Animal Reproduction Science 60–61 Ž2000.

481–492
www.elsevier.comrlocateranireprosci

The causes of reduced fertility with cryopreserved

semen
P.F. Watson
Department of Veterinary Basic Sciences, Royal Veterinary College, Royal College Street,
London NW1 0TU, UK

Abstract

Cryopreserved mammalian semen is generally acknowledged to have an impaired fertility by

comparison with fresh semen. The reduction arises from both a lower viability post-thaw and
sublethal dysfunction in a proportion of the surviving subpopulation. The reasons for the loss of
fertility are various. In this paper, factors affecting the proportion of survivors Že.g., cold shock
susceptibility, cooling rate, diluent composition and osmotic stress. and factors influencing
functional status of survivors Že.g., membrane stability, oxidative damage, membrane receptor
integrity, nuclear structure. are briefly reviewed. The possible effects of cryopreservation on the
role of spermatozoa in the early stages of embryogenesis are considered. In the light of this
review, indications for new approaches for improving the performance of cryopreserved semen are
offered. q 2000 Elsevier Science B.V. All rights reserved.

Keywords: Spermatozoa; Cryopreservation; Fertilizing capacity; Sublethal damage; Capacitation; Cryoprotec-

tant

1. Introduction

Cryopreservation of semen has long been seen as a means of benefiting the breeding
of animals of agricultural importance, and has been recognised as contributing to the
conservation of endangered species and to overcoming aspects of male infertility in
humans. Nevertheless, with the possible exception of bull semen, a lower fertility is

E-mail address: pwatson@rvc.ac.uk ŽP.F. Watson..

0378-4320r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 4 3 2 0 Ž 0 0 . 0 0 0 9 9 - 3
482 P.F. Watsonr Animal Reproduction Science 60–61 (2000) 481–492

generally accepted as a consequence of cryopreservation. In this paper I want to offer a

perspective on the causes of lower fertility.
Cryopreserved bull semen has been used commercially in dairy cattle for decades and
conception results are now comparable or better than with natural mating. However, this
is not so for the majority of mammalian species where fertility is clearly reduced by the
cryopreservation protocol. Indeed, the losses from cryopreservation are compensated by
the insemination of larger numbers of spermatozoa, and this is true for cattle semen as
well as other species. The difference between species is explained by the extent of the
compensation that can be achieved in cattle, where relatively few spermatozoa are
required for good fertility, complete compensation can be achieved by inseminating of
the order of 20 million total spermatozoa, still allowing for sufficient dilution of semen
to make the process commercially viable. With other species many more spermatozoa
are necessary to achieve reasonable fertility, and the losses associated with cryopreserva-
tion are such as to preclude the process being commercially viable. As a generalisation,
some 40–50% of the population do not survive cryopreservation even with optimised
protocols. When comparisons are made on the basis of similar numbers of motile
Žassumed viable. spermatozoa, results are still generally poorer than with fresh semen,
indicating that even the viable subpopulation after cryopreservation is compromised. An
approach to overcome the problem in some species has been to utilise surgical
insemination permitting the inseminate to be introduced high in the reproductive tract to
achieve a more satisfactory fertility, but at the higher cost of surgery.
This phenomenon can be explained by the need to have a sufficient number of fully
competent spermatozoa capable of achieving fertilisation over the period when ovulation
is likely to occur. The isthmus of the oviduct acts as a functional sperm reservoir
providing a source of potentially fertilising spermatozoa over the ovulatory period
ŽHunter, 1984; Hunter and Nichol, 1983.. Only a minute proportion of spermatozoa
introduced into the lower reaches of the female tract enter the oviducal reservoir, the
majority being expelled through the vulva or phagocytosed in the tract. In the ampulla at
the time of fertilisation, the sperm:oocyte ratio approaches unity ŽHunter, 1996.. The
numerical and temporal competency of this sperm cohort depends on both the number
and quality of spermatozoa introduced into the lower reaches of the tract. Apart from
maternally-related aspects, conception at artificial insemination can be seen as a
probability event determined by considerations of the inseminate. When the sperm
number or quality in the inseminate is reduced fertility declines on an exponential curve
ŽFig. 1.; normally, insemination is performed with sufficient competent spermatozoa to
achieve results on the asymptote of this curve. Both the slope of the curve and the
asymptote vary with individual sire and can be influenced by cryopreservation proce-
dures ŽAmann and Hammerstedt, 1993.. If the total number of fully functional spermato-
zoa in a cryopreserved inseminate falls below the number needed to achieve a high
probability of fertilisation, then fertility is reduced.
When the inseminate is placed higher in the female tract than is normally the case
with artificial insemination, fewer spermatozoa are required to achieve the same
probability of fertility since a greater proportion will survive to colonise the oviduct.
Thus, with sheep, intrauterine insemination is far more effective than posterior cervical
insemination. Maxwell Ž1986. showed that only 20 million motile cryopreserved ram
 P.F. Watsonr Animal Reproduction Science 60–61 (2000) 481–492 483

Fig. 1. The theoretical relationship between sperm number in the inseminate and fertility. Normally, for
artificial insemination, the sperm dose should be sufficient to reach the asymptote. Both the slope and the
maximum response are determined by semen characteristics.

spermatozoa were required to achieve greater than 50% fertility, whereas it is recognised
that cervical insemination requires 10 times that dose. If the insemination was made into
the oviduct itself, less than 1 million spermatozoa were needed ŽMaxwell et al., 1993..
Another observation relating to cryopreserved semen is manifested with boar semen.
In the pig, ovulation can occur over an extended period of oestrus such that spermatozoa
may be required to survive up to 40 h in the oviduct. Waberski et al Ž1994. have noted
that fertility with cryopreserved semen may be high providing the insemination is made
in the period 4 h before ovulation. Outside this period, fertility with cryopreserved
spermatozoa declined dramatically, but fresh semen maintained its fertility for a much
longer period. Cryopreserved spermatozoa do not survive in the female tract compared
with fresh spermatozoa.
In summary, the cryopreservation process results in reduced fertility compared with
fresh semen. It has been shown that this arises from a combination of both loss of sperm
viability and an impairment of function in the population of survivors. This situation
needs to be borne in mind when strategies to improve the results are contemplated. We
need to consider not only the cryopreservation protocol to optimise the number of
survivors, but also the functional ability of the surviving population.

2. Increasing the proportion live

The cryopreservation protocol has a number of potentially damaging stresses: firstly,

the change in temperature; secondly, the osmotic and toxic stresses presented by
exposure to molar concentrations of cryoprotectants; and thirdly, the formation and
dissolution of ice in the extracellular environment. We shall discuss these in turn.

2.1. Change in temperature

It has long been known that cooling ungulate semen too rapidly between 308C and
08C induces a lethal stress in some of the cells proportional to the rate of cooling, the
temperature interval and the temperature range ŽWatson, 1981.. This phenomenon,
484 P.F. Watsonr Animal Reproduction Science 60–61 (2000) 481–492

known as cold shock, also variably affects a number of other species and, as a result,
cooling in this range prior to cryopreservation is usually conducted carefully.
In boar spermatozoa, the phenomenon is mostly manifested immediately after
ejaculation but the cells become progressively less sensitive over the next few hours
ŽPursel et al., 1972.. We have investigated this phenomenon, and found that boar sperm
incubated at room temperature in their own seminal plasma become quite resistant to
cold shock over 16 h after ejaculation ŽTamuli and Watson, 1994.. The membranes are
altered such that they do not respond to the cold stress. This observation suggests that
the ideal time to cryopreserve boar semen may not be as soon after ejaculation as
possible, i.e. within 6 h, as is commonly practised, but after 18–24 h when the resistance
is at a maximum. Modern diluents now permit the maintenance of a high proportion of
spermatozoa in a viable state for at least 24 h.
Even with slow cooling, however, temperature change induces stresses on mem-
branes. It is probable that these are related to phase change in lipids and altered
functional state of membranes. Cold shock is then seen merely as the extreme state of a
continuum of stress, influenced by the rate of onset of the phenomenon. Such stresses on
the membranes may be continued below 08C since phase changes are not complete at
08C, but our preliminary studies Žunpublished. were unable to show cold shock injury in
bull spermatozoa below 08C. However, it is well known that a major phase change
occurs in the vicinity of 5–158C ŽDrobnis et al., 1993., and this may well be the prime
temperature range for temperature dependent injury.
The suggestion that membrane injury results from phase events in the lipid bilayer is
well attested. Freeze fracture studies of membranes before, during and after cooling
show clear evidence of phase separation events, which are only partially reversed after
rewarming ŽHolt and North, 1984; de Leeuw et al., 1990.. Pettitt and Buhr Ž1998. have
shown the importance of modulation of the lipid environment of the plasma membranes
during cooling, implicating the lipid component in mechanisms of injury.
Nevertheless, one should not overlook the other membrane elements that may be
altered by temperature stress. Integral membrane proteins are clustered by lipid phase
separation, and this may be expected to alter function, especially of proteins which
undergo a structural modulation to carry out their function, such as ion channel proteins.
Indeed, it is known that membrane permeability is increased after cooling ŽRobertson
and Watson, 1986; Robertson et al., 1988. and this may be due to a generally increased
membrane leakiness, but could be due to effects on specific protein channels. Calcium
regulation is clearly affected by cooling and this undoubtedly has serious consequences
in terms of cell function ŽBailey and Buhr, 1994.; in severe cases, the change may be
incompatible with continuing cell viability. The uptake of calcium during cooling
contributes both to capacitative changes Žsee below. and fusion events between the
plasma membrane and underlying outer acrosomal membrane. There are strong similari-
ties between membrane damage during cooling and the acrosome reaction, the one being
a disorganised version of the other.
In addition, cytoskeletal elements are temperature-sensitive. The cooling of other
cells results in a premature depolymerisation of actin filaments ŽHall et al., 1993;
Saunders and Parks, 1999.. Spungin et al Ž1995. have postulated that the depolymeriza-
tion of cytoskeletal F-actin is a necessary step permitting the approximation of plasma
 P.F. Watsonr Animal Reproduction Science 60–61 (2000) 481–492 485

membrane and the underlying outer acrosomal membrane promoting acrosomal exocyto-
sis. Perhaps, this also could contribute to a disorganised fusion of membranes following
cooling or cryopreservation.

2.2. CryopreserÕatiÕe stresses

The addition and removal of cryoprotectant in molar proportions applies a substantial

but transient osmotic stress to the plasma membrane of spermatozoa, depending upon
the relative permeability of the cryoprotectant ŽGao et al., 1993.. Generally, the
cryoprotectant of choice for spermatozoa is glycerol Žor occasionally, dimethyl sulphox-
ide., which induces osmotic stresses. Gao et al Ž1995. have shown that when human
spermatozoa were exposed to 1 M glycerol in a single step, the volume excursion
exceeded tolerable limits both on addition and removal. They showed that the stress
could be reduced to tolerable limits by stepwise addition and removal and this
substantially improved the proportion of spermatozoa surviving.
Furthermore, spermatozoa are also sensitive to toxic effects of cryoprotectants,
resulting in the unsuitability of some compounds commonly used for other cells being
less useful for spermatozoa ŽStorey et al., 1998.. Even with glycerol, care should be
exercised in its use with spermatozoa ŽKatkov et al., 1998..

2.3. Ice crystal formation and dissolution

The stresses induced by ice crystal formation are mainly associated with the
accompanying osmotic pressure changes in the unfrozen fraction ŽWatson and Duncan,
1988.. When a solution is cooled below the freezing point, ice crystals are nucleated and
pure water crystallises out as ice. The solutes are dissolved in the remaining liquid water
fraction and the osmotic strength of the solution rises. The proportion of the water
crystallising out as ice, and hence the osmotic strength of the remaining solution,
depends on the temperature — the lower the temperature, the smaller the unfrozen
fraction and hence the higher the osmotic strength of the solution. It is generally
recognised that the duration of exposure to such events should be minimised for optimal
cell survival implying that the cooling rate should be rapid. However, the cooling rate
must be slow enough to allow water to leave the cells by osmosis preventing intra-
cellular ice formation which is lethal. Sperm cells are generally frozen at quite rapid
rates in the range 15–608Crmin, which have been empirically determined as giving the
best survival rates. Obviously, if the cell water permeability and its activation energy
were known it should be possible to predict the maximal cooling rate compatible with
osmotic equilibrium and so determine optimal cryopreservation protocols. Such consid-
erations have been shown to be important for other cell types ŽMazur, 1984..
We and others have measured the water permeability of the membranes of spermato-
zoa from a number of species. With the exception of the rabbit ŽCurry et al., 1995a.,
most species seem to have a rather high water permeability in comparison to the
permeability of other cell types ŽWatson et al., 1992; Noiles et al., 1993, 1997; Gilmore
et al., 1996.. We have also shown that modulating the glucose channels of the sperm
membrane affects water permeability ŽCurry et al., 1995b.. Further studies of this
phenomenon may offer novel ways to modify sperm cell responses to cryopreservation.
486 P.F. Watsonr Animal Reproduction Science 60–61 (2000) 481–492

However, when these figures for water permeability are used in equations to calculate
the maximum cooling rate compatible with approximate osmotic equilbrium during
cooling, the rate works out much higher than is known empirically to be optimal ŽCurry
et al., 1994.. We have, therefore, examined the assumptions on which the calculations
are based. The first assumption is that the water permeability of the membrane remains
unchanged in the presence of cryoprotectant, an assumption recently shown to be
questionable ŽGilmore et al., 1998. although the magnitude of the change by no means
accounted for all the discrepancy. Another possibility is that the permeability of the
sperm plasma membrane is regionally variable. The assumption that a single value
applies to the whole cell is unlikely to be true given the known regionalization of the
sperm plasma membrane. Thus, a measured value may not represent, say, the sperm
head when the majority of the water transport may occur across the tail membrane ŽHolt
and North, 1994.. Alternatively, the techniques for estimating water permeability simply
overestimate the true value. More recent methodology showed this to be the case but
again, the magnitude did not rectify the discrepancy ŽGilmore et al., 1996; Curry and
Watson, unpublished.. Fourthly, the activation energy measured above 08C may not
reflect the activation energy below 08C, an hypothesis shown to be true for human
spermatozoa but still inadequate to account for the discrepancy between calculated and
empirical optimal cooling rates ŽNoiles et al., 1993.. Perhaps, the discrepancy is
explained by the sum of all these errors in the assumptions on which the theoretical
calculations are based. A recent study with mouse spermatozoa has suggested that water
permeability is markedly less at subzero temperatures in the presence of ice crystals and
cryopreservatives, and in this instance, the discrepancy was largely eliminated ŽDe-
vireddy et al., 1999..
Nevertheless, it is possible that there are other stresses that are additional to those
included in these calculations. Holt and North Ž1994. have shown that the signs of
distress were manifested during rewarming and that these were related to osmotic stress,
although not necessarily implying that the damage occurred during rewarming phase.
These considerations led us to hypothesise that there were indeed other factors
determining the optimal cooling rate independent of the risk of intracellular ice
formation. We and others have shown the extreme sensitivity of sperm membranes to
osmotic stresses. We believe these may determine the optimum cooling rate, not by
permeability to water but by the rate of displacement required of the plasma membrane
to accommodate the volume change, and we suggested that this may stress the
attachments of the cytoskeleton ŽWatson, 1995.. In support of this hypothesis, when
mouse or Koala spermatozoa were exposed to cytochalasin D Žwhich disrupts f-actin
filaments. they were more able to withstand extremes of osmotic stress without
membrane rupture ŽNoiles et al., 1997; Holt and Johnston, 1999.. It appears that we
need a more elaborate theory of plasma membrane disruption during cryopreservation
than is currently in vogue.
The proportion of cells, which survive the cryopreservation protocol, is determined
by the sensitivity to osmotic stress during cryopreservative addition and removal, and
during cooling and rewarming. While there may be species differences in overall sperm
sensitivity to cryopreservation, the ejaculate is heterogeneous with a variable resistance
to osmotic stress amongst the cells. Those that succumb to lethal membrane damage,
 P.F. Watsonr Animal Reproduction Science 60–61 (2000) 481–492 487

however, are not determined stochastically. The fact that indiÕiduals can often be
classified as ‘‘good freezers’’ or ‘‘bad freezers’’ implies that certain characteristics of
membrane structure, which may be genetically determined, predispose towards survival
under cryopreservation stress. Attempts to modify lipid composition have not demon-
strated any dramatic benefits, perhaps implying that other membrane elements may be
more important, e.g. cytoskeleton. This is an area for further investigation.
Nevertheless, even if we optimise the process and minimise the cell death, there will
still be a proportion of cells which fail to survive. We need, therefore, to concentrate on
the function of the surviving population.

3. Increasing the quality of survivors

3.1. Capacitation-like changes
A significant breakthrough in understanding has come with the recognition that
cooled and rewarmed spermatozoa behave as if they were capacitated ŽWatson, 1995..
However, we are unsure at present whether spermatozoa manifest the changes of
capacitation or simply are enabled to by-pass capacitation and proceed directly to
acrosomal exocytosis. We have begun to study this phenomenon and have shown that
cooled spermatozoa display chlortetracycline staining and an increase in intracellular
free Ca2q, typical of capacitated spermatozoa, but that the tyrosine phophorylation
patterns are somewhat different in cooled and rewarmed spermatozoa ŽWatson and
Green, in press.. Fig. 2 shows the free Ca2q in cooled spermatozoa compared with that
seen in capacitated spermatozoa. We are continuing to investigate these events to
determine the nature of the membrane changes resembling capacitation.

Fig. 2. The intracellular free Caq response of boar spermatozoa either to incubation in a capacitating medium
or to cooling. The illustrations are of ‘‘dot plots’’ from the flow cytometer of spermatozoa labelled with the
viability probe, propidium iodide Žvertical axis, FL3 LOG. and a calcium-sensitive indicator, Fluo-3 AM ester
Žhorizontal axis, FL1 LOG.. ŽA. Control at time zero, ŽB. after 2-h incubation at 398C in modified Tyrode’s
medium Žincubation at ambient temperature does not produce this change., and ŽC. after slow cooling at
0.188Crmin to 58C and rapid rewarming to 398C. Area P represents dead cells, area Q represents cells with a
low calcium concentration and area R represents those with high calcium concentration. A strong similarity
can be seen in the responses in ŽB. and ŽC., indicating a rise in intracellular free calcium in both situations in
the majority of live cells.
488 P.F. Watsonr Animal Reproduction Science 60–61 (2000) 481–492

3.2. Motility impairment

One obvious characteristic of cryopreserved spermatozoa is the decline in motility of

the cells. While a minority seem to exhibit vigorous forward progression, the majority
show a variable degree of impairment. This would seem to be an important contribution
to their relatively poor fertilising potential when introduced into the reproductive tract at
artificial insemination. A study of cryopreserved human spermatozoa under IVF condi-
tions found that both motility and forward progression were important factors ŽKelly et
al., 1997.. However, one recent observation reached a different conclusion; in a study of
in vitro fertility of asthenoteratozoospermic dogs, the quality of motility though poor did
not impair fertility ŽHewitt and England, unpublished.. These studies relate only to
events associated with the final contact with the oocyte; transport to the site of
fertilisation may still require a minimum capability of forward motility, which may be
compromised in the majority of cryopreserved spermatozoa.

3.3. OxidatiÕe damage

More important is the observation that oxidative damage could impair sperm func-
tion. This subject is difficult to tackle because of the recognition that a degree of
superoxide radical formation is necessary for the events preceding fertilisation Žde
Lamirande et al., 1997.. However, it is now generally accepted that cryopreservation
induces the formation of reactive oxygen species that are detrimental to subsequent
performance ŽAlvarez and Storey, 1992; Bell et al., 1993; O’Flaherty et al., 1997.. Much
of the published work on lipid peroxidation and cryopreservation relates to work on
human spermatozoa.
The use of antioxidants in cryopreservation diluents is not commonplace, partly
because there are a number of possible agents from which to select, and partly because
different pathways Žand hence, different antioxidants. are utilised in different species
ŽAskari et al., 1994; O’Flaherty et al., 1997.. Moreover, it is clear that oxidative damage
is only one of a number of stresses the cryopreserved sperm cell experiences ŽAlvarez
and Storey, 1993.. This is clearly an area for further investigation.

3.4. Surface changes affecting recognition of receptors

An issue that has been recently examined is the extent to which the surviving
population is impaired in its ability to interact with the oviductal epithelium ŽEllington et
al., 1999.. These interactions are now recognised as receptor–ligand interactions, often
involving intracellular signalling mechanisms, which are actively being explored. A
similar interaction involves spermatozoa and the oocyte and its vestments ŽBwanga et
al., 1991; Oehninger et al., 1993.. The freeze-fracture observations, suggesting that the
clustering of membrane proteins during lipid phase separations induced by cooling are
not entirely reversible Žsee above., may well have implications for receptor–ligand
interactions. Although the evidence from IVF studies generally indicates that cryopre-
served spermatozoa are capable of effecting fertilisation Že.g., Donaghue et al., 1992.,
this is an artificial situation when hundreds of spermatozoa are present. In vivo, the
 P.F. Watsonr Animal Reproduction Science 60–61 (2000) 481–492 489

reality may be quite different when only a few spermatozoa reach the site of fertilisation
Žsee above..

3.5. Ability of sperm to sustain embryonic deÕelopment

Finally, another area that has not been adequately studied is the ability of cryopre-
served spermatozoa to sustain embryonic development. Technically, this is difficult to
investigate since the evidence of early embryonic death often passes without overt signs.
Nevertheless, a persistent suggestion is that frozen–thawed spermatozoa are associated
with an increased incidence of early embryonic mortality ŽSalamon and Maxwell, 1995..
The potential mechanisms can now be studied more effectively. DNA damage can be
investigated with the COMET assay ŽHughes et al., 1996. or with flow cytometry
ŽKarabinus et al., 1990; Royere et al., 1991. and may indicate functional damage to the
nuclear structures ŽEllington et al., 1998.. Moreover, the suggestion that the spermato-
zoon’s contribution to the zygote is more than the haploid male genome ŽNavara et al.,
1995. implies that there are more subtle ways in which the spermatozoon’s function
could be disrupted during cooling and cryopreservation. The post-syngamy fate of other
sperm structures is now known better than ever before ŽSutovsky et al., 1996; Sathanan-
than et al., 1997., and the possible importance of sperm RNA ŽRohwedder et al., 1996;
Miller, 1997. to the events before the embryonic genome is activated cannot be
disregarded. Any of these structures may suffer alteration during cryopreservation and
can all now be investigated.

4. Conclusion

The effects that cryopreservation can induce in spermatozoa, ranging from lethal
injury to those which merely impair subsequent function, are numerous. In the last few
years, the considerable increase in our understanding both of the cell physiology of
spermatozoa and of the stresses of cryopreservation has contributed to a renewed interest
in improving the performance of cryopreserved semen.
Today, the biotechnological applications of cryopreservation enjoy an interest which
is unprecedented. Sperm freezing for human infertility, for addressing adverse fertility
consequences of other life-threatening diseases, for conservation and for animal agricul-
ture all clamour for successful cryopreservation techniques. We can look forward to a
very productive period in our studies.

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