Journal of Andrology, Vol. 21, No.
1, January/February 2000
Copyright q American Society of Andrology
Semen Cryopreservation in
Domestic Animals: A Damaging and
Capacitating Phenomenon
JANICE L. BAILEY, JEAN-FRANÇOIS BILODEAU,
AND NATHALY CORMIER
From the Centre de Recherche en Biologie de la
Reproduction, Département des Sciences Animales,
Université Laval, Québec, Canada.
The genetic improvement of agriculturally important spe-
cies and disease control are of fundamental importance to
the success of a sustainable agri-food industry. In this
sense, artificial insemination (AI) is arguably the most
important tool contributing to the advancement of modern
animal production. Through AI, 1 ejaculate from a ge-
netically superior male can be used to impregnate multi-
ple females to maximize the distribution of agriculturally
favorable genes. As well, AI eliminates physical contact
between animals, thus limiting the spread of sexually
transmitted diseases. Successful semen cryopreservation
enhances these advantages of AI over natural breeding.
Long-term storage facilitates semen transport over dis-
tances, permits the quarantine of semen, and enables ex-
tended use of superior germplasm, even after the sire’s
death. For agriculturally important animals, semen cryo-
preservation is an established industry worldwide, partic-
ularly for dairy cattle. Genome resource banking to pre-
serve the biodiversity of endangered species or valuable
transgenic lines also would benefit from sperm cryopres-
ervation.
For many mammals; however, effective semen cryo-
preservation is not a reality because a large number of
sperm are apparently infertile following freezing and
thawing. Compared to fresh, 8 times more cryopreserved
bovine sperm were required to achieve equivalent fertil-
ization rates in vivo (Shannon and Vishwanath, 1995).
Although AI in pigs with fresh, cooled semen is increas-
ingly popular, the fertility of cryopreserved semen re-
mains about half that of fresh semen (Crabo, 1991). Fol-
lowing either transuterine or oviductal insemination of su-
perovulated ewes, conception rates and percentage of fer-
Correspondence to: Janice L. Bailey, PhD, Centre de Recherche en
Biologie de la Reproduction, Département des sciences animales, Univ-
ersité Laval, Québec, Québec, Canada G1K 7P4 (e-mail:
janice.bailey@crbr.ulaval.ca).
Received for publication July 23, 1999; accepted for publication Sep-
tember 22, 1999.
1
Minireview
tilized ova with frozen-thawed ram semen were
approximately 20% less than with fresh semen (Maxwell
et al, 1993). The fertility of cryopreserved semen plum-
mets to unacceptable levels with vaginal or cervical in-
semination (Eppelston and Maxwell, 1993). In stallions,
satisfactory techniques for semen cryopreservation have
not yet been developed (Samper and Morris, 1998). In-
terest in conserving transgenic lines has stimulated a
surge of effort toward mouse sperm conservation; how-
ever, in vitro fertilization rates with cryopreserved epi-
didymal sperm were, at best, 62% that of unfrozen con-
trols (Storey et al, 1998).
At present, it is generally accepted that the consequenc-
es of sperm cryoinjury caused by the cryopreservation
procedure are impaired transport and poor survival in the
female reproductive tract (Salamon and Maxwell, 1995).
This concept has led to the routine use of oviductal in-
semination by laparoscopy rather than vaginal or even
transcervical insemination in sheep. Similarly, surgical in-
semination of cryopreserved porcine semen directly into
the oviducts achieved fertility rates comparable to those
with fresh semen (Polge et al, 1970). When the interval
between insemination and ovulation is reduced, fertiliza-
tion rates increase (Parrish and Foote, 1986; Kemp and
Soede, 1997), supporting the hypothesis that sublethal
cryodamage is an effect of the freeze-thaw procedure. The
goal of this minireview is to describe recent findings on
the mechanisms by which cryopreservation initiates
‘‘cryo-capacitation,’’ thus reducing the functional life
span of sperm.
Sperm Damage Due to Cryopreservation
The sperm plasma membrane is the primary site of dam-
age induced by cryopreservation (Hammerstedt et al,
1990, Parks and Graham, 1992, Watson, 1995). Cryomi-
croscopic examination of ram sperm loaded with a marker
for membrane integrity revealed that exposure to low tem-
peratures followed by warming differentially affected the
plasma membranes over the principal-piece, midpiece,
and head, with the head membrane inevitably damaged
(Holt and North, 1994). Further investigation showed sig-
nificant membrane permeabilization after sperm were ex-
posed to a high salt concentration followed by restoration
of osmotic equilibrium as would be generated during a
freeze-thaw cycle. Because of the temperature and os-
2 Journal of Andrology · January/February 2000
motic effects, both freezing and thawing induce tremen-
dous alterations in cell water volume, which confer con-
siderable mechanical stress on the cell membranes (Ham-
merstedt et al, 1990; Noiles et al, 1993, 1995).
Noiles et al (1995) observed a temperature-dependent
discontinuity of the water permeability of mouse sperm
plasma membranes between 4 and 08C. These data sug-
gest that a membrane-phase transition occurs between 4
and 08C in mouse sperm during the freezing process,
which is associated with elevated membrane fragility due
to imbrittlement. At physiologic temperatures, freeze-
fracture electron micrographs of bull and boar head plas-
ma membranes exhibit a random distribution of mem-
brane proteins (de Leeuw et al, 1991). On cooling, phase
transitions change sperm membrane ultrastructure such
that particle-free regions in the bull and protein aggre-
gation in the boar replace the random distribution. Be-
cause boar sperm are known to be sensitive to cooling,
the difference between these species may indicate differ-
ing intensities of damage. These ultrastructural modifi-
cations are not fully reversible on rewarming. Head plas-
ma membranes isolated from cryopreserved porcine
sperm are less dynamic than those from unfrozen sperm
and do not undergo the fluidity changes observed in mem-
branes from fresh or even cooled cells (Buhr et al, 1989).
Morphometry measurements indicate that cryopreserved
bull sperm have reduced head size compared with un-
frozen sperm, possibly reflecting permanent modification
of membrane architecture (Gravance et al, 1998). Cryo-
preservation does affect lipid composition and organiza-
tion of sperm plasma membranes in rams (Hinkovska-
Galcheva et al, 1989) and boars (Buhr et al, 1994). Trans-
bilayer-phospholipid asymmetry also is disrupted in ram
sperm plasma membranes after cryopreservation (Hin-
kovska-Galcheva et al, 1989; Muller et al, 1999). Ultra-
structural damage to membranes due to cryopreservation
destabilizes them, predisposing sperm to gross morpho-
logic defects, such as missing and abnormal acrosomes.
Phase transitions and other ultrastructural modifications
of the plasma membranes during cooling and rewarming
may play a role in the poor fertility of cryopreserved
sperm. It seems plausible that reorganization of sperm
membrane lipids disturbs the lipid-lipid and lipid-protein
interactions required for normal membrane function
(Parks and Graham, 1992). Sperm from domestic mam-
mals do not survive rapid cooling to 08C (cold shock).
Therefore, sperm are always protected from sudden tem-
perature shifts from the time of semen collection and dur-
ing processing until the cryoprotectant is added. Plasma
membrane disruption due to cooling or freezing favors
the loss of cations and enzymes from sperm (Harrison
and White, 1972), providing some explanation for early
observations that cold shock irreversibly depresses sperm
motility and metabolic activity (White, 1993). Cold shock
also destroys the selective permeability of sperm mem-
branes to calcium, thus leading to excessive intracellular
levels, which reduce motility and lead to necrosis (Simp-
son and White, 1986; Robertson et al, 1990).
Cryopreservation (in the presence of a cryoprotectant,
typically glycerol) is considered to be a more moderate
treatment than cold shock, since a subpopulation of sperm
survives and maintains fertilizing capacity, notwithstand-
ing an overall reduction in the percentage of motile and
viable cells. In contrast to what might be a logical asso-
ciation, the computer-assessed motility of bovine sperm
immediately after thawing is not correlated with fertility
(Bailey et al, 1994). Furthermore, despite preservation of
adequate motility, cryopreserved human sperm exhibit
significant membrane damage as indicated by subnormal
hypo-osmotic swelling tests (Check and Check, 1991).
Indeed, after cryopreservation, surviving bull and human
sperm contain more intracellular calcium than before, re-
flecting impaired membrane selective permeability mech-
anisms (Bailey and Buhr, 1994; McLaughlin and Ford,
1994).
Once sperm approach the site of fertilization, they at-
tach transiently to oviductal epithelial cells, experience
capacitation and hyperactivation, bind to the oocyte zona
pellucida, undergo the acrosome reaction, penetrate the
zona, and finally fuse with and penetrate the oolemma.
Impaired sperm membrane function due to cryopreser-
vation inevitably affects each of these processes and so
diminishes the likelihood of successfully fertilizing an oo-
cyte in vivo. Certainly poorly motile sperm are less likely
to arrive at the site of fertilization in vivo or penetrate the
oocyte vestments. Moreover, the motile sperm population
itself is adversely affected by cryopreservation; when
equal numbers of motile cells are inseminated, the fertility
of fresh semen is superior to that of frozen-thawed semen
(Watson, 1995).
The structural reorganization of sperm head plasma
membranes after cryopreservation appears to disrupt the
ability of the sperm to interact normally with cells of the
female genital tract. Dobrinski et al (1995) used differ-
ential fluorochrome labeling to distinguish the binding of
fresh and cryopreserved stallion sperm to oviductal epi-
thelial cells in vitro. Fewer cryopreserved sperm attached
to the cells than fresh semen, and similar findings were
obtained with fresh and frozen-thawed bull sperm (Gold-
man et al, 1998). Moreover, cryopreserved equine sperm
were less able to bind homologous zonae pellucidae than
those stored at room temperature (Dobrinski et al, 1995).
In pigs, sperm stored at 188C penetrated fewer zona-free
hamster eggs than did fresh sperm, and penetration by
cryopreserved sperm was markedly compromised (Clarke
and Johnson, 1987). Reduced sperm binding is likely a
consequence of membrane injury, possibly by structural
Bailey et al · Mechanisms of Sperm Cryo-Capacitation
damage to the sperm receptors or by incomplete receptor
aggregation.
The elevated intracellular calcium levels of cryopre-
served sperm and their reduced capacity to maintain nor-
mal concentrations of this cation (Bailey and Buhr, 1993,
1994; McLaughlin and Ford, 1994) may also partly ex-
plain the poorer fertility of sperm post-thaw. Intracellular
calcium levels increase in sperm during capacitation (Par-
rish et al, 1999), hyperactivation (Suarez et al, 1993), and
the zona pellucida–induced acrosome reaction (Bailey
and Storey, 1994; Florman, 1994). Recent research sug-
gests that the calcium regulatory mechanisms during ca-
pacitation (Parrish et al, 1999) and the acrosome reaction
(Florman et al, 1998) are complex, involving intracellular
stores and voltage-dependent calcium channels. Disrup-
tion of capacitation and/or the acrosome reaction would
severely compromise the fertilizing potential of sperma-
tozoa, which may account for observations that the in
vivo fertility rates of cryopreserved bovine semen are cor-
related with the ability of the sperm to moderate internal
calcium levels (Bailey et al, 1994).
Mechanisms of Membrane Damage
Much of cryopreservation sperm damage depends on the
structural stability of the plasma membrane (de Leeuw et
al, 1993). Differences among species in the sensitivity of
their sperm to cooling are largely attributable to compo-
sitional variations of the sperm plasma membranes. The
susceptibility of the plasma membrane to undergo lipid-
phase transitions during cooling is inversely related to the
proportion of cholesterol present (Drobnis et al, 1993).
Lower cholesterol levels are present in bull and ram
sperm, which are considered to be sensitive to cooling,
than in rabbit and human sperm, which are less suscep-
tible. Moreover, the effectiveness of glycerol as a cryo-
protectant is partially attributed to its ability to prevent
some of the phase transitions during cooling by increasing
the water permeability and fluidity of sperm plasma mem-
branes (Noiles et al, 1995). Susceptibility to cold tem-
peratures also seems to be linked to a high ratio of un-
saturated to saturated fatty acid content of the sperm plas-
ma membrane. Again, species can be segregated in 2 clas-
ses, those with high levels of saturated fatty acids and
those with lower quantities (White, 1993). Bull, ram, and
boar sperm (sensitive), have a higher ratio of unsaturated
to saturated fatty acids (.2.5), whereas more resistant
sperm from rabbits, dogs, and human beings have lower
ratios (;1).
An increasing number of studies show that reactive ox-
ygen species (ROS) play an important role in fertility/
infertility. Several reports indicate significant physiologic
roles of ROS during normal sperm function, including
hyperactivation, capacitation and the acrosome reaction,
and zona binding (Kodama et al, 1996; de Lamirande et
3
al, 1997). Conversely, when the balance between ROS
production and detoxification by antioxidants is disrupted,
an excess of ROS creates oxidative stress. ROS such as
H2O2 are known to arrest motility and block oxidative
metabolism in sperm (Tosic, 1947). Also, ROS decrease
oocyte penetration by sperm and block sperm-egg fusion
in the mouse via a mechanism involving oxidation of
sperm -SH groups (Mammoto et al, 1996). Sperm DNA
damage by ROS also has been reported, which has serious
consequences for postfertilization development (Aitken et
al, 1998).
The generation of ROS by several potential sources
occurs during cryopreservation. Increased ROS produc-
tion by both human sperm and seminal leukocytes during
cooling to 48C was reported by Wang et al (1997). In
somatic cells, ROS leakage from the mitochondrial re-
spiratory chain has been evaluated at 2 to 5% (Boveris,
1977), and this is probably a major source of ROS pro-
duced by sperm. Contaminating leukocytes in the semen
can produce large amounts of ROS if stimulated (Krausz
et al, 1992). Aitken et al (1997) hypothesized that an
NADH/NADPH oxidase could exist on sperm as occurs
on many other cell types. Specifically, the activation of
an aromatic amino acid oxidase following the death of
ram and bull sperm has been identified as a major source
of ROS production in the semen of these animals (Tosic,
1947; Upreti et al, 1998). The release of this oxidase from
dead sperm in egg yolk extender reduces the motility and
viability of the remaining living bull sperm (Shannon and
Curson, 1972).
Unsaturated fatty acids, which predominate in sperm
membranes, are susceptible to peroxidation (Halliwell and
Gutterridge, 1984), and the consequences are numerous,
ranging from membrane damage, inhibition of respiration,
and leakage of intracellular enzymes (White, 1993). Lipid
peroxidation derivatives, such as 4-hydroxynonenal, have
been shown to be mutagenic and abolish sperm motility
in ram semen (White, 1993). Slaweta et al (1988) reported
that lipid peroxidation in bull sperm increases after cryo-
preservation. Moreover, Trinchero et al (1990) observed
that frozen-thawed bull sperm are more easily peroxidized
than fresh sperm. Recent studies indicate that cryopres-
ervation decreases the antioxidant defenses of whole se-
men. Loss of superoxide dismutase activity occurs in
cryopreserved human and bull semen when compared
with fresh semen (Lasso et al, 1994; Bilodeau et al, in
press). Moreover, cryopreservation reduced bull sperm
glutathione levels by 78% (Bilodeau et al, in press).
Observations that addition of antioxidants to semen im-
proves sperm quality provide indirect evidence for the
damaging effects of ROS on sperm function. Studies to
preserve unfrozen semen revealed that controlling oxi-
dation by exogenous antioxidants in the extender, such as
catalase, greatly helped maintain sperm quality (Foote,
4 Journal of Andrology · January/February 2000
1967). Inclusion of natural antioxidants such as a-to-
copherol and ascorbate had a protective effect on meta-
bolic activity and cellular viability of cryopreserved bo-
vine sperm (Beconi et al, 1991; 1993).
Capacitationlike Changes Due to Cryopreservation
Watson (1995) suggested that cryopreservation-induced
modifications to sperm membranes make them more re-
active to their environment after thawing such that cryo-
preserved sperm are in a partially capacitated state. In-
deed, numerous groups have reported that cooled or cryo-
preserved sperm exhibit capacitationlike behavior and,
even a decade ago, it was observed that freezing and
thawing reduced the time required for tiger sperm to pen-
etrate zona-free hamster eggs (Byers et al, 1989). More
recently, Pérez et al (1996) showed that cryopreserved
ram sperm undergo capacitation more quickly than fresh
controls as assessed by reactivity to calcium ionophore
and the chlortetracyline (CTC) fluorescent assay. CTC
fluorescence also revealed a greater proportion of pattern
B (‘‘capacitated’’; Ward and Storey, 1984) sperm due to
cooling and immediately after thawing in mice (Fuller and
Whittingham, 1996), bull (Cormier et al, 1997), ram (Gil-
lian et al, 1997), and boar (Maxwell and Johnson, 1997).
Furthermore, sperm were confirmed to be functionally ca-
pacitated by cooling or cryopreservation, as reflected by
their ability to fertilize homologous oocytes in vitro with-
out any pretreatment normally required by uncooled
sperm (Cormier et al, 1997; Fuller and Whittingham,
1997).
The mechanism by which this cryo-capacitation occurs
is not fully understood and is complicated by the fact the
onset of normal capacitation has yet to be elucidated. Ca-
pacitation of mammalian sperm is associated with reor-
ganization of the head plasma membranes due to phos-
pholipid redistribution and cholesterol removal (Langlais
and Roberts, 1985; Lin and Kan, 1996). Watson (1995)
speculated that cryopreservation introduces a sublethal
modification of the sperm membranes, which renders
them more sensitive to the environment after thawing. As
outlined above, cooling and cryopreservation also modify
sperm membrane architecture and behavior. Similarities
in the membrane changes associated with capacitation and
cooling or cryopreservation are evident, based on the in-
creased incidence of sperm displaying the CTC pattern B
after either of these phenomena. Furthermore, based on
flow cytometric analysis of CTC stained sperm, Maxwell
and Johnson (1997) suggested that pattern B reflects
membrane destabilization and calcium influx to the cell.
Thus, the CTC pattern B fluorescence induced by cooling
and cryopreservation appears to be related to sperm ca-
pacitation in the functional sense, as these treatments fa-
vor sperm fertilization in vitro.
In bovine sperm, an increase in intracellular calcium
Sperm changes associated with capacitation and cryopreservation*
Capacitation Cryopreservation
CTC fluorescence pattern B CTC fluorescence pattern B
Plasma membrane reorganiza- Plasma membrane reorganiza-
tion and fluidization tion and destabilization
Elevated intracellular calcium Elevated intracellular calcium
Generation of reactive oxygen Generation of reactive oxygen
species species
cAMP-mediated protein tyrosine Appearance of tyrosine-phos-
phosphorylation phorylated proteins
Able to fertilize oocytes in vitro Able to fertilize oocytes in vitro
* CTC indicates chlortetracycline; cAMP, cyclic adenosine monophos-
phate.
accompanies heparin-induced capacitation in vitro (Par-
rish et al, 1999), although its mechanism of entry is un-
clear. As previously discussed, restructured membranes
and altered lipid-protein associations are thought to favor
calcium influx during cryopreservation. Visconti et al
(1998) speculated that during capacitation, membrane
modifications stimulate adenylyl cyclase to initiate cyclic
adenosine monophosphate–mediated tyrosine phosphor-
ylation of sperm proteins. Furthermore, elevated sperm
calcium levels are thought to trigger an intracellular sig-
naling cascade that has recently been associated with ca-
pacitation. To test whether cryopreservation induces the
signal transduction events associated with capacitation,
the presence of phosphotyrosine-containing proteins was
evaluated in bull sperm immediately after thawing. In
support of this hypothesis, a series of tyrosine phosphor-
ylated proteins was evident in cryopreserved sperm im-
mediately after thawing (Bailey and Bérubé, 1998). In
contrast, fresh bovine sperm require incubation for at least
4 hours with heparin for these phosphorylated proteins to
be visualized (Galantino-Homer et al, 1997).
Given their importance in fertility/infertility, a role for
ROS in sperm cryo-damage is not unlikely. In vitro ca-
pacitation and tyrosine phosphorylation of sperm proteins
are correlated with superoxide anion generation in human
sperm (de Lamarinde et al, 1998). Conversely, both su-
peroxide dismutase and catalase inhibit capacitation and
tyrosine phosphorylation (Leclerc et al, 1997). It is tempt-
ing to speculate that cryo-capacitation is at least partly
induced by ROS activity generated during sperm pro-
cessing.
Conclusions
In summary, it is clear that cryopreserved and capacitated
sperm share several characteristics (Table 1). Harrison
(1996) described capacitation as a ‘‘window of destabi-
lization’’ within which sperm acquire fertilizing ability
but are susceptible to membrane degeneration and spon-
taneous acrosome reactions should they not fertilize. In
Bailey et al · Mechanisms of Sperm Cryo-Capacitation
addition, cooling and cryopreservation reduce the hetero-
geneity of the sperm population. Consequently, a number
are killed or grossly damaged, leaving a subpopulation of
partially or fully capacitated sperm. In species where se-
men can be frozen with some success, a noncapacitated
subpopulation with good fertilizing potential also remains
after thawing. Cryo-capacitation would then produce a
sperm subpopulation with a shortened life span in vivo,
effectively reducing the fertilization efficiency of the pop-
ulation as a whole. Based on this theory of cryopreser-
vation-induced damage, future studies toward improving
the fertility of frozen-thawed semen should endeavor to
either prevent or reverse cryo-capacitation. Possible ap-
proaches could include improving membrane stability
during cryopreservation to prevent elevated sperm calci-
um levels and continued efforts to control ROS produc-
tion during processing. Finally, a further understanding of
fundamental cryobiology is needed to develop optimal
cryopreservation conditions for sperm of individual spe-
cies.
Acknowledgments
The authors thank Drs Bayard Storey, Robert Sullivan, and Marc-André
Sirard for critically reading this manuscript and providing helpful com-
ments.
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