Semen cryopreservation: Successes and persistent

problems in farm species

J. Bailey1, A. Morrier, and N. Cormier
Centre de recherche en biologie de la reproduction, Département des sciences animales,
Université Laval, Québec, Québec, Canada G1K 7P4 (Janice.Bailey@crbr.ulaval.ca).
Received 17 February 2003, accepted 20 May 2003.

Bailey, J., Morrier, A. and Cormier. N. 2003. Semen cryopreservation: Successes and persistent problems in farm species. Can. J.
Anim. Sci. 83: 393–401. Artificial insemination has arguably been the most important practice contributing to the advancement of ani-
Can. J. Anim. Sci. Downloaded from cdnsciencepub.com by 103.162.62.46 on 11/15/22

mal production. The numerous advantages of artificial insemination are augmented when the semen is cryopreserved and conserved for
long periods. Unfortunately, the utility of cryopreserved semen is limited because for most mammals, even cattle, a considerable pro-
portion of sperm loose their fertility during freezing-thawing. In many species, this loss of fertility is substantial, rendering cryopreserved
semen impractical for routine use. Our goals are to characterize the nature of the sublethal sperm damage caused by cryopreservation,
and to develop a method to improve the functional fertility of thawed semen. This review describes our theory that sperm are prematurely
activated by cryopreservation (“cryo-capacitation”) and discusses strategies for preventing this damage.

Key words: Sperm, artificial insemination, capacitation, fertility

Bailey, J., Morrier, A. et Cormier, N. 2003. La cryoconservation de la semence : les succès et les problèmes persistants chez les
espèces de la ferme. Can. J. Anim. Sci. 83: 393–401. L’insémination artificielle a grandement contribué à l’avancement des pro-
ductions animales. Les nombreux avantages qu’offre cette technique sont augmentés lorsque la semence est cryoconservée et entre-
posée pour de longues périodes. Malheureusement, l’utilisation de la semence cryoconservée est limitée puisque chez la majorité
For personal use only.

des mammifères, incluant la vache, une large proportion des spermatozoïdes perdra leur motilité pendant le processus de congéla-
tion-décongélation. Chez plusieurs espèces, cette perte de fertilité est suffisamment substantielle pour rendre impraticable l’utilisa-
tion routinière de la semence cryoconservée. Les objectifs de notre laboratoire portent sur la caractérisation des dommages sous
létaux subis par les spermatozoïdes lors de la cryoconservation et le développement de méthodes permettant d’améliorer la fertilité
des spermatozoïdes décongelés. Cette revue décrit notre théorie selon laquelle les spermatozoïdes seraient prématurément activés
par la cryoconservation (“cryo-capacitation”). Différentes stratégies permettant de prévenir ce dommage y sont également abordées.

Mots clés: Spermatozoïde, insémination artificielle, pouvoir fécondant, capacitation

An overview of the use of artificial insemination
Artificial insemination (AI) has arguably been the most impor-
tant practice contributing to the advancement of animal pro-
duction. The benefits of AI are numerous, including better herd
health, and improved safety and less costs resulting from fewer
male animals on the farm. However, the primordial benefit is
an accelerated rate of genetic improvement stemming from the
insemination of multiple females with the semen from a male
of superior genetic quality. In Canada, the dairy and swine
industries have profited from the genetic gains facilitated by
AI; about 75–80% of calves in Canada (Semex Alliance, per-
sonal communication) and 85–90% of pigs in the province of
Quebec (Jean-Paul Laforest, Université Laval, personal com-
munication) are produced through AI. Other industries such as
beef, sheep, goat and even fish are well positioned to use this
selection tool to optimize production.
In particular, dairy production worldwide has dramatically
improved because of the highly successful distribution of
Presented at the session “Améliorer les productions ani-
males grâce aux technologies reproductives. Improving
Animal Production with Reproductive Physiology” during
the CSAS/ADSA/ASAS 2002 Joint Meeting, Québec City,
QC, 20–25 July 2002.
393
frozen semen. Indeed, the advantages of AI are augmented
when the semen can be conserved for long periods and trans-
ported great distances, enabling the use of semen when the
female is in oestrus, even long after the sire has died. Semen
cryopreservation is also a valuable tool outside of animal pro-
duction. Numerous assisted reproduction techniques in human
fertility clinics require frozen-thawed semen (Mortimer 1994).
Cryopreserved semen is an important aspect of genome
resource banking to conserve the biodiversity of endangered
animal species. Controlled reproduction in these animals also
benefits from cryopreserved semen (Pukazhenthi et al. 1999);
semen can be collected from males in their natural habitat and
frozen for later use with captive females. In the laboratory,
transgenic mouse lines are economically preserved in the form
of cryopreserved sperm (Storey et al. 1998).
Limitation of the Use of Cryopreserved Semen
Unfortunately, the full potential of cryopreserved semen is
limited. In most mammals, a considerable proportion of
Abbreviations: AI, artificial insemination; CTC, chlorte-
tracycline; NCM, non-capacitating medium, EYT, egg
yolk-Tris diluant, EYTG, EYT containing 7% glycerol,
EYTG-4, EYTG cooled to 4°C
 394 CANADIAN JOURNAL OF ANIMAL SCIENCE
sperm loose their fertility during cryopreservation, render-
ing frozen-thawed semen impractical for routine use.
Porcine sperm are notoriously sensitive to cold temperatures
and the fertility of frozen-thawed pig semen (farrowing rates
between 40 and 70% and 8–10 piglets per litter) is unac-
ceptably low for commercial application (Hofmo and
Grevle 1999). In sheep, satisfactory conception rates with
cryopreserved semen are only achieved when laporoscopy is
used to deposit sperm directly in the oviduct, indicating that
cryopreserved sheep semen cannot effectively transit the
female reproductive tract (Salamon and Maxwell 1995).
When using cryopreserved stallion semen, careful mare
management to ensure sound breeding condition and the use
of ovulatory inducing agents to reduce the interval between
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insemination and ovulation are recommended to counter the
reduced number of sperm reaching the oviduct and their
short life span (Samper 2001). Even bovine semen suffers a
major loss, despite that it is widely considered to be cryo-
resistant. Shannon and Vishwanath (1995) showed that
eight-to tenfold more frozen-thawed than fresh bull sperm
are required to achieve equivalent fertility rates in vivo,
clearly demonstrating that cryopreservation markedly
reduces the fertilising potential of the sperm.
The lower fertility of cryopreserved semen is due, in part,
to a reduced number of live sperm after thawing.
Furthermore, post-thaw sperm motility is poorly correlated to
For personal use only.
semen fertility. Clearly, a considerable proportion of the
sperm experience sublethal damage, in that they are viable,
motile and morphologically normal, yet poorly fertile. Our
goals are to characterize the nature of the sublethal sperm
damage caused by cryopreservation and thereby develop a
method to improve the functional fertility of thawed semen.
Therefore, the purpose of this review is to describe our theo-
ry that sperm are prematurely “activated” by cryopreservation
and discuss strategies for preventing the cryo-damage.
ACQUISITION OF SPERM FERTILITY
Spermatogenesis occurs in the seminiferous tubules that are
looped within the testicle. It is a remarkable cascade of cell
divisions during which the diploid spermatogonia are trans-
formed into haploid spermatozoa. Although spermatogenesis
is extremely complex, the end results are sperm with tightly
condensed chromatin bound to protamines. In addition, the
newly formed sperm have a flagellum and contain little
excess cytoplasm, which facilitates their primary function: to
deliver the genetic contribution of the sire to the oocyte.
The sperm head contains a specialized structure derived
from the Golgi apparatus, the acrosome, which is filled with
hydrolytic enzymes. The loss of the endoplasmic reticulum
and the ultracondensed DNA render the spermatozoa to be
non-transcriptional. Consequently, the synthesis of new pro-
teins and lipids cannot occur, although their modification cer-
tainly occurs in post-testicular sperm during transit through the
male reproductive tract [reviewed by Eddy (1988)]. As well,
the sperm plasma membrane acquires peripheral proteins dur-
ing passage through the female tract (Lapointe et al. 1998).
Testicular sperm, though morphologically developed, are
not yet fertile and at ejaculation, mammalian sperm are still
unable to fertilise an ovum. The potential for forward motility
and fertility is acquired during passage through the epididymis
[reviewed by Yanagimachi (1994)]. Physiologically, however,
modifications must occur during transit through the female
reproductive tract in vivo (Austin 1951; Chang 1951), or in
defined media in vitro (Yanagimachi 1994). Collectively,
these post-ejaculatory modifications are termed “capacita-
tion” (Austin 1952) and, despite the discovery of this phe-
nomenon over 50 yr ago, the molecular mechanism of
capacitation is still not fully understood (Bailey et al. 2001).
It is generally accepted that capacitation is initiated at the
sperm plasma membrane (Visconti et al. 1998), during
which cholesterol is removed, altering protein-lipid organi-
zation (Langlais and Roberts 1985; Lin and Kan 1996) and
increasing membrane fluidity (Wolf et al. 1986; Smith et al.
1998). Other events coinciding with capacitation are mem-
brane hyperpolarisation (Zeng et al. 1995), calcium uptake
(Parrish et al. 1999; Dubé et al. 2001), increased cyclic
adenosine 5’monophosphate (Parrish et al. 1994; Lefievre et
al. 2000), kinase activation (Tardif et al. 2001), protein
phosphorylation (Visconti et al. 1995a; Leclerc et al. 1996;
Galantino-Homer et al. 1997; Tardif et al. 1999), and the
production of reactive oxygen species (de Lamarinde and
Gagnon 1993).
At present, capacitation can be defined as a series of
events that enables the sperm to bind the oocyte and under-
go the acrosome reaction in response to specific glycopro-
teins of the zona pellucida, which is the acellular matrix
surrounding the oocyte. During the acrosome reaction, a
massive influx of calcium occurs, provoking exocytosis of
the acrosome and liberation of its enzymes (Bailey and
Storey 1994). The acrosome reaction is obligate for fertili-
sation as these enzymes favour sperm penetration of the
oocyte (Saling and Storey 1979).
THE CRYOPRESERVATION PROCESS
Semen cryopreservation involves several general steps,
dilution, cryoprotection, cooling and freezing, storage, and
thawing, which affect sperm structure and function
(Hammerstedt et al. 1990): .
Immediately after collection, the semen is diluted in an
appropriate medium, often based on egg yolk or milk,
although there is a market for novel extenders containing no
animal-derived components. At high dilution levels, as for
bull and ram semen, sperm function declines, a phenomenon
called the “dilution effect” [Mann and Lutwak-Mann (1981)
in Maxwell and Johnson (1999)], which may be due to
reduced contact between sperm and components of the male
reproductive tract (Garner et al. 2001).
Traditionally, sperm are diluted at a temperature close to
body temperature (30 to 39°C) then cooled to 5°C at which
point a cryoprotectant (glycerol) is added (Polge et al.
1949). For most domestic species, cooling from body tem-
perature to near freezing represents a major stress to the
sperm plasma membrane resulting in the rearrangement and
destabilization of membrane components, and calcium
influx (de Leeuw et al. 1993; Noiles et al. 1995; Maxwell
and Johnson 1997; Collin et al. 2000). White (1993) specu-
lated that this increased permeability is due to subtle irre-
versible structural changes to the membrane architecture

caused by cooling below the thermotropic phase transition
temperature of membrane phospholipids. At the transition
temperature, there is an abrupt change from the gel to the
liquid-crystal phase of membrane phospholipids, and this
would lead to irreversible structural changes to the mem-
brane architecture and accounts for this increased perme-
ability. Since sperm membranes are composed of many
phospholipids in a species-specific manner (White 1993),
with each phospholipid having a precise phase transition
temperature, the degree of structural damage depends on the
temperature and the lipid composition of the membrane.
The plasma membrane is the primary site of injury to cry-
opreserved sperm and the principal damage occurs during
freezing and thawing (Hammerstedt et al. 1990; Parks and
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Graham 1992). As sperm are frozen, ice crystals form in the
extracellular medium, thereby increasing the osmolality of
the unfrozen water. Intracellular water diffuses out of the
sperm in response to this osmotic gradient, thus dehydrating
the cell and plasma membrane. At thawing, the phenomenon
is repeated in reverse as the extracellular ice crystals melt
and water diffuses into the sperm. Ultrastructural deforma-
tion of the plasma membrane occurs as a consequence of
osmotic stress and drastic change in volume during freezing
and thawing. The inclusion of a cryoprotectant, commonly
glycerol for mammalian sperm, is essential for cell survival
during cryopreservation. The efficacy of glycerol is partial-
For personal use only.
ly due to its osmotic effects that dramatically increase the
osmolality of the medium, which retards ice formation
(Hammerstedt et al. 1990). The osmotic effects of glycerol
are marked, but transitory, and such rapid change in cell vol-
ume does not appear to be detrimental to sperm function
(Liu and Foote 1998). Furthermore, glycerol acts directly on
the sperm plasma membrane, possibly to reduce some of the
phase transitions, and to increase membrane fluidity during
cooling (Noiles et al. 1995).
CRYOPRESERVATION AND PREMATURE
CAPACITATION
Cryopreservation is a substantial procedure and it is remark-
able that so many sperm actually survive the trauma
(Hammerstedt et al. 1990). However, a study conducted
using four ejaculates from each of six different bulls and
4482 inseminations showed that the in vivo fertility of cryo-
tolerant sperm was reduced in a manner not correlated with
motility assessed by computer-assisted sperm motility
analysis (Bailey et al. 1994). Interestingly, in vivo fertility
outcome was associated with intracellular calcium regula-
tion of frozen-thawed sperm (Bailey and Buhr 1994).
Calcium is a critical signalling molecule and is integrally
involved in the onset of capacitation and the acrosome reac-
tion. Therefore, our general hypothesis is that poor calcium
control in cryopreserved sperm inhibits normal cell physiol-
ogy. We used the chlortetracycline fluorescence (CTC)
assay and in vitro fertilisation to determine if capacitation,
the acrosome reaction and fertilisation are similar between
cryopreserved and unfrozen bull semen (Cormier et al.
1997). As evaluated by the CTC assay, more than twice as
many cryopreserved bull sperm were capacitated immedi-
ately after thawing than fresh sperm diluted in buffer and
BAILEY ET AL. — SEMEN CRYOPRESERVATION 395
fresh or cooled sperm extended in an egg yolk-glycerol dilu-
ant (Fig. 1). These data were supported by the in vitro fer-
tilisation assay that showed cryopreserved sperm fertilised
over 20% more oocytes than did fresh sperm (Fig. 2).
Unexpectedly, the unfrozen sperm that had been diluted in
extender and cooled to 4°C were also able to penetrate a sig-
nificant number of oocytes, showing that the molecular
changes to the sperm caused by the egg yolk-Tris-glycerol
extender and cooling effectively mimic capacitation. It is
important to note that this “cryo-capacitation” (Bailey et al.
2000) did not occur in the entire population of sperm, since
cryopreserved sperm supplemented with heparin, an estab-
lished capacitating agent for bull sperm (Parrish et al. 1988),
penetrated nearly twice as many oocytes as the heparin-free
cryopreserved sperm (Fig. 2). Regardless, these data provid-
ed evidence that partial or full cryopreservation induces pre-
cocious capacitation of bull sperm. Similar findings have
also been reported for different species, including mice
(Fuller and Whittingham 1996), sheep (Pérez et al. 1996;
Morrier et al. 2002), and pigs (Gillan et al. 1997).
Capacitation, though a necessary step toward fertilisation, is
known to destabilise the sperm membranes (Harrison 1996).
The capacitated sperm plasma membrane is fragile and sus-
ceptable to deterioration or spontaneous acrosome reactions
and death if fertilisation has not occurred. Therefore, the fer-
tile lifespan of sperm would be restricted in vivo and the fer-
tility of the entire population jeopardised by the proportion
of cryo-capacitated sperm present in the insemination dose
(Bailey et al. 2000).
Cryo-capacitation and the Role of Calcium
To elucidate the mechanism(s) by which cryo-capacitation
is induced, we speculated that cryo-capacitation and semen
fertility are associated with abnormal intracellular sperm
calcium levels. Because cryopreservation modifies sperm
membrane structure and function to favour calcium influx,
we used flow cytometry and a fluorescent intracellular cal-
cium indicator (indo–1) to compare the sperm from bulls of
proven low and high fertility (Collin et al. 2000). As expect-
ed, sperm from the most fertile bulls contained less calcium
than did sperm from the poor fertility bulls (Fig. 3).
Furthermore, among 10 bulls with a broad range of fertility,
sperm intracellular calcium concentrations (three ejaculates
per bull) were inversely correlated with non-return rate
(r2 = 0.15; P = 0.04; n = 10 sires with three ejaculates per sire).
To determine at which point during the cryopreservation
process the calcium influx occurs (and thereby develop a
method to prevent it), fresh semen was divided into six treat-
ments: neat undiluted (control); diluted in non-capacitating
medium (NCM); extended in egg yolk-Tris diluant (EYT);
diluted in EYT containing 7% glycerol (EYTG); diluted in
EYTG and cooled to 4°C (EYTG-4); fully cryopreserved
according to commercial cryopreservation procedures
(frozen-thawed). Flow cytometry was used to measure the
internal calcium levels of the sperm with indo-1 and sam-
ples were probed with propidium iodide to identify live cells
with intact membranes (stained with indo-1 only), moribund
cells (double stained with indo-1 and propidium iodide) and
dead cells (stained with propidium iodide only; Collin
 396 CANADIAN JOURNAL OF ANIMAL SCIENCE

Fig. 1. The effect of pre-exposure for 4 h to non-capacitating medium (NCM) at 23°C or egg yolk-Tris-glycerol extender (EYTG) (4°C,
23°C, or complete freezing and thawing) on capacitation of bull sperm as assessed by pattern B of the chlortetracycline fluorescence assay
(CTC). *Frozen-thawed sperm display more pattern B fluorescence than fresh sperm (P < 0.05) indicating that full cryopreservation induces
capacitation-like modifications to the sperm surface (from Cormier et al. 1997).
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Fig. 2. The effect of pre-exposure for 4 h to non-capacitating medium (NCM) at 23°C or egg yolk-Tris-glycerol extender (EYTG) (4°C,
23°C, or complete freezing and thawing) on the ability of bull sperm to fertilise bovine cumulus-oocyte complexes in vitro. a, b, c
Proportions with different letters are different (chi-square P < 0.001). Sperm that were cooled to 4°C in EYTG and those that were frozen-
thawed were able to fertilise in the absence of heparin, which is necessary for in vitro capacitation of bull sperm (Parrish et al. 1988). Sperm
that were frozen-thawed (F-T) and supplemented with 2 µg mL–1 heparin served as a positive control (F–T + heparin). These data indicate
that both cooling in the extender and complete cryopreservation induce premature capacitation of bull sperm (from Cormier et al. 1997).

2000). Frozen-thawed semen consistently contained more
moribund and fewer live sperm than did the non-frozen
samples and among those, no differences in the percentages
of live or damaged sperm were evident (Fig. 4). However,
for the live sperm, higher intracellular calcium levels were
apparent during cooling and freezing-thawing than after the
dilution steps (Fig. 5). We speculate that this high-calcium
subpopulation corresponds to sperm that are fully function-
al, but are slightly compromised in their calcium-regulating
mechanism. These sperm may in fact be undergoing early
steps of cooling-induced capacitation. It is tempting to spec-
ulate that cooling acts as a primer for the inability of sperm
to properly regulate internal calcium concentration, which is
then exacerbated by freezing and thawing.
Interestingly, Thundahil et al. (1999) reported that the
proportion of uncapacitated sperm (pattern F) was correlat-
ed to bull fertility in vivo using the CTC assay. Taken
together with our observations, these results support the
hypothesis that cryopreservation somehow precociously
triggers the signal transduction pathway leading to capacita-
tion, and that cryo-capacitation is partly responsible for the
reduced fertility of thawed semen. Physiological capacita-
tion is now accepted to be regulated by intracellular sig-
nalling pathways that result in the tyrosine phosphorylation
of various sperm proteins. The signalling pathways are not
completely elucidated, although protein kinase A and pro-
tein tyrosine kinases appear to be involved (Visconti et al.
1995b; Tardif et al. 2001). The glycosanimoglycan heparin
favours in vitro capacitation of bull sperm after 4–5 h of
incubation (Parrish et al. 1988) and the simultaneous
appearance of a cohort of tyrosine phosphoproteins
(Galantino-Homer et al. 1997). Results from our laboratory
show that immediately after thawing numerous tyrosine
phosphoproteins are already present (Fig. 6), suggesting that
the signalling cascade leading to protein tyrosine phospho-
rylation is already activated (Cormier and Bailey 2003).
Furthermore, the addition of heparin does not further
increase tyrosine phosphorylation. This lack of response
might be because the sperm “substrate” for heparin is no
longer reactive/present following the physicochemical stress
of cryopreservation, or because the membrane modifica-
tions during processing already triggered the heparin “sub-
strate” in its absence.
In support of our hypothesis that the signalling path-
ways leading to capacitation are activated by sperm
manipulation, Green and Watson (2001) recently reported
that cooling and storage also result in the appearance of a
tyrosine phosphoprotein that is associated with capacita-
 BAILEY ET AL. — SEMEN CRYOPRESERVATION 397

Fig. 3. Intracellular calcium levels in cryopreserved sperm from bulls of low and high fertility according to their adjusted non-return rates
(n = 3 ejaculates from each of three different bulls per group). Measures of intracellular calcium were conducted hourly for 4 h after thaw-
ing; only levels at 0 and 4 h post-thaw are shown. At least 30 000 sperm per measure were analysed for calcium level as a function of indo-
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1 fluorescence measured by flow cytometry. *Sperm from high fertility bulls contained less intracellular calcium than sperm from high
fertility sires at all times (P < 0.05; from Collin et al. 2000).
For personal use only.

Fig. 4. Percentages of live (labelled with indo-1 only) and moribund (double-stained) sperm following a 4 h pre-incubation (n = 6). Fresh
semen was divided into six treatments: undiluted (Control); diluted in noncapacitating medium (NCM); extended in egg yolk-Tris diluant
(EYT); diluted in EYT containing 7% glycerol (EYTG); diluted in EYTG and cooled to 4°C (EYTG-4); fully cryopreserved according to
commercial cryopreservation procedures (frozen-thawed). *Frozen-thawed semen had fewer live and more moribund sperm following freez-
ing-thawing (P < 0.05; from Collin 2000).

Fig. 5. Relative intracellular calcium levels of viable sperm (i.e., labelled with indo-1 only and not stained with propidum iodide) following
a 4 h pre-incubation (n = 6). Fresh semen was divided into six treatments: neat undiluted (Control); diluted in noncapacitating medium
(NCM); extended in egg yolk-Tris diluant (EYT); diluted in EYT containing 7% glycerol (EYTG); diluted in EYTG and cooled to 4°C
(EYTG-4); fully cryopreserved according to commercial cryopreservation procedures (Frozen-Thawed). a, b, c Intracellular calcium levels
with different letters are different (P < 0.05, protected LDS test). Cooled and fully cryopreserved sperm contained more calcium than the
other treatments, with the frozen-thawed sperm containing the highest amount (from Collin 2000).

tion in pig sperm. These authors pointed out that despite
many similarities between “true” capacitation and capaci-
tation induced by cooling and/or cryopreservation, certain
physiological differences exist, and one type does not mir-
ror the other. Regardless, the characterisation of cryo-
capacitation (or capacitation due to cooling) provides
novel insight as to the mechanism by which the fertility
of processed sperm is compromised, as well as an oppor-
tunity to develop strategies to prevent this type of
sublethal damage.
 398 CANADIAN JOURNAL OF ANIMAL SCIENCE

1 2 3 4 5 6
Fresh, extended semen

56 kDa
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Cryopreserved semen
For personal use only.

56 kDa

Fig. 6. The pattern of phosphotyrosine-containing proteins associated with capacitation of bovine sperm induced by heparin or cryopreser-
vation in (top panel) fresh-extended and (bottom panel) cryopreserved semen (n = 14). Ejaculated sperm were extended with egg yolk-Tris-
glycerol and half were cryoperserved then incubated for 5 h in capacitating medium with 0 (lanes 1, 3 and 5) or 15 µg mL–1 heparin (lanes
2, 4 and 6). Sperm proteins were extracted at 0, (lanes 1, 2) 2.5 (lanes 3, 4) and 5 h (lanes 5, 6) and separated by SDS-PAGE. Tyrosine phos-
phorylated proteins were detected by immunoblot analysis using a monoclonal antiphosphotyrosine antibody and immunoreactivity was
revealed using a peroxidase-conjugated goat antimouse IgG. Note the heparin-dependent appearance of a Mr 56 000 tyrosine phosphopro-
tein in fresh sperm at 5 h. In cryopreserved sperm, a band at Mr 56 000 appears immediately after thawing (0 h), with and without heparin.
Moreover, its intensity does not change due to heparin over time. These data indicate (1) that cryopreserved sperm have a tyrosine phos-
phoprotein profile that partly resembles that of heparin-capacitated sperm and (2) that cryopreserved sperm do not respond to heparin as do
fresh sperm (from Cormier and Bailey 2003).

HOW CAN SPERM BE PROTECTED? prevent the cryopreservation-dependent capacitation

It is known that glucose blocks heparin-induced capacitation
of fresh bull sperm by preventing intracellular alkalinasation
(Parrish et al. 1994). Tyrosine phosphorylation of sperm
proteins is also prevented by the inclusion of glucose in the
heparin-based capacitation medium (Galantino-Homer et al.
1997). In our laboratory, bull semen has been typically cry-
opreserved in an egg yolk-Tris-glycerol extender containing
fructose. To test the hypothesis that the presence of glucose
during cryopreservation inhibits cryo-capacitation of bull
sperm, we replaced the fructose in the extender with glu-
cose. In contrast to our hypothesis, however, glucose did not
(Fig. 7), internal calcium concentration (data not shown) or
tyrosine phosphorylation of sperm proteins (data not shown).
These results, confirmed that the mechanism of cryopreserva-
tion-induced capacitation is different from that induced by
heparin (“true” capacitation; Green and Watson 2001).
Since the primary site of damage induced by cooling and
cryopreservation is the plasma membrane, a reasonable
approach to protect the sperm is to better stabilise this part
of the cell. Certainly the phospholipids in egg yolk are con-
sidered to support the sperm plasma membrane during the
stress of processing [reviewed in Parks and Graham (1992)],
 BAILEY ET AL. — SEMEN CRYOPRESERVATION 399

Fig. 7. The effect of replacing fructose with glucose in egg-yolk-Tris-glycerol extender on the proportion of capacitated sperm after thaw-
ing (n = 3 ejaculates from different bulls). Capacitation was detected using the chlortetracycline assay. Whole ejaculates were divided into
two aliquots, one in egg-yolk-Tris-glycerol containing fructose (Control) and the other in egg-yolk-Tris-glycerol containing glucose
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(Glucose). Semen was processed identically. Straws were thawed, sperm incubated in capacitating medium with or without 15 µg mL–1
heparin for up to 5 h. No effect of the glucose treatment is evident as similar percentages of sperm are capacitated at thawing (0 h; P > 0.05;
from Cormier and Bailey 2003).
For personal use only.

Fig. 8. The effect of cholesterol-saturated methyl-β-cyclodextrin on the motility and viability of cryopreserved ram sperm. Cholesterol-sat-
urated methyl-β-cyclodextrin (4.5 mg mL–1) was added to a commercial egg yolk-glycerol extender. Freshly ejaculated ram semen (n = 3
ejaculates from different rams) was spit into two aliquots and diluted in extender with or without the cholesterol-saturated methyl-β-
cyclodextrin. Semen was then cryopreserved by standard industry procedures. Supplementation of the extender with cholesterol-saturated
methyl-β-cyclodextrin appears to be beneficial to sperm function immediately after thawing as the proportions of viable (determined by
eosin-nigrosin exclusion), motile and progressively motile (assessed by computer-assisted sperm analysis) sperm were greater than controls.
According to the chlortetracycline fluorescence assay, the cyclodextrin treatment yielded more non-capacitated and capacitated sperm (pat-
terns F and B, respectively) and markedly fewer acrosome reacted sperm (pattern AR). These data suggest that a larger population of func-
tional sperm survive the cryopreservation procedure in the presence of cholesterol-saturated methyl-β-cyclodextrin.

and as discussed earlier, the cryoprotecting properties of
glycerol are, in part, attributable to effects on the sperm
membrane. He et al. (2001) incubated fresh boar sperm with
small unilamellar liposomes composed of five specific
phospholipids selected to improve membrane resistance to
cryopreservation. This approach successfully resulted in
liposome fusion with the sperm and subsequent lipid trans-
fer. Moreover, the liposome-treatment improved sperm sur-
vival and motility after thawing, while maintaining the
ability of the sperm to undergo “true” capacitation and the
acrosome reaction (induced by ionophore).
Currently, we are testing the use of cyclodextrin to rein-
force the plasma membrane. Cyclodextrins are cyclic
oligomers of glucose that form water soluble complexes with
other organic molecules that may not be water
soluble. We hypothesised that methyl-β-cyclodextrin could
be used to deliver cholesterol to the plasma membranes of
ram sperm during cryopreservation, and plasma membranes
stabilised by the presence of cholesterol would be more resis-
tant to cryo-capacitation. Our results, though preliminary, are
promising (Fig. 8). Post-thaw sperm quality was improved
when a standard egg yolk-glycerol extender was supplement-
ed with cholesterol-saturated methyl-β-cyclodextrin.
Immediately after thawing, sperm viability, total motility and
progressive motility were higher than for controls. According
to the CTC assay, slightly more cyclodextrin-treated sperm
showed pattern B staining, which is indicative of capacitation.
However, about twofold more sperm showed pattern F fluo-
rescence, non-capacitated sperm, indicating that the
cyclodextrin treatment better conserved the sperm in a “non-
 400 CANADIAN JOURNAL OF ANIMAL SCIENCE
activated” state. Furthermore, the acrosome reaction (pattern
AR) occurred prematurely in at least half as many cyclodex-
trin-treated sperm than controls. These preliminary data sug-
gest that a larger population of functional sperm survive
cryopreservation in the presence of cholesterol-saturated
methyl-β-cyclodextrin, presumably because plasma mem-
branes stabilised by the presence of cholesterol more effec-
tively tolerate the phase transitions, osmotic stress and
volume changeassociated with cooling, freezing and thawing.
CONCLUSION
In conclusion, we and others have shown that premature
capacitation-like modifications occur in a substantial pro-
portion of cooled and/or cryopreserved sperm. This form of
Can. J. Anim. Sci. Downloaded from cdnsciencepub.com by 103.162.62.46 on 11/15/22
sublethal damage may reduce in vivo fertility of the semen
by shortening the functional lifespan of the sperm, since the
capacitated sperm membrane is subject to rapid deteriora-
tion. Strategies to prevent cryo-capacitation may improve
the post-thaw efficacy of semen and increase membrane sta-
bility during cryopreservation. Other interesting approaches
not discussed here are calcium chelators, calcium channel
blockers and specific inhibitors of the signalling pathway
leading to capacitation.
ACKNOWLEDGEMENTS
We are grateful for collaboration with Drs. Mary Buhr,
For personal use only.
Marc-André Sirard and François Castonguay. Thanks to
Simon Collin for his excellent work as a graduate student and
to Dr. Maurice Dufour for his expertise in flow cytometry.
Financial support was obtained from NSERC, CORPAQ,
Alliance Semex and the Centre d’expertise ovine du Québec
(CEPOQ). Technical assistance was provided by CEPOQ
and the Centre d’insémination artificielle du Québec.
Austin, C. R. 1951. Observations on the penetration of the sperm
into the mammalian egg. Aust. J. Biol. Sci. 4: 581–596.
Austin, C. R. 1952. The “capacitation” of the mammalian sperm.
Nature 170: 326.
Bailey, J. L. and Buhr, M. M. 1994. The impact of cryopreserva-
tion on Ca2+ regulation by bovine spermatozoa. Can. J. Anim. Sci.
74: 45–52.
Bailey, J. L. and Storey, B. T. 1994. Calcium influx into mouse
spermatozoa activated by solubilized mouse zonae pellucidae,
monitored with the calcium indicator, fluo-3. Inhibition of the
influx by three inhibitors of the zona pellucida induced acrosome
reaction: tyrphostin A48, pertussis toxin, and 3-quinuclidinyl ben-
zilate. Mol. Reprod. Dev. 39: 297–308.
Bailey, J. L., Bilodeau, J. F. and Cormier, N. 2000. Semen cry-
opreservation in domestic animals; a damaging and capaciting phe-
nomenon. J. Androl. 21: 1–7.
Bailey, J. L., Robertson, L. and Buhr, M. M. 1994. Calcium reg-
ulation, computerized motility parameters and the fertility of
bovine spermatozoa. Can. J. Anim. Sci. 74: 53–58.
Bailey, J. L., Tardif, S. and Dubé, C. 2001. The nature of sperm
capacitation: current concepts after 50 years of research. Pages
237–246 in B. Robaire, H. Chemes, and C. Morales, eds.
Andrology in the 21st Century: Proceedings of the VIIth
International Congress of Andrology: Plenaries, Symposia and
Workshops. Medimond Publ., Englewood, NJ.
Chang, M. C. 1951. Fertilizing capacity of spermatozoa deposited
into fallopian tubes. Nature 168: 697–698.
Collin, S. 2000. La régulation du calcium intracellulaire chez les
spermatozoïdes bovins cryopréservés : un élément clef de la qual-
ité de la semence cryopréservée. Master’s Thesis presented to
Université Laval, QC. 107 pp.
Collin, S., Sirard, M. A., Dufour, M. and Bailey, J. L. 2000.
Sperm calcium levels and chlortetracycline fluorescence patterns
are related to the in vivo fertility of cryopreserved bovine semen. J
Androl. 21: 938–943.
Cormier, N. and Bailey, J. L. 2003. A differential mechanism is
involved during heparin- and cryopreservation-induced capacita-
tion of bovine spermatozoa. Biol. Reprod. 69: 177–185.
Cormier, N., Sirard, M.-A. and Bailey, J. L. 1997. Premature
capacitation of bovine spermatozoa is initiated by cryopreserva-
tion. J. Androl. 18: 461–468.
de Lamarinde, E. and Gagnon, C. 1993. Human sperm hyperac-
tivation and capacitation as parts of an oxidative process. Free
Radical Biol. Med. 14: 157–166.
de Leeuw, F. E., Chen, H. C., Colenbrander, B. and Verkleij,
A. J. 1993. Cold-induced ultrastructural changes in bull and boar
sperm plasma membranes. Cryobiology 27: 171–183.
Dubé, C., Tardif, S., Leclerc, P. and Bailey, J. L. 2001.
Importance of calcium during porcine sperm capacitation. Pages
115–119 in B. Robaire, H. Chemes, and C. Morales, eds.
Andrology in the 21st Century: Proceedings of the VIIth
International Congress of Andrology: Short Communications.
Medimond Publ., Englewood, NJ.
Eddy, M. 1988. The physiology of reproduction. Vol. 1. The
spermatozoon. Pages 27–68 in E. Knobil, J.D. Neill, L.L. Ewing,
G. S. Green, C.L. Markert, and D.W. Pfaff, eds. Raven Press, New
York, NY.
Fuller, S. J. and Whittingham, D. G. 1996. Effect of cooling
mouse spermatozoa to 4 degrees C on fertilization and embryonic
development. J. Reprod. Fertil. 108: 139–145.
Garner, D. L., Thomas, C. A., Gravance, C. G., Marshall,
C. E., DeJarnette, J. M. and Allen, C. H. 2001. Seminal plasma
addition attenuates the dilution effect in bovine sperm.
Theriogenology 56: 31–40.
Galantino-Homer, H. L., Visconti, P. E. and Kopf, G. S. 1997.
Regulation of protein tyrosine phosphorylation during bovine
sperm capacitation by a cyclic adenosine 3’, 5’-monophosphate-
dependent pathway. Biol. Reprod. 56: 707–719.
Gillan, L., Evans, G. and Maxwell, W. M. C. 1997. Capacitation
status and fertility of frozen-thawed ram spermatozoa. Reprod.
Fertil. Dev. 9: 481–487.
Green, C. E. and Watson, P. F. 2001. Comparison of the capaci-
tation-like state of cooled boar spermatozoa with true capacitation.
Reproduction 122: 889–898.
Hammerstedt, R. H., Graham, J. K. and Nolan, P. 1990.
Cryopreservation of mammalian sperm: What we ask them to sur-
vive. J. Androl. 11: 73–88.
Harrison, R. A. P. 1996. Capacitation mechanisms, and the role of
capacitation as seen in eutherian mammals. Reprod. Fertil. Dev. 8:
581–594.
He, L., Bailey, J. L. and Buhr, M. M. 2001. Incorporating lipids
into boar sperm alters chilling sensitivity but not capacitation
potential. Biol. Reprod. 64: 69–79.
Hofmo, P. O. and Grevle, I. S. 1999. Page 9 in Proceedings of the
IV International Conference of Boar Semen Preservation.
Development and commercial utilisation of frozen boar semen in
Norway. 8–11 August, Beltsville, MD.
Langlais, J. and Roberts, K. D. 1985. A molecular membrane
model of sperm capacitation and the acrosome reaction of mam-
malian spermatozoa. Gamete. Res. 12: 183–224.

Leclerc, P., de Lamirande, E. and Gagnon, C. 1996. Cyclic
adenosine 3’, 5’ monophosphate-dependent regulation of protein
tyrosine phosphorylation in relation to human sperm capacitation
and motility. Biol. Reprod. 55: 684–692.
Lapointe, S., Sullivan, R. and Sirard, M. A. 1998. Binding of a
bovine oviductal fluid catalase to mammalian spermatozoa. Biol.
Reprod. 58: 747–753.
Lefievre, L., de Lamirande, E. and Gagnon, C. 2000. The cyclic
GMP-specific phosphodiesterase inhibitor, sildenafil, stimulates
human sperm motility and capacitation but not acrosome reaction.
J Androl. 21: 929-1037.
Lin, Y. and Kan, F. W. 1996. Regionalization and redistribution
of membrane phospholipids and cholesterol in mouse spermatozoa
during in vitro capacitation. Biol. Reprod. 55: 1133–1146.
Liu, Z. and Foote, R. H. 1998. Osmotic effects on volume and
Can. J. Anim. Sci. Downloaded from cdnsciencepub.com by 103.162.62.46 on 11/15/22
motility of bull sperm exposed to membrane permeable and non-
permeable agents. Cryobiology 37: 207–218.
Maxwell, W. M. and Johnson, L. A. 1997. Chlortetracycline
analysis of boar spermatozoa after incubation, flow cytometric sort-
ing, cooling or cryopreservation. Mol. Reprod. Dev. 46: 408–418.
Maxwell, W. M. C. and Johnson, L. A. 1999. Physiology of sper-
matozoa at high dilution rates: the influence of seminal plasma.
Theriogenology 52: 1353–1362.
Morrier, A., Castonguay, F. and Bailey, J. L. 2002. Glycerol
addition and conservation of fresh and cryopreserved ram sperma-
tozoa. Can. J. Anim. Sci. 82: 347–356.
Mortimer D. 1994. Practical laboratory andrology. Oxford
University Press, New York, NY. 393 pp.
Noiles, E. E., Bailey, J. L. and Storey, B. T. 1995. Temperature
For personal use only.
dependence of the water permeability, Lp, of murine sperm shows
a discontinuity between 4° and 0°C. Cryobiology 32: 220–238.
Parks, J. E. and Graham, J. K. 1992. Effects of cryopreservation
procedures on sperm membranes. Theriogenology 38: 209–222.
Parrish, J. J., Susko-Parrish, J., Uguz, C. and First, N. L. 1994.
Differences in the role of cyclic adenosine 3’,5’-monophosphate
during capacitation of bovine sperm by heparin or oviduct fluid.
Biol. Reprod. 41: 1099–1108.
Parrish, J. J., Susko-Parrish, J., Winer, M. A. and First, N. L.
1988. Capacitation of bovine sperm by heparin. Biol. Reprod. 38:
1171–1180.
Parrish, J. J., Susko-Parrish, J. L. and Graham, J. K. 1999. In
vitro capacitation of bovine spermatozoa: role of intracellular cal-
cium. Theriogenology 51: 461–472.
Pérez, L. J., Valcárce, I. A., de las Heras, M. A., Moses, D. F.
and Baldassarre, H. 1996. Evidence that frozen/thawed sperma-
tozoa show accelerated capacitation in vitro as assessed by chlorte-
tracycline assay. Theriogenology 46: 131–140.
Polge, C., Smith, A. U. and Parkes, A. S. 1949. Revival of sper-
matozoa after vitrification and dehydration at low temperature.
Nature 164: 666.
Pukazhenthi, B., Pelican, K., Wildt, D. and Howard, J. 1999.
Sensitivity of domestic cat (Felis catus) sperm from normospermic
versus teratospermic donors to cold-induced acrosomal damage.
Biol. Reprod. 61: 135–141.
Salamon, S. and Maxwell, W. M. C. 1995. Frozen storage of ram
semen II. Causes of low fertility after cervical insemination and
methods of improvement. Anim. Reprod. Sci. 38: 1–36.
BAILEY ET AL. — SEMEN CRYOPRESERVATION 401
Saling, P. M. and Storey, B. T. 1979. Mouse gamete interactions
during fertilization in vitro. Chlortetracycline as a fluorescent
probe for the mouse sperm acrosome reaction. J. Cell. Biol. 83:
544–555.
Samper, J. C. 2001. Management and fertility of mares bred with
frozen semen. Anim. Reprod. Sci. 68: 219–228.
Shannon, P. and Vishwanath, R. 1995. The effect of optimal and
suboptimal concentrations of sperm on the fertility of fresh and
frozen bovine semen and a theoretical model to explain the fertili-
ty differences. Anim. Reprod. Sci. 39: 1–10.
Smith, T. T., McKinnon-Thompson, C. A. and Wolf, D. E.
1998. Changes in lipid diffusibility in the hamster sperm head plas-
ma membrane during capacitation in vivo and in vitro. Mol.
Reprod. Dev. 50: 86–92.
Storey, B. T., Noiles, E. E. and Thompson, K. A. 1998.
Comparison of glycerol, other polyols, trehalose, and raffinose to
provide a defined cryoprotectant medium for mouse sperm cryop-
reservation. Cryobiology 37: 46–58.
Tardif, S., Dubé, C., Chevalier, S. and Bailey, J. L. 2001.
Capacitation is associated with tyrosine phosphorylation and tyro-
sine kinase-like activity of pig sperm proteins. Biol. Reprod. 65:
484–792.
Tardif, S., Sirard, M. A., Sullivan, R. and Bailey, J. L. 1999.
Identification of capacitation-associated phosphoproteins in
porcine sperm electroporated with ATP-γ-32P. Mol. Reprod. Dev.
54: 292–302.
Thundahil, J., Gil, J., Januskauskas, A., Larsson, B.,
Soderquist, L., Mapletoft, R. and Rodriguez-Martinez, H.
1999. Relationship between the proportion of capacitated sperma-
tozoa present in frozen-thawed bull semen and fertility with artifi-
cial insemination. Int. J. Androl. 22: 366–373.
Visconti, P. E., Bailey, J. L., Moore, G. D., Pan, D., Olds-
Clarke, P. and Kopf, G. S. 1995a. Capacitation in mouse sper-
matozoa. I. Correlation between the capacitation state and protein
tyrosine phosphorylation. Development 121: 1129–1137.
Visconti, P. E., Galantino-Homer, H., Moore, G. D., Bailey, J.
L., Ning, X. P., Fornes, M. and Kopf, G. S. 1998. The molecular
basis of sperm capacitation. J. Androl. 19: 242–248.
Visconti, P. E., Moore, G. D., Bailey, J. L., Leclerc, P.,
Connors, S. A., Pan, D., Olds-Clarke, P. and Kopf, G. S. 1995b.
Capacitation in mouse spermatozoa. II. Protein tyrosine phospho-
rylation and capacitation are regulated by a cAMP-dependent path-
way. Development 121: 1139–1150.
White, I. G. 1993. Lipids and calcium uptake of sperm in relation
to cold shock and preservation: A review. Reprod. Fertil. Dev. 5:
639–658.
Wolf, D. E., Hagopian, S. S. and Ishijima, S. 1986. Changes in
sperm plasma membrane lipid diffusibility after hyperactivation
during in vitro capacitation in the mouse. J. Cell. Biol. 102:
1372–1377.
Yanagimachi R. 1994. Mammalian fertilization. Pages 189–319
in E. Knobil, and J.D. Neill, eds. The physiology of reproduction.
2nd ed. Raven Press, New York, NY.
Zeng, Y., Clark, E. N. and Florman. H. M. 1995. Sperm mem-
brane potential: hyperpolarization during capacitation regulates
zona pellucida-dependent acrosomal secretion. Dev. Biol. 171:
554–63.
Can. J. Anim. Sci. Downloaded from cdnsciencepub.com by 103.162.62.46 on 11/15/22
For personal use only.