FUNDAMENTAL

ASPECTS OF SPERM CRYOBIOLOGY:

THE IMPORT ANCE OF
SPECIES AND INDIVIDUAL DIFFERENCES
W.V.Holt

Institute ofZoology,

Zoological

Society ofLondon,

Regent's

Park, London NWl 4RY, UK

ABSTRACT
While semen cryopreservation
is successfully used for a few species, application to other
species can be problematic. Here, 1 argue that species differences in female tract anatomy, subtle
differences in sperm transport mechanisms, ability to time inseminations and deliver spermatozoa
effectively are powerful determinants of fertility with cryopreserved
spermatozoa.
Poor sperm
survival represents one major aspect of the problem and determining biophysical characteristics of
the sperm plasma membrane is an established approach to solving it. However, this approach is
unable to account for the consistent differences in post-cryopreservaton
sperm quality between
individual males, an effect that is recognized in many species although only documented in a few.
Searching for genetic differences between these individuals might offer a genomically-based
direction in semen cryopreservation research. Cryopreservation
of spermatozoa and spermatogenic
cells for intracytoplasmic sperm injection has been developed primarily to deliver an intact genome
and presents a very different set oftechnical problems.
1999 by Elsevier Science Inc.

Key words: semen cryopreservation, spermatozoa, sperm transport, artificial insemination.

INTRODUCTION
Semen storage technology was revolutionized approximately 50 years ago by the discovery
that glycerol could act as a cryoprotectant (36). This important observation enabled spermatozoa to
be frozen, stored for prolonged periods, and then used successfully for artificial insemination.
Fortunately for the pioneers ofthis technology they chose to work with bull and fowl spermatozoa.
Had they elected to begin with rodent, marsupial or porcine spermatozoa instead, they would have
encountered a series of species-specific problems that even today remain difficult to solve. While
sorne species differences, such as sperm head shape, are obvious or measurable, other factors are
more complexo Sperm transport in the femaIe tract is one such species-specific difference; access to
the site of fertilization within the oviduct involves overcoming a series of obstacles that may have
evolved specifically to impede poor quality spermatozoa. Since even the best cryopreservation
techniques cause extensive lethal and sublethal cryoinjury, it would not be surprising that the
efficiency of sperm transport may be severely reduced after cryopreservation.
Acknowledgments:
Healthbred Ltd, PIC International and the UK Pig Reproduction Research Liaison Group for I
am grateful to the Ministry of Agriculture, Fisheries and Foods (United Kingdom), JSR financial
and moral support.
Correspondence and reprint requests to W. V. Holt, Institute of Zoology, Regent's Park,
London NWl 4RY. E-mail;bill.holt@ioz.ac.uk
Theriogenology 53:47-58, 2000

1999 by Elsevier 5cience Inc.

oo93-691X100~ee Iron! maller

PII 50093-691 X(99)00239-3

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Theriogenology

The field of semen cryopreservation research has, over the past few decades, been
weighted heavily towards empirical studies. Many papers have been published in which the effects
of varying a few technical parameters, such as the concentration of cryoprotectants and other
additives, or cooling and warming rates, have been examined. While these may help improve postthaw sperm quality under a particular set of circumstances, they fail to identiy important cornmon
themes in sperm cryobiology, and are of limited use in predicting the way forward. Sorne research
groups have recognized the limitation of this approach and have focused on modeling the
hydraulic fluxes that spermatozoa experience during the freeze-thaw process. For this, they have
undertaken detailed studies of membrane permeability in spermatozoa and then used biophysical
approaches to predict minimally damaging cooling protocols. Sorne of this work, especially that
relating to the addtion and removal of cryoprotectant, has been very successful in allowing
optimal sperm recovery (14), although the accurate prediction, on theoretical grounds, of optimal
cooling rates is still problematc. In this review I surnmarze sorne of this fundamental work.
However, 1 will also highlight other issues that probably affect the success of sperm
cryopreservation techniques and that may be species- or taxon-specific. These issues present
major problerns for the development of sperm storage methods for threatened species where
background data are often sparse and opportunities for research may be limited.
Cellular Responses to Cryopreservation and Cryoprotectants
When cells are frozen, they are subjected to stresses resulting from the water-solute
interactions that arise through ice crystallzation. Crystallzation induces the formation ofunfrozen
pockets of hyperosmotic solution while cooling to approximately -50C is in progress. Freezesubstitution electron microscopic studies (3) of spermatozoa within frozen diluent have shown
that sorne cells become excluded from the space occupied by the ice crystals and are, therefore,
preferentially exposed to the anisosmotic environment. This results in withdrawal of intracellular
water, consequent cell shrinkage and possible influx ofions (4,30). Thawing involves a reversal
of these effects, and the consequent inward water flux may cause cell membrane disruption. Since
overly rapid freezing causes lethal intracellular ice formation, the optimal cooling rate is thought
to be slow enough to prevent this lethal effect, but fast enough to minimze the harmful effects of
prolonged exposure to high salt concentrations ("solution effects"). High salt treatments are
standard textbook techniques used preparatively and analytically to release peripheral proteins
from cell surfaces. Exposing sperm to the hypertonic solutions formed by ice crystal development
means that they will be injured by the removal of membrane proteins during this phase of the
cryopreservation process if it is too prolonged.
As the magnitude of the water flux is highly dependent on cooling rate, optimal values for
water fluxes should be achievable by calculating optimal freezing rates. These argurnents have
been applied to embryo and oocyte freezing (e.g., 29, 38), where theoretical predictions matched
observations with a considerable degree of success, and their relevance to spermatozoa has been
studied in some detail (e.g., 8, 13, 15). At present, with one exception which employed a novel
differential scanning calorimetric technique to measure biophysical parameters of mouse
spermatozoa (10), the theoretically-derived optimal cooling rates for spermatozoa do not match
the observations. Several likely reasons for this discrepancy can be suggested. Calculation of
water flux depends upon the derivation of membrane-specific parameters, hydraulic conductivity

Theriogenology

49

and activation energy, but as the spermatozoon is such a complex cell, these parameters may vary
over the various rnembrane domains of the sperm surface. Furthermore, changes in rnembrane
lipid packing may induce temperature-dependent variation in the derived parameters and
cryoprotectants, especially glycerol, are known to interact with rnembrane lipids and may
themselves affect the derived parameters (for references, see 20).
As the estimates of sperm surface area needed in order to develop the theoretical
approach have typically been derived from models which regard the spermatozoon as a
combination of simple geometric shapes such as cylinders and cubes, considerable experimental
error may also have been introduced. It should be possible to improve the accuracy of these
measurements by using novel techniques such as atomic force microscopy. This method, which
essentially involves the use of a tapping stylus to create a molecular scale 3-D representation of
the cell surface, has the added advantage that the spermatozoa do not require pretreatment other
than irnmobilization by mild fixation. This avoids the artifacts introduced when cells are processed
for electron microscopy.
Determinants of Species Differences in the Susceptibility of Sperm to Cryoinjury
While this theoretically based approach is a logical way forward for developing
cryopreservation methods for particular species, it might be argued that if cooling and warming
rates could be controlled appropriately, the problem would be solved. As the theory and practice
do not match, other factors must also be important. The major significance of the empirical work
is the demonstration that the chemical and osmotic environment of the spermatozoa is of sorne
consequence in obtaining good sperm survival. Choice of buffer system, nature of cryoprotectants
and additives such as sugars, calcium chelators, antioxidants and milk or egg yolk proteins have
all been shown to influence sperm survival profoundly. It is unlikely that the fundamental role of
these substances is to modify the water permeability of the sperm plasma membrane and,
therefore, other explanations for their different and species-species effects must be sought.
The implications of species variability in both the beneficial and detrimental effects of
glycerol are worth considering in this respect. Bull spermatozoa are routinely cryopreserved using
4-8% glycerol, and acceptable results have generally been obtained with wild ruminants when this
range is used (red deer, scirnitar horned oryx and gaur; references 12, 16 and 24, respectively).
Similarly, studies of primate sperm cryopreservation have focused upon this range as optimal.
However, spermatozoa from other species do not tolerate exposure to these concentrations. For
example, porcine spermatozoa suffer severe acrosornal damage if the glycerol concentrations
exceed 3%, and although little comparative work has been done on rodents, it is known that
mouse spermatozoa are damaged by glyceroI concentration exceeding 1.75%. Indeed, sorne
recent techniques for mouse sperm cryopreservaton avoid glycerol completely. That this is a
species-specfc effect, and not a taxon-specifc effect, can be deduced from studies of chinchilla
spermatozoa, where optimal re~lts were obtained with 6% glycerol (37). Further inconsistency is
detected when marsupial studies are considered. Taggart and colleagues (44) showed that 4-8%
gIyceroI allowed very good post-thaw recovery of sperm motility (70-90%) in sorne marsupials
(brush-tailed possum, northern brown bandicoot), while only 3% motility was recovered in fattailed dunnart. Surprisingly, in sorne species (e.g., koala, brush-tailed possum) there was

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considerable benefit from increasing the glycerol concentrations to levels (15-20%) which would
normally be considered excessively high (25, 26, 31).
Such comparative data show that the effective range of glycerol concentrations in
unfrozen diluent spans an order of rnagnitude 0.2 M to approxirnately 2 M). It seems
implausible that this is exclusively attributable to species differences in membrane permeability,
and more reasonable to consider all aspects of membrane structure and function. The molecular
interaction of gIyceroI with the sperm plasma membrane was reviewed in sorne detail by
Hammerstedt and Graham (20), who suggested that it might both alter membrane fluidity, by
becoming intercalated into the lipid bilayer, and change the intracellular cytoplasmic viscosity thus
affecting all metabolic reactions. Disruption of cytoskeIetal protein-membrane interactions in
mouse spermatozoa by the action of glycerol has also been proposed (34). In this context, the
potential dsruptive effects were regarded as beneficial because tolerance to the osmotic stresses
associated with water fluxes may be enhanced. This hypothesis may help explain the need for such
high glycerol concentrations for sorne marsupial spermatozoa, as the group of species in which
cryopreservation is both probIematic and require the highest glycerol concentrations in the
cryodiluents (kangaroos and wallabies), are also those with elaborate submembranous cytoskeletal
structures (43).
ObviousIy, cryopreservation invoIves exposure to non-physiologically low temperatures
even before freezing occurs. This process is known to induce changes in two-dimensional
membrane lipid organization or "packing" (lipid phase transitions) and, in tum, to modify the
kinetic properties ofintramembranous enzymes (11, 22, 23). Thus membrane fusogenicityand the
responses of signaI transduction pathways couId also be affected by such changes, contributing to
the possibility that post-thaw sperm longevity is reduced through accelerated capacitation (49).
Efforts to correlate susceptibility to cryoinjury with membrane lipid composition across species
have suggested that cold-shock, a phenomenon by which cryoinjury is induced by sudden cooling
but without freezing, is more severe when sperm membrane sterol concentrations are low and
polyunsaturated fatty acid concentrations are high (9, 50). This classification distinguished fowl
and human spermatozoa as more cold-shock resistant than bull and ram spermatozoa. However,
it cannot explain the major differences in post-freeze/thaw survival and fertility between, e.g.,
porcine and bovine spermatozoa. Technical procedures such as slow cooling, and additives such
as egg yolk or milk proteins, are routinely incIuded in cryopreservation protocols to minimize
cold-shock effects. The most significant cryoinjuries are therefore probably sustained during the
freezing and thawing process; this suggests that differences in membrane lipid composition are not
as important as was once thought. Despite this concIusion, sorne attempts to manipulate the lipid
composition ofspermatozoa through diet have been undertaken in the beliefthat this would alIow
the generation of more cryoresistant cells. So far, this research has shown that, as one might
expect physiologically, sperm lipid composition' is unresponsive to manipulation (28); however,
this work is being continued.
Attempting to understand the source of differences in sperm cryosusceptibility among
species is complicated because there are so many potentially confounding factors to considero One
way to simplify this analytical approach is to look instead at differences between individuals ofthe
same species. Many practitioners of artificial insemination with frozen spermatozoa relate

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51

anecdotes about individual males whose spermatozoa either consistently fail to survive the
freeze/tbaw process or altematively, survive particularly well. Although such observations are
generally anecdotal, they apply across a range of species, including dogs, bulls, boars, stallions
and humans. The clearest demonstrations of these inter-individual differences come from two
series of experiments. Heterospermic insemination trials using mixed samples of bull spermatozoa
showed tbat the cryopreservation process changed the proportions of conceptions attributable to
particular sires when the sperm were in competition with each other (2). This implies tbat those
spermatozoa tbat are normally advantaged in their ability to reach and fertilize oocytes are not
necessarily those most suited to withstand cryopreservation. This is an important observation
because the experimental design eliminated many factors, especially those related to female tract
anatomy and ovulation tirning, which ofien confound inter-specific comparisons. The second
series of experiments to reveal the importance of inter-individual differences comes from
experiments on mouse sperm cryopreservation. Working with three strains ofmice Songsasen and
Leibo (40) showed tbat simple exposure of spermatozoa to cryoprotectve medium, wthout
freezing, reduced fertilization rates in two strains while the third strain tested (B6D2Fl) remained
unaffected by this treatment. After freezing, spermatozoa from the B6D2Fl strain produced
61.2% 2-cell embryos afier IVF, while fertility was severely depressed in the other two strains
(17.2 and 3.0% 2-cell embryos for 129/J and C57BL/6J, respectively). Further experiments
showed tbat these differences were mainly attributable to differential sensitivity to the osmotic
shocks associated with the addition and removal of cryoprotectant. These observations not only
point once more to plasma membrane properties as highly influential determinants of post-tbaw
sperm survival and fertility, but are especially valuable because gross species difference effects are
clearly not relevant.
These results also formally test and support the hypothesis that inter-individual differences
in sperm "freezability" are genetically inherited rather than being random. Previous experiments
on mouse sperm cryopreservation had suggested the existence of genetically determined strain
differences (42). Indeed, the difficulty of working with a transgenic mouse strain (C57BL/6J)
prompted the use of partial zona dissection to improve the fertilization rate (33). These findings
appear to provide the basis for a molecular approach to sperm cryobiology. Within species, and
even within strains or breeds, identiying groups of males that only differ in the response to sperm
cryopreservation protocols might be possible. Comparisons of pooled genomic DNA among the
groups should highlight gene differences, whose function may be correlated with sperm
cryopreservation. This is a potentially powerful approach, although novel and yet untested in this
contexto
The observation that tolerance to osmotic stresses during the addition and removal of
cryoprotectant is highly predictive of sperm survival after cryopreservation can be viewed in two
ways. On the one hand it shows that differences in membrane structure, permeability and elasticity
determine the extent to which sperm will survive the cryopreservation process. However, at a
technical level this information suggests that sperm survival can be optimized by avoiding or
minimizing osmotic shock as much as possible. Detailed biophysical studies of membrane
permeability characteristics of human spermatozoa have allowed prediction of strategies that
minimize volume excursions during the addition and removal of cryoprotectants (14). The optimal
technique, which involved the gradual addition and removal of glycerol in a series of

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approximately eight incremental or decremental steps, permitted recovery of approximately 90%

live spermatozoa. This considerably outperformed the results obtained when cryoprotectant was
added or diluted all at once, which is the procedure routinely used in most laboratories. The more
painstaking and elaborate approach to cryoprotectant addition and removal is, of course, less
practical in a busy laboratory. However, the availability ofthis technique for human spermatozoa
is worthy of note, especially for those dealing with samples from chemotherapy patients and
which may be irreplaceable or of extremely poor quality.
Owing to space limitations, the aboye discussion has considered only a limited number of
characteristics which may be relevant to the relative success of cryopreservation protocols in
different species. However, many different aspects ofreproduction undoubtedly contribute to the
ease of both cryopreserving spermatozoa and subsequently using them successfully for
inseminations. The following paragraphs aim to provide a brief indication of these topics, without
necessarily going into much detail.
There are major differences among species in sperm morphology, especially in acrosomal
shape and flagellar length, but their relevance to cryopreservation is unclear. Many rodents have
elaborate sperm heads, often being hook-shaped and possibly more susceptible to distortion and
damage. Bull, ram and boar sperm heads are very similar, i.e., "paddle-shaped", but the boar
spermatozoa are more susceptible to cryoinjury. Sorne of the macropodid marsupials (kangaroos
and wallabies) have elaborate submembranous cytoskeletal assemblies (43) that may render them
particularly susceptible to cryoinjury. Other marsupial spermatozoa pair by head-head attachment
during sperm maturation in the epididyrnis (e.g., south American opossurns) and cannot swim
progressively if this mutual arrangement is disrupted (32); cryopreservation is likely to be
particularly difficult with such species.
Insemination tirning is critical when using frozen spermatozoa as the duration of sperm
survival is typically OOIfthat of fresh sperm. Success of Al programs is thus highly dependent
upon the availability of good techniques for predicting, controlling or detecting ovulation. In
general, insemination prior to ovulation is better than post-ovulation insemination. These technical
issues are compounded by anatomical variations and sperm transport mechanisrns. Intrauterine
insemination is difficult in species such as sheep, where the cervix efIectively blocks catheter
insertion, thereby reducing the number of spermatozoa reaching the uterus and oviduct. In other
species, such as cattle and pigs, insemination devices can easily pass through the cervix, increasing
the efficiency of sperm deposition. Entry of sperm into the oviduct is, however, restricted by the
utero-tubal junction; sperm showing defective motility or advanced capacitation-like phenomena
may be selectively prevented from entering the oviducto Direct intrauterine deposition of sperm by
laparoscopy is often more successful than intra- or transcervical insemination, demonstrating that
defective sperm transport is a major consequence of sublethal cryoinjury. Seminal volume and
sperm numbers in the ejaculate reflect anatomical difIerences in the female tract. In pigs, semen
volume exceeds 300 ml and normally causes uterine distention and pressure; consequently, large
volumes of fluid and large numbers of cryopreserved spermatozoa are required for high
conception rates. In cattle and many large ungulates semen volume is <10 ml and is naturally
deposited into the anterior vagina. Consequently, efficiency of sperm cryopreservation can be
lower in these species without significant loss of fertility, because smaller absolute numbers of

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fertile spennatozoa can stilI achieve high conception rates if deposited beyond the cervix, While
these remarks are especially relevant to large eutherian mammals, the principIes also apply to
other groups of species. Marked differences in femaIe reproductive tract anatomy are especially
notable in the marsupials, where two separate uteri are each connected to lateral vaginae by twin
cervices.
Watson (49) linked observations tbat exposure of spermatozoa to low temperatures
shortens their capacitation time, with changes in membrane lipid architecture, membrane
permeability and the reduced efficiency of homeostatic enzymes, especially those extruding
calcium ions. Together, these changes resemble capacitation, and are likely to reduce long-term
sperm viability and alter physiological functions such as motility. Capacitation-like changes may
also influence the nature of interactions between sperrnatozoa and immune cells or epithelial cells
in the female reproductive tracto Within tbis context, species differences in sperm membrane
physiology, structure and biochemistry would probably induce different responses to cooling and
freezing, thus influencing the subsequent fertility of spermatozoa.
Cryopreservation is directly letbal to a significant proportion (circa 50%) of spermatozoa
in a typical semen sample and consequently the number of sperrnatozoa required for successful
inseminations has to be increased aboye tbat needed for fresh spermatozoa. However, the
requisite increase may be ten or a hundred fold, suggesting tbat many spermatozoa, although live
and motile, are unable to reach and fertilize the egg. The avoidance of sperm transport effects by
laparoscopic insemination shows tbat some of the inseminated spermatozoa can achieve
fertilization, even though they may bave sustained sublethal darnage impairing their ability to
penetrate cervical mucus or pass through the utero-tubal junction. Whether these spermatozoa
possess innate genetic advantages over other sperm in the ejaculate is unclear. There is also a
strong possibility tbat sperrnatozoa selectively bind to oviductal epithelia, where they are stored
and protected until ovulation occurs (for review, see 41).
As the source of semen and the collection method affects sperm concentration, motility
and probably also fertility, differences in ease of collection can bave major implications for the
success of cryopreservation. The use of artificial vaginae and trained semen donors provides the
best and most natural samples for cryopreservation. Electroejaculation under general anaesthesia
is widely used with wild animals, but the quality of semen obtained is variable. Recovery of semen
from the vagina after natural mating is sometimes useful where electroejaculation is inadvisable;
however, vaginal secretions are detrimental to sperrnatozoa. Although seminal plasma contains
many components wbich are beneficial to sperrnatozoa and may complete their maturation
process, it should be noted tbat during natural ejaculation the sperrnatozoa do not usually stay in
prolonged contact with it. This is, however, an almost inescapable drawback of electroejaculation
and spermotoxic effects of seminal plasma bave been noted.
Recent Developments in the Preservation ofMale Gametes
When the aim of sperm preservation is for use with artificial insemination, few options are
available except to freeze appropriately high numbers of sperrnatozoa and ensure tbat they retain
functionality as much as possible. Ibis is true for insemination procedures applied to agricultura!

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species on an industrial scale, or with wild species where the ability to recover oocytes and
perfonn intensive procedures is limited by practical and ethical considerations, However, the
development of techniques for helping the penetration of eggs by functionally defective
spermatozoa (e.g., zona drilling, subzonal insemination and intracytoplasmic sperm injection
(ICSI)) has, for sorne purposes, eliminated the need to retain physiological integrity in stored
spermatozoa. Inital observations that isolated spenn nuclei can support embryonic development
in cattle, human and hamster (19,21) prompted more recent investigations on mouse sperm
cryopreservation without cryoprotectant.
This direct approach to mouse sperm preservation was tested (45, 46) and shown to be
feasible. Spermatozoa were suspended in culture media, with and without the inclusion of
raffinose as a debydrating agent. Aliquots (50 IlL) ofsperm suspension were dispensed into 1 mL
cryotubes tbat were then placed in a freezer (-20C, -50C or directly into liquid nitrogen).
Viability assays confirmed tbat these spermatozoa had damaged plasma membranes and were
therefore immotile. ICSI performed with tbese spermatozoa, baving first separated the heads and
flagellae, resulted in 95-100% of injected oocytes being activated and fertilized normally wbatever
the final storage temperature. The authors noted sorne differences among the various
modifications ofthe sperm freezing procedure, but qualified their remarks by saying tbat all oftbe
treatments were successful, and tbat females produced litters of normal size with no apparent
abnormalities. In view of these results and the limitation tbat cryopreserved mouse spermatozoa
must, in any case, be used for in vitro fertilization, it seems likely tbat storage of mouse
spermatozoa may in the future be undertaken routinely by this simple, glycerol-free, method.
Intracytoplasmic sperm injection is being increasingly adopted for human clnical use in
special cases where sperm quality is very poor or sperm numbers are very low due to testicular
failure or epididymal obstruction. In the last two years at least twenty-five publications bave
described the use of ICSI with immotile human testicular or epididymal spermatozoa recovered
from frozenltbawed tissue samples (e.g., 1,11). ICSI seems to offer an effective means ofusing
these recovered spermatozoa. To date, no deleterious effects bave been attributed to the
cryopreservation technique itself. Despite the intention to use ICSI, sorne authors bave been
concemed with optimizing the post-tbaw recovery of sperm motility in minced testicular tissue.
Glycerol has therefore been included as a cryoprotectant for this purpose (11). Following similar
logic, Cohen et al. (6) developed an ingenious technique for the cryopreservation of single
spermatozoa. Their technique involved encapsulation of spermatozoa, individually or in small
groups, within empty zonae pellucidae preloaded with cryoprotective media. After
cryopreservation the spermatozoa were recovered with considerable success (14/15 spermatozoa
recovered). Ten of these spermatozoa were used for ICSI and eight successfully fertilized the
oocytes. Given tbat ICSI bypasses many physiological processes involved in fertilization, the
justification for using cryoprotectants for the preservation of motility and cellular integrity seems
questionable. Nevertheless, recent studies bave indicated tbat human fertilization and pregnancy
rates obtained using ICSI are considerably reduced if immotile, rather than motile, spermatozoa
are used. Shibahara et al. (39) obtained high fertilization rates when epididymal spermatozoa
showing normal motility were used (68.6% and 68.4%; fresh and frozen spenn, respectively).
However, the fertilization rate was halved (31.6%) when immotile frozen spermatozoa were used.
This result suggests tbat a correlation exists between motility and other important qualities of

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individual spermatozoa. Use of cryoprotectants to maintain optimal sperm motility seems

therefore highly advisable. However, there is an alternative way of viewing this data which is
reminiscent of the arguments used in support of sperm competition theory (18). This suggests that
the spermatozoa which are most effective in reaching and fertilizing oocytes also convey superior
genetic qualities into the next generation. The ICSI studies cited above imply that cryoinjuries
affecting motility may be more severe when nuclear structures are defective; alternatively they
could indicate that individual spermatozoa with the most eryosuseeptible motility-generatng
struetures carry the poorest quality DNA. Given the complex meehanisms whieh have evolved in
the female reproductive traet to seleet the fertilizing spermatozoa from among the millions present
in the ejaculate, the absence of selectivity when performing ICSI should be a matter of concern
and vigilanee, whether using fresh or frozen spermatozoa.
An extension to the use of ICSI with cryopreserved testicular spermatozoa is the direet
intracytopIasrnic injeetion of round spermatids and even spermatocytes into oocytes. This
approach was pioneered using mouse testicular cells (35), and has since been applied to human
patients. A potential demand for round spermatid cryopreservation thus exists; however, as they
are only likely to be used with ICSI, the retention of nuclear integrity may be the only important
requirement Nevertheless, successful cryopreservation of human spermatogenic cells has been
reported (1). In a parallel development the feasibility of cryopreserving rat sperrnatogonia and
transpIanting them into irradiated mouse testes was demonstrated experirnentally (5). A standard
somatic eell cryopreservation procedure was used in this case. The eryopreservation probably
succeeded because, being stem cells, sperrnatogonia can multiply to regenerate a viable population
even if many sustain lethal damage. A1though the original aim of the sperrnatogonial
transpIantation was the study of spermatogenesis, this approach clearly offers sorne potential for
the reseue of the male germline in, for example, the preservation of genetic resources in
agrieulture or wildlife conservation. At present, if spermatozoa are unavailable, there are no other
options for the storage of germpIasm from genetically valuable individuals. Considerable care will
be required to ensure that functional markers of DNA integrity are developed for monitoring the
effectiveness of this approach.
CONCLUSIONS
Since the literature on sperm eryopreservation is vast and has been reviewed extensively
(21,47-49), the aim ofthis review was to explore sorne aspects ofthe topie which are crucially
important but frequently overlooked. One of my intentions in writing this article was to emphasize
that while preserving sperm integrity during freezing and thawing is clearly important, the in vivo
fertilizing ability of cryopreserved spermatozoa also depends 00 the biologieal eontext in which it
is used. Thus, anatornical features of the femaIe reproductive traet, and the ability to detect or
control ovulation aecurately, will ultimately govern the sueeess of artificial inserninations just as
powerfully as the percentage of acrosome-intact or motile spermatozoa. In fact, it can be argued
that there will nearly always be enough spennatozoa which are capable of fertilizing the egg,
provided they are able to reach it at the appropriate time. Another intention was to develop the
idea that one potentially fruitful route to understanding the basis of cryoinjury may be through the
study of differences between individuals of the same species. Studies in the mouse are beginning
to reveal genetically-based trends in the success of sperm cryopreservation procedures, and rnight

Theriogenology

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ultimately pro vide clues which point to specific differences between strains. Ideally, one would
hope to see that the differences corre late with the presence of particular gene products in the
spennatozoa, such as membrane channels or structural proteins. The extraordinarily rapid
developments which are now taking place in functional genomics, especially of the mouse and the
pig, mean that this is now a realistic avenue of study.
REFERENCES
1. Aslam 1, Fishel S. Short-term in-vitro culture and cryopreservation of spennatogenic cells
used for human in-vitro conception. Hum Reprod 1998;13:634-638.
2. Beatty RA, Stewart DL, Spooner RL, Hancock JL. Evaluation by the heterospermic
insemination technique of the differential effect of freezing at -196C on the fertility of
individual bull semen. J Reprod FertilI976;47:377-379.
3. Bwanga CO, Ekwall H, Rodriguez-Martinez H. Cryopreservation of boar semen. III.
Ultrastructure of boar spermatozoa frozen ultra-rapidly at various stages of conventional
freezing and thawing. Acta Vet Scand 1991;32:463-471.
4. Castellini C, Bizzarri AA, Cannistraro S. Water volume o rabbit spennatozoa measured by
electron paramagnetic resonance at different temperatures. 6th World Rabbit Congress
Toulouse 1996;55-58.
5. Clouthier DE, Avarbock MR, Maika SD, Hammer RE, Brinster RL. Rat spennatogenesis in
mouse testis. Nature Lond 1996;381:418-421.
6. Cohen J, Garrisi GJ, Congedo-Ferrara TA, Kieck KA, Schimmel TW, Scott RT.
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