the
advantage of using low concentrations of CPA, which can reduce
chemical toxicity and osmotic shock. Vitrification is a rapid
method, which decreases chilling injury; however, it requires
high concentrations of CPA (Rall and Fahy 1985). Recent emerg-
ing ultra-rapid vitrification techniques have stimulated more
research on the cryopreservation of female gametes, including
various endangered species and indigenous livestock breeds, as
there has been greater success in the cryopreservation of female
gametes using methods such as OPS, SSV, cryoloop, cryotop
and Vitmaster (Arav et al. 2002).
Novel gamete storage
Advantages of gamete cryopreservation
Sperm cryopreservation has been used extensively in human
reproductive medicine and sperm banking (Critser and Linden
1995). It allows donor semen to be screened for sexually trans-
mitted diseases such as HIV and hepatitis before clinical use
(Mascola and Guinan 1986), whereas cryopreservation of male
gametes at various developmental stages (i.e. spermatozoa, elon-
gated or round spermatids) can be used to provide more advanced
treatment options in the case of spontaneous or iatrogenic infer-
tility (Hallak et al. 2000). Mouse spermatozoa and embryo cry-
opreservation are major tools for maintaining specific transgenic
or knockout mouse strains and lines that are crucially important
to biomedical research (Critser and Mobraaten 2000). Modern
ART, especially AI and to a lesser extent in vitro fertilisation
(IVF) and ICSI supported by gamete and embryo cryopreserva-
tion, have revolutionised domestic animal breeding programs.
These technologies have accelerated the progress of genetic
improvement and have enabled distribution of germplasm world-
wide (Watson 1990). Gradually, these technologies are being
used in conservation efforts for endangered species and captive
breeding programs (Johnston and Lacy 1995).
Oocyte cryopreservation in mammalian and endangered
species has a series of potential benefits and applications aimed
at maintaining gene pool diversity, regenerating populations after
disease outbreaks, rescuing rare or endangered species, provid-
ing a source of genetic material for research purposes, supplying
germplasm for new line and breed development, and global
germplasm trading. Therefore, successful cryopreservation of
oocytes provides insurance for existing genetic diversity within
a species.
Complications of gamete cryopreservation
Complications of cryopreserving spermatozoa
Protocols for cryopreservation of spermatozoa from various
species have been established, including those for stallion
(Graham 1996; Ecot et al. 2000), boar (Johnson et al. 2000),
goat (Leboeuf et al. 2000), ram (Salamon and Maxwell 2000)
and dog (Rodriguez-Martinez et al. 1993). Although these proto-
cols have integrated the optimised freezing procedures, they do
not achieve much more than 50% motility after thawing (Watson
2000; Vishwanath and Shannon 2000), whereas the fertilising
ability of the spermatozoa is reduced approximately seven-fold
(Watson 2000; Sullivan 2004).
There is a vast diversity in the cryobiological response of
spermatozoa among different mammalian species and among
different individuals within a species (Holt 2000). According to
Woods et al. (2004) the unavoidable variations in the success of
freezing are due to essential biological variability. Establishing
cryopreservation procedures for a specific cell requires optimi-
sation for that cell type within a species, and among individuals
in that species. Cryosurvival requires that freezing and thawing
are carried out within specific biophysical and biological limits
defined by the underlying fundamental cryobiological charac-
teristics of each species’ spermatozoa. In the 1950s, soon after
the positive effect of glycerol was discovered, a great increase in
the use of AI with frozen spermatozoa for dairy and beef cattle
followed. Since then, attempts to optimise the cryopreservation
Reproduction, Fertility and Development 723
of spermatozoa have been carried out empirically, resulting in
many improvements, including the development of semen exten-
ders such as egg yolk and skim milk, and cholesterol-loaded
cyclodextrins (Watson 1995; Moce and Graham 2006). However,
considerable loss of viability is still observed. Attempts to adapt
effective methods from one species to another has resulted in lim-
ited success (Kumar et al. 2003). In some species, methods with
efficiency acceptable for the breeders are yet to be determined,
whereas in other species, such as mouse, methods that seem
adequate for some strains fail in others (Critser and Mobraaten
2000). Spermatozoa of carnivores cannot be cryopreserved effi-
ciently using standard livestock methodologies (Farstad 2000).
These results indicate that fundamental cryobiological proper-
ties of spermatozoa and examination of the exact nature of sperm
cryoinjury must include a species-specific design in order to
develop appropriate cryopreservation protocols (Holt 2000).
Various aspects of sperm cryopreservation, such as chemical
composition of extenders and their effects on the sperm plasma
membrane, osmotic tolerance limits, hydraulic conductivity and
CPA permeability have also been studied (Gilmore et al. 1997,
1999, 2000).Apart from the damage associated with freezing and
thawing that has been discussed in previous sections, oxidation
of sperm membrane lipids (Brouwers et al. 2005) and damage to
the selective permeability mechanisms of the membrane are the
most damaging factors in freezing. In addition, the most sensitive
part of the sperm cells are their special acrosomal membranes
(Friend and Rudolf 1974; Grondahl et al. 1994). Freeze-fracture
studies of membranes before, during and after cooling show clear
evidence of phase separation, which is only partially reversed
after warming (Holt and North 1984; de Leeuw et al. 1990).
Many detrimental effects of freezing and thawing on sperm
capacitation (Watson 1995) and DNA denaturation (Gillan and
Maxwell 1999) have been reported. Studies suggest that sper-
matozoa do indeed have different cryobiological properties and
various degrees of sensitivity to cold shock, manipulation (e.g.
pipetting, centrifugation) and osmotic tolerance in every species
(Katkov and Mazur 1999; Phelps et al. 1999).
Dessicated (lyopreserved) spermatozoa would provide long-
term storage without the need for expensive and burdensome
cryogenic conditions, and it may provide a new perspective in
genome banking and become part of developing ART. In mouse,
Wakayama andYanagimachi (1998) were the first to produce live
offspring after ICSI from frozen-dried spermatozoa. However,
the procedure is still in its experimental stages, and requires
further investigation for both efficiency and safety. Some of
the previous studies have examined the cytogenic status of
zygotes (fertilised by ICSI), such as chromatin decondensation,
pronucleus formation, sperm aster formation and chromosome
abnormalities, in non-human primates, rabbit, as well as ham-
ster oocytes injected with human spermatozoa (Goud et al. 1998;
Hewitson et al. 2000; Terada et al. 2000).
One potential difficulty in the application of lyopreserved
spermatozoa is aberrant decondensation of sperm heads and
oocyte activation. In rats, Hirabayashi et al. (2005) reported that
viable offspring can be obtained by intracytoplasmic injection
of freeze-dried and rehydrated spermatozoa and that the ultra-
sonic treatment of spermatozoa is effective in increasing the
production of ICSI-derived offspring. They had established
724 Reproduction, Fertility and Development
protocols for microinsemination, including ICSI, elongating
spermatid injection and round spermatid injection. The effi-
ciency of bovine ICSI is limited because of the necessity for
additional oocyte activation after the ICSI procedure. Studies
with activation protocols have demonstrated that the develop-
mental competence of bovine embryos could be improved by
treatment with ionomycin followed by 6-dimethylaminopurine
after ICSI (Rho et al. 1998; Keskintepe et al. 2002).
Storing spermatozoa in liquid nitrogen can preserve their bio-
logical function for many years (Mazur 1984); in bovine, good
fertilisation rates have been achieved using even 37-year-old cry-
opreserved bovine spermatozoa (Leibo et al. 1994). However,
long-term effects of dessication on sperm genetic stability dur-
ing storage in various media is unknown. The long-term effects
of desiccated spermatozoa are estimated theoretically by acceler-
ated degradation kinetics. Calculated extrapolation of Arrhenius
plots indicates that mouse development rate to the blastocyst
stage would not decline significantly for 100 years of storage
at −80◦ C. The chromosome integrity could be maintained by
adding calcium-chelating agents during freeze drying (Kawase
et al. 2005).
Indeed, it was found that structural chromosomal abnor-
malities were significantly higher in mouse oocytes injected
with spermatozoa freeze-dried in Chatot, Ziomet and Bavister
medium. However, chromosome integrity during freeze dry-
ing can be maintained using Tris-HCl-buffered solution with
high concentrations of a calcium-chelating agent (Kaneko and
Nakagata 2005).
Vitrification of human spermatozoa in the absence of conven-
tional cryoprotectants is feasible. The DNA integrity of vitrified
spermatozoa is comparable with that shown by standard slow-
frozen–thawed spermatozoa, yet the method is quick and simple
and does not require special cryobiological equipment. This
technology has great potential for application in companion ani-
mals, non-domestic and endangered species (CANDES) in field
conditions, although so far only human spermatozoa have been
vitrified successfully. Other shortcomings could be the limita-
tions of the amount of sample held by each cryoloop and sperm
prewashing. Considering that many CANDES have low sperm
concentrations in their collected ejaculates, it might be imprac-
tical to use many cryoloops to vitrify one AI dosage; thus, the
method might be more suitable when combined with ICSI.
In situ cryostorage of spermatozoa by freezing testis, or even
whole animals, could be an interesting option and potentially
very advantageous in CANDES. Recently Ogonuki et al. (2006)
reported high success rates of ICSI from frozen testis and epi-
didymidis. Surprisingly, progeny were generated by ICSI with
spermatozoa recovered from whole mouse stored at −20◦ C for
15 years. This not only shows the resistance of sperm DNA
to damage, but would offer a very practical option for stor-
ing smaller CANDES cadavers or reproductive organs under
conditions available even in remote areas and non-specialised
facilities.
Complications of cryopreserving female gametes
Despite the extensive research conducted during the past 20
years, progress in the cryopreservation of oocytes remains
A. Dinnyes et al.
(a) (b)
PB
ZP
MT
Nuclear
Spindle
membrane
ZP
GV with DNA
MII oocyte
GV oocyte
Fig. 1. The typical structure of immature and mature oocytes. (a) The
immature oocyte at the germinal vesicle (GV) stage is full size but its
chromatin remains at the diplotene stage of prophase one with a nuclear
membrane and zona pellucida (ZP), and it does not possess a spindle appa-
ratus. (b) In matured oocyte (MII stage), the oocyte has undergone nuclear
and cytoplasmic maturation, the first polar body (PB) has been expelled and
the chromosomes are condensed and arranged on the delicate MII spindle.
The meiotic spindles consist of microtubules (MT). Microtubules start from
microtubular organising centres at both poles and anchor chromosomes at
the kinetochores, forming a barrel shape. The chromosomes align at the
equatorial plane of the meiotic spindles.
limited, and the current live birth rate is low (Rutherford and
Gosden 1999). Although female gametes have been cryopre-
served using various protocols, the fertilisation and in vitro
development rates achieved are much lower than those obtained
with fresh oocytes.
Difficulties in the cryopreservation of mammalian oocytes
are due to their complex structure (Massip et al. 1987; Friedler
et al. 1988; Rall 1992; Arav et al. 1993; Smorag and Gajda
1994; Kuwayama and Kato 2000). Oocytes are among the largest
mammalian cells, with a low surface-to-volume ratio. Further-
more, the oocyte’s cytoskeleton structure can be sensitive to
cooling. At the time of ovulation, in most species oocytes are
in metaphase stage of the second division (MII). The dichro-
matid chromosomes aligned on the equatorial axis are bound
to the microtubules in the meiotic spindle (Fig. 1). This stage
can be problematic for cryopreservation (Bernard and Fuller
1996; Wood et al. 2001) with temperature and CPA sensitiv-
ity of the spindle and microtubules (Newton 1998; Arav et al.
2002). In addition, the lipid content of the membranes can change
CPA and water permeability. The large size of oocytes with
consequent low surface-to-volume ratios make it more diffi-
cult for water and cryoprotectants to enter or leave the cells
across plasma membranes (Leibo and Songsasen 2002), thus
increasing the chances of ice crystal formation and CPA tox-
icity. Low temperature causes depolymerisation of the spindle
and microtubules. As a result, fertilisation may not take place
because normal separation of chromatids might be prevented,
resulting in aneuploidy following extrusion of the second polar
body. Zona pellucida hardening due to premature cortical gran-
ule release can be triggered by some CPA and eventually can
prevent normal fertilisation.
Collection of oocytes from secondary follicles of ovaries
results in germinal vesicle (GV)-stage oocytes. There have been
theoretical grounds for favouring GV-stage oocyte cryopreserva-
tion. Before GV breakdown, chromosomes are still surrounded
Novel gamete storage
by a nuclear membrane (Wood et al. 2001), making them poten-
tially more cryotolerant (Agca et al. 2000). However, after
cryopreservation at this stage, there is a need for GV-stage
oocytes to mature efficiently in vitro up to the stage where nor-
mal fertilisation can be achieved. Moreover, cryopreservation
procedures have been reported to have deleterious influences
on chromatin and other cytoplasmic organelles of GV oocytes.
In some cases in vitro maturation following cryopreservation
of GV oocytes led to higher incidences of chromosomal and
spindle abnormalities and adversely affected postfertilisation
development (Van Blerkom and Davis 1994; Son et al. 1996;
Park et al. 1997). The present efficiency and reliability of using
cryopreserved oocytes for generating offspring is still much
lower compared with cryopreserved embryos or spermatozoa.
Cryopreservation of oocytes of avian and fish species is not yet
successful, largely because of the large size, high lipid content
and polar organisation (vegetal and animal pole) of bird and
fish oocytes (Douard et al. 2005). Fish eggs are sensitive to
osmotic changes, which can result in spontaneous activation.
However, recent reports on cryopreservation of fish primordial
germ cells followed by their transfer into the abdominal cav-
ity resulted in offspring (Kobayashi et al. 2007), demonstrating
that success can be achieved with such an approach. If inter-
species transfer of fish primordial germ cells works, this would
help endangered species too. However, in most cases such proce-
dures would be complicated to carry out. Similarly, although the
current very inefficient approach for cryopreservation of avian
genetics occurs via germinal disk stem cell isolation, cryopreser-
vation then introduction of the stem cells into a developing egg
would be possible. For example, chimeric chicken and ducks
have been produced with this method using cryopreserved ger-
minal cells, and this technology is under development owing
to its relevance for poultry biotechnological applications (Kino
et al. 1997).
Storage of ovarian tissue
Considering the complexity of cryopreserving female gametes,
ovarian tissue cryostorage may be an alternative solution. Preser-
vation of female gametes may be best achieved by storing pieces
of ovarian tissue that contain numerous small follicles. Cryop-
reservation of ovarian tissue has been reported to be possible
in humans, mice, sheep, rats, marmosets, elephants, wombats,
tammar wallabies and rabbits (see review by Amorim et al.
2003).
Early-stage oocytes in primordial follicles are smaller, posses
fewer organelles and have no zona pellucida or cortical granules;
hence, they are potentially easier to cryopreserve (Oktay et al.
1998; Paynter 2000). In addition, xenografting of the ovaries of
3-week-old mice into rats was successful in a generation of pups
(Shaw et al. 2000). Interspecies transplantation of ovaries from
large mammals, including humans, monkeys, cows, pigs, dogs
and marsupials, to recipient mice has resulted in the develop-
ment of antral follicles and even matured oocytes (see review
by Katska-Ksiazkiewicz et al. 2006). This technique might also
be used in wildlife conservation, through the formation of gene
banks to maintain gene pool diversity in captive breeding (Wildt
2000; Amorim et al. 2003).
Reproduction, Fertility and Development 725
Cryostorage of ovarian preantral follicles would also pro-
vide a source of viable oocytes that could be used to propagate
endangered or other species (Van den Hurk et al. 2000; Biron-
Shental et al. 2004). However, in vitro growth of early follicles
is still very inefficient.
Packaging and biocontainment
Usually, the cryopreservation of gametes of laboratory and
domestic animals is conducted in a laboratory where sophis-
ticated equipment is available and environmental conditions
and the risk of contamination are controllable. There are issues
of packaging and biocontainment that require more serious
consideration for CANDES than for laboratory and domestic
species. From a cryobiological point of view, the packaging
would affect the cooling and warming rates due to different
dimensions and materials used in various packaging systems
and appropriate adjustments of parameters in cryopreserva-
tion protocols are usually needed to compensate for these
variables.
Basic choices of packaging include cryovials or straws. Cry-
ovials can hold a larger volume of sample (beneficial for sperm
preservation); however, cooling rates and actual temperatures in
the vials vary during cooling and warming. Moreover, such vials
are only safe to store in the gas phase of nitrogen tanks, as storage
in liquid nitrogen (although a common practice) usually results
in leakage of the liquid nitrogen into the vials. This can result in
cross contamination between vials, and can even blow the vials
up during warming.
Plastic straws (usually 0.25- and 0.5-mL volumes) are more
homogeneous during cooling and warming, with higher cool-
ing and warming rates achievable. Powder or metal ball sealings
are not fully leak proof, and regular straws are easy to break
at cryogenic temperatures. Heat sealing can prevent leakage,
and high-security straws (CryoBioSystem, Paris) are especially
suitable for leakage-proof storage and are not fragile at low tem-
perature (Mortimer 2004). These samples can be stored without
any concerns in liquid nitrogen.
Packaging can affect the effectiveness of biocontainment,
which is particularly important for CANDES due to the high
risk of contamination and cross-contamination in the field
conditions. Precautionary steps can reduce risk and damage
substantially, such as disinfecting the outside of the packaging,
sterilising the cryopreservation equipment and dividing storage
locations (Mortimer 2004). However, some novel technologies,
such as the vitrification of human spermatozoa or oocytes in
minimum volume systems (OPS, cryoloop, droplets, pellets),
require direct exposure of samples to liquid nitrogen in order
to achieve sufficiently rapid cooling rates, and this usually pre-
cludes effective biocontainment. There are efforts to overcome
these problems (e.g. OPS mini-straws packed into 0.5-mL sealed
straws for storage). Also, the SSV method avoids direct contact
with liquid nitrogen.
During the design of cryopreservation protocols to pre-
serve CANDES, strategic planning must include experimental
conditions as well as risk assessment of contamination and cross-
contamination depending on the choice of packaging, storage
and cryogenic equipment.
726 Reproduction, Fertility and Development
Conclusion
In the last 50 years, the need for breeding management and
genetic improvement for domestic livestock industries, more
advanced treatments for human infertility, efficient preservation
of laboratory animal species, and better conservation of bio-
diversity for endangered species have motivated scientists and
breeders to utilise empirical and theoretical research approaches
in developing efficient cryopreservation procedures for a broad
range of species. Systematic research on conventional freezing
methods have lead to improved CPA use, cooling and warm-
ing procedures and avoidance of damaging ice formation for
humans and laboratory and domestic animals. Evidently, these
techniques have been modified and adapted for preserving com-
panion, non-domestic and endangered species. Many CANDES
spermatozoa have been cryopreserved successfully (Blanco et al.
2000; Robeck and O’Brien 2004; Si et al. 2004), although in
some species there are still major difficulties (Pukazhenthi et al.
2006b).
With the progress of overall ART, new preservation technolo-
gies are emerging too. Vitrification of human spermatozoa in
the absence of conventional CPA has been shown to be feasible;
however, more research is needed to demonstrate the long-term
effects of such an approach on fertilisation, fetal development
and progeny in animal models. Dessicated (lyopreserved) sper-
matozoa would provide a beneficial method for genome banking
and ART. To date, embryonic development after ICSI with
freeze-dried sperm heads has been reported in large domestic
animals, including cattle and pigs, and live offspring in mice,
rat, rabbits and fish have been born.
Despite progress in freezing and vitrification methods, cry-
opreservation of mammalian oocytes remains a major challenge
in ART. Matured oocytes have major problems in terms of
temperature and CPA sensitivity, and in some species GV
oocytes might be easier to obtain and cryopreserve. However,
in vitro maturation following cryopreservation of GV oocytes
needs species-specific studies as incidences of chromosomal and
spindle abnormalities that occur during maturation can reduce
postfertilisation development. Cryopreservation of oocytes of
avian and fish species is not yet successful, largely because of
the large size, high lipid content and polar organisation, although
novel approaches using germ cells offer some alternatives.
Cryopreserving ovarian preantral follicles or ovarian tissue
would provide a great number of developmentally competent
oocytes for protocols such as IVF, embryo transfer, cloning and
transgenesis (Van den Hurk et al. 2000).
Future directions
The challenges in development of optimal cryopreservation tech-
nology for CANDES spermatozoa are due to the diversity of
cryopreservation conditions and availability of resources in dif-
ferent species. The challenge is to determine the optimal protocol
among an almost infinite number of possibilities (Karlsson and
Toner 2000). To replace traditional empirical methods, such as
modifying technical conditions in freezing trials, novel models
have been proposed to predict the likelihood of freezing-induced
injury or mortality (Muldrew and McGann 1990, 1994; Toner
et al. 1990). Membrane permeability to water and permeating
A. Dinnyes et al.
CPA, time of exposure, resistance to osmotic changes, minimal
critical volume, cell size and morphology greatly influence the
occurrence of freezing injury. Taking into account these vari-
ables has laid the foundation for developing routine and effective
cryopreservation protocols for human, bull and stallion sper-
matozoa. Accumulated knowledge in cryobiology and cellular
characteristics made it possible to rationally design cooling pro-
cedures using computer simulations (Karlsson et al. 1994; Liu
et al. 2000).
Simulations in the development of cryopreservation meth-
ods for CANDES would be highly beneficial as they would save
limited resources. In order to achieve this, further fundamental
research is required to elucidate the mechanisms of damage dur-
ing cryopreservation, and to develop predictive models for these
processes. Likewise, measurements of the biophysical proper-
ties of CANDES spermatozoa should be developed in order to
obtain necessary model parameters.
One of the promising attempts for CANDES spermatozoa
was made by Blanco et al. (2000) investigating potential factors
of spermatozoa cryosurvival for several avian species including
chicken, turkey, golden eagle, Bonelli’s eagle, imperial eagle
and peregrine falcon. These studies focused on sperm toler-
ance toward osmotic stress, concentrations of CPA, equilibration
times at 4 and 21◦ C, and cryopreservation by rapid or slow
freezing, demonstrating the necessity for species-specific cryop-
reservation protocols for avian species. Thirumala et al. (2003)
calculated that canine sperm freezing rates based on membrane
permeability parameters differ significantly from those in other
mammalian species. The optimal rate of freezing for canine
sperm cells was in the order of tens of ◦ C min−1 , in good
accordance with rates obtained empirically.
Vitrification of human spermatozoa in the absence of con-
ventional cryoprotectants has been proven experimentally to be
feasible. However, more research is needed to test whether the
technology can be applied to other species, especially CANDES,
as it would be very advantageous in eliminating the need for
sophisticated equipment. As discussed in the previous section,
this technology might require better AI and IVF efficiency to
compensate for the small sample size cryopreserved.
Although the technology of desiccated (lyopreserved) sper-
matozoa as an alternative to cryopreservation is in the experi-
mental stage, it has been investigated for laboratory and domestic
animals. It would provide a convenient method of long-term
storage, transportation, and an effective way of storing sper-
matozoa for some laboratory animals (inbred rat and mouse
strains) in which conventional cryopreservation results in very
poor fertility.
However, further investigations are required to improve its
efficiency and feasibility for CANDES. This method is rather
inconvenient and inpractical, as the procedures of microinjec-
tion, oocyte activation, IVF and embryo transfer are essential
parts of the whole process of preservation and rederivation and
should be used only as a last resort for CANDES.
Cryopreservation is used widely in the fields of human
medicine, biological research, domestic livestock industries and
preservation of CANDES. Although seeking effective and effi-
cient cryopreservation procedures are goals for each scientist
in these areas, different areas might define the ‘effectiveness
Novel gamete storage
and efficacy’ differently due to their scientific and economical
priority and resources. Progress in both conventional and novel
cryopreservation methods would definitely provide new choices
for CANDES researchers to select accordingly.
Historically, methods for female gamete storage were devel-
oped using an empirical approach and the parameters to assess
the success of a given protocol have typically been morphologi-
cal survival rate, fertilisation and blastocyst formation. Although
this approach has certainly resulted in improved cryopreserva-
tion techniques and protocols, it has provided limited informa-
tion regarding the actual physiology of the female gamete and
complex events such as interactions with CPA, effects of cool-
ing and warming rates used and impact on cell function. Gene
expression changes due to cryopreservation have been reported
for embryos (Boonkusol et al. 2006; Mamo et al. 2006) but very
little is known about changes in gene expression and protein
profiles due to oocyte cryopreservation. More molecular bio-
logical data might help to model mathematically the oocyte as a
‘system’ and to make reliable predictions for cryobehaviour and
create fundamentally new avenues for cryopreservation taking
into account long-term biological consequences as well.
As reviewed, there is still much to be done to optimise pro-
tocols for successful cryostorage of female gametes, especially
from CANDES. These gametes would be a valuable resource
contributing to the preservation of genetic material essential for
the maintenance and captive management of endangered species.
There are still many obstacles to overcome, such as the improve-
ment of cryopreservation protocols for both male and female
gametes and establishment of good and optimised in vitro mat-
uration protocols for post-thawing use. Furthermore, effective
techniques in domestic species are not necessarily applicable to
wild species. Reproductive mechanisms are highly varied among
animal groups.
Storage of ovarian tissue and early-stage follicles might
become an alternative resource for the foundation for cryogene
banks, and for direct storage of CANDES individuals killed acci-
dentally or by hunting. However, it is too early to predict when
this technology will become practical. Some of these problems
might eventually be overcome by growing cryopreserved folli-
cles and oocytes to maturity in vitro or in temporary hosts, such
as ectopic sites of severe combined immunodeficiency diseased
mice (Kagawa et al. 2007).
Female gamete storage has been done mostly by vitrification
rather than the slow-freezing method. Future female gamete cry-
opreservation might be based mainly on vitrification (Vajta and
Kuwayama 2006), although some of the most efficient vitrifica-
tion methods are lacking the possibility for direct transfer and
there are concerns of possible disease transmission through liq-
uid nitrogen during vitrification and storage. This might be a
particularly important issue in the case of CANDES where full
record of health status is often not available. Another area worth
investigating is stabilising cell membranes with trehalose. The
drawback is how to transfer trehalose into the cytoplasm of the
cell, where it could protect internal membranes as well (Seidel
2006).
Oocyte cryostorage is improving, especially in cattle, humans
and some other species, and one day may replace embryo
cryopreservation offering an alternative to animals at risk of
Reproduction, Fertility and Development 727
losing their reproductive capacity or for rescuing endangered
species.
Acknowledgements
The work was supported by the European Union (MEDRAT-LSHG-CT-
2005-518240 and MRTN-CT-2006-035468), by the Hungarian Govern-
ment (NKTH/KPI Kozma Ferenc TUDAS-1-2006-0005 project), and a
Hungarian–South African Bilateral Scientific and Technological (TET)
collaborative project.
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Manuscript received 26 February 2007, accepted 8 June 2007