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- Original Article -
Cryopreservation of cynomolgus monkey (Macaca
fascicularis) spermatozoa in a chemically defined extender
Ya-Hui Li1,2,3, Ke-Jun
Cai1, Lei Su1, Mo
Guan1, Xie-Chao He1, Hong
Wang1, Andras Kovacs4, Wei-Zhi
1Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
2Graduate School of the Chinese Academy of Sciences, Beijing 100039, China
3College of Food Science and Technology, Yunnan Agricultural University, Kunming, Yunnan 650201, China
4Research Institute for Animal Breeding and Nutrition, 2053 Herceghalom, Hungary
Aim: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender.
Methods: Semen samples were collected by electro-ejaculation from four sexually mature male
cynomolgus monkeys. The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free
Tes-Tris (mTTE) synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1 %,
3 %, 5 %, 10 % and 15 % [v/v]) and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw
spermatozoa were examined by sperm motility and sperm head membrane integrity.
Results: The post-thaw motility and head membrane integrity of spermatozoa were significantly higher
(P<0.05) for 5 % glycerol (42.95¡À2.55 and 50.39¡À2.42, respectively) than those of the other groups (1 %: 19.19¡À3.22
and 24.84¡À3.64; 3 %: 34.23¡À3.43 and 41.37¡À3.42; 10 %: 15.68¡À2.36 and 21.39¡À3.14; 15 %:
7.47¡À1.44 and 12.90¡À2.18). The parameters for 30 min equilibration (42.95¡À2.55 and 50.39¡À2.42) were
better (P < 0.05) than those of the other groups (10 min: 31.33¡À3.06 and 38.98¡À3.31; 60 min:
32.49¡À3.86 and 40.01¡À4.18; 90 min: 31.16¡À3.66 and 38.30¡À3.78). Five percent glycerol and 30 min
equilibration yielded the highest post-thaw sperm motility and head membrane integrity. Conclusion: Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which
is related to the concentration and the equilibration time of glycerol.
(Asian J Androl 2005 Jun; 7(2): 139_144)
Keywords: cryopreservation; Macaca
fascicularis; chemically defined extender; glycerol
Correspondence to: Dr Wei-Zhi Ji, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan 650223, China.
Received 2004-07-22 Accepted 2005-02-04
Although spermatozoa of many mammalian species can be frozen successfully, a high proportion of
spermatozoa lose their motility or other functions after thawing due to damage during the freezing-thawing process
and the recovery of motile spermatozoa is less than
50 % in most mammals . This low freezing efficiency may be due to the lack of information on the
mechanisms of sperm injury during cryopreservation and
cryoprotective mechanisms of certain compounds.
Several hypotheses such as cold shock, osmotic stress, ice crystal formation  and oxidative damage
 have been put forward to explain the cause of sperm
cryoinjury, but the mechanisms of cryodamage largely
remain unclear. Therefore, sperm cryopreservation has remained empirical and, at present, even with the most
advanced equipment and the optimal freezing protocol,
only about 50 % of spermatozoa can survive cryopreservation.
Glycerol is a preferable cryoprotectant for sperm
freezing in most mammals. Additionally, complex agents
such as egg yolk, skim milk, milk and even serum are
used in sperm freezing extenders for many different
species in order to provide maximal cryoprotection for
spermatozoa. As complex components of freezing extenders such as egg yolk contain a large number of
undefined molecules, it is difficult to analyze the roles of a
particular compound played in sperm preservation. In
addition, these complex compounds not only contain
potential infectious agents (viruses, bacteria, fungi,
prions) but are with difficultly-controlled quality.
Therefore, a chemically defined extender would be
helpful to understand the mechanism of both sperm cryodamage and cryoprotection. Previous studies have
described how the spermatozoa of a mouse [4, 5], goat
 and man  were successfully frozen in a
chemically defined extender, but not yet those of nonhuman
Sperm motility and functional intact membrane are
interrelated and both are required for fertilization .
Sperm membranes may be irreversibly damaged by the
process of freezing-thawing that leads to a marked
decline in sperm motility.
In the present study, efforts have been made to
develop a method for the cryopreservation of the
cynomolgus monkey sperm in a synthetic extender.
Frozen-thawed sperm's function was evaluated by sperm
motility and sperm head membrane integrity, which are two
important factors related to fertilization.
2 Materials and methods
All chemicals were obtained from Sigma Chemical
Co. (St Louis, MO, USA), unless indicated otherwise.
2.1 Dilution and freezing extenders
The extender for dilution of cynomolgus monkey sperm was based on Tes-Tris-egg yolk (TTE) , but in
this study it did not contain egg yolk and was
abbreviated to mTTE (Table 1). The extender was prepared as
follows: after dissolving all the ingredients in Milli-Q water
and adjusting the pH to 7.0_7.2 with 1 mol/L NaOH or
HCl, the extender was divided into 4-mL aliquots and
stored at 30 ¡æ for no more than 2 weeks and thawed in
a 37 ¡æ water bath before use. The freezing extender
was made by adding glycerol to the mTTE to reach final
concentrations of 1 %, 3 %, 5 %, 10 % and 15 % (v/v),
2.2 Semen collecting and processing
Four sexually mature male cynomolgus monkeys, aged 5_11 years, were provided by the Laboratory
Animal Center of the Kunming Institute of Zoology. The
animals were individually caged with lights on from
06:00 to 18:00 at a temperature of 20_25 ¡æ. Animals were
anesthetized using 5 mg of ketamine hydrochloride
(Xin-Gang Co. Shanghai, China)/kg body weight i.m. and
stimulated with penile electrodes. Ejaculated semen was
collected into a disposable plastic test tube containing
2 mL of pre-warmed Tyrode's albumin lactate pyruvate
(TALP)-Hepes . The diluted semen was kept at 37
¡æ water bath for 30 min to allow liquefaction. After
that, the semen was transferred into a 15-mL disposable
plastic tube. A small sample was taken to examine the
sperm motility and head membrane integrity (see below)
and the rest was washed twice with TALP-Hepes at a
rate of 1:9 (1 part of semen for 9 parts of TALP-Hepes)
and centrifuged at 200 ¡Á g for 10 min. The supernatant
was aspirated and the sperm pellet was dispersed and
mixed with a Pasteur pipette. Volume and concentration
of sperm were determined, only ejaculates with a
concentration of 2.0_4.0
¡Á 109 sperm/mL and containing
¡Ý 70 % motile spermatozoa were used for freezing.
2.3 Freezing and thawing of spermatozoa
The washed spermatozoa were diluted 1:9 slowly in
mTTE and kept at room temperature (12_18 ¡æ) for 60
min. An equal volume of mTTE containing glycerol was
added in steps at 6_7 min intervals at room temperature
within 30 min to reach a final glycerol concentration of
1 %, 3 %, 5 %, 10 % and 15 %, respectively.
The spermatozoa were equilibrated in the glycerol
extender for some period and the equilibration times tested
at room temperature, were 10, 30, 60 and 90 min in
mTTE extender containing 5 % glycerol, respectively.
After equilibration, spermatozoa were drawn into
0.25-mL plastic straws (IMV, L'Aigle, France), sealed
with a straw heater (Tew Impulse Sealer, Tish-200, Tew
Electric Heating Equipment Co. Ltd., Taiwan ) and then
kept in a 4 ¡æ refrigerator for 45 min. Straws were
frozen by putting them horizontally on a rack 3 cm above
the surface of liquid nitrogen. Ten minutes later, they
were submerged directly into liquid nitrogen
(LN2) for storage.
After storage in LN2 for more than 7 days, straws
containing frozen spermatozoa were placed in a 37 ¡æ
water bath for 2 min for thawing. Thawed sperm
suspension was diluted in a disposable sterile test tube with
1.25-mL pre-warmed TALP-Hepes, which was added in five parts at 30 s intervals. The diluted sperm was washed
twice by centrifugation at
200 ¡Á g for 10 min, and the sperm pellet was dispersed immediately.
2.4 Examination of sperm motility and head membrane
2.4.1 Sperm motility
Using a pre-warmed hemocytometer counting chamber, fresh and thawed sperm samples were assessed
for percentage of forward progressive motility by
counting 200 spermatozoa in duplicates. This evaluation was
conducted by another operator who did not know the
identity of the sperm samples offered.
2.4.2 Sperm motility recovery rate
The sperm motility recovery rate was calculated by
comparing the motilities of pre-freeze
(Mpr) and post-thaw (Mps) spermatozoa. If
Mpr and Mps are the sperm motility percentages before and after freezing, then the
recovery rate would be:
Mps/Mpr ¡Á 100 %.
2.4.3 Sperm head membrane integrity
Sperm head membrane integrity was measured by counting non-damaged spermatozoa, defined as
non-stained by Hoechst 33258, as described by Cross
et al. . Shortly, 1 ¦ÌL of 0.5
mg/mL Hoechst 33258 was added to 500 ¦ÌL fresh or thawed sperm specimen (diluted
with warm TL-Hepes without protein) and stained at 37
¡æ for 10 min in the dark. Then, spermatozoa were
centrifuged at 250 ¡Á g for 5 min to remove excess stains.
The sperm pellet was resuspended in 100 ¦ÌL pre-warmed
TL-Hepes; 10_20 ¦ÌL of the suspension was smeared on
a microscope slide with a cover slip. Under ultraviolet
(UV) light, membrane intact spermatozoa showed little
or no blue fluorescence in the head, while spermatozoa
with damaged membrane showed bright blue fluorescence. At least 200 spermatozoa were scored for
each sample and the identities of the samples were
hidden from the observer to obtain an objective assessment.
2.5 Statistical analysis
A total of 16 ejaculates (four ejaculates per male)
were studied and experiments were conducted with
n = 16 replicates. All data are expressed as
mean ± SD. Data of sperm motility and head membrane integrity were
subjected to arcsine square root transformation and analyzed
by ANOVA and Fisher's protected least significant
difference (LSD) test. Differences were considered
statistically significant at P < 0.05.
3.1 Effect of glycerol concentration
The effect of glycerol concentration on post-thaw
sperm motility and sperm head membrane integrity is
shown in Table 2 and Figure 1. With the increase of
glycerol concentrations from 1 % to 5 %, the parameters of thawed spermatozoa rose significantly
(P < 0.05). However, all the examined values of sperm
decreased significantly when glycerol concentration
increased from 5 % to 15 % (P < 0.05). The highest
percentages of sperm motility and head membrane integrity
were achieved when the spermatozoa were frozen in mTTE extender containing 5 % glycerol
(P < 0.05).
3.2 Effect of equilibration time
The effect of equilibration time on post-thaw sperm
motility and head membrane integrity was presented in
Table 3 and Figure 2. The highest sperm motility and
head membrane integrity data were achieved when the
spermatozoa were equilibrated in mTTE extender containing 5 % glycerol for
30 min, more than those of the other groups
(P < 0.05). There was no significant
difference for sperm parameters among the 10, 60 and 90
min groups (P > 0.05).
TTE extender and the simple "straw freezing" procedure were successfully used for cryopreservation of
cynomolgus monkey spermatozoa . In the present
study, TTE was modified by omitting egg yolk to obtain
a chemically defined medium, in which glycerol
concentration was tested and the cryopreservation protocol
(duration of exposure to mTTE, glycerol equilibration
temperature) had been improved accordingly.
Glycerol has been widely used as a cryoprotectant
for freezing mammalian spermatozoa to protect them
against cryoinjury, but it can also damage sperm cells at
high concentration, depending on different species. For
example, marsupial spermatozoa can tolerate as high as
10 %_20 % glycerol, whereas mouse spermatozoa will
be dramatically damaged if the glycerol proportion
exceeds 1.75 % . For nonhuman primate spermatozoa,
the concentration of glycerol used in sperm
cryopreservation varied in a range of 3 %_14 % [13, 14]. In addition,
the optimal glycerol concentration for sperm
cryopreservation varies with other factors even within a certain
species. For instance, 3 % or 5 % glycerol was found
to be best for freezing cynomolgus monkey spermatozoa in straw [9, 13], whereas 5 % or 7 %_10 % glycerol
was optimal for pellet freezing on dry ice . Similarly,
Morrell  observed that the optimal concentration of
glycerol for cryopreserving marmoset sperm is lower
(2.5 %_5.0 %) for straw freezing than that for pellet
freezing (5.0 %_7.5 %). The result of this study showed
that the optimal concentration of glycerol was 5 % in the
mTTE medium, the same as the results reported by Sankai
et al. , but the temperature of adding extender with
glycerol was different. In the study of Sankai
et al. , the extender containing glycerol was added at a lower
temperature (i.e. 5 ¡æ) to minimize the toxicity of glycerol.
In the present study, however, exposure to glycerol was
performed at room temperature (12 %_18 ¡æ) to match
the temperature of the mTTE medium, avoiding
changeful temperature that may damage sperm cells. Previous
studies on rhesus  and vervet monkey  sperm
cryopreservation indicated that extenders containing
glycerol were added at 37 ¡æ or 32 ¡æ rather than 5 ¡æ and
the recovery of post-thaw motility was more than 60 %.
These results suggested that glycerol permeability might
have a wide range of temperature dependency.
Equilibration time with glycerol in cryoprotectant
extender is another factor to affect the survival of
frozen-thawed spermatozoa, because the speed and degree
of glycerol penetration of sperm is species-dependent.
Glycerol can penetrate into sperm membrane very quickly
for some species but slowly for others. Berndtson and
Foote  found that glycerol was able to permeate bull
spermatozoa within 3_4 min either at 25 ¡æ or 5 ¡æ and
maximum post-thaw motility occurred when they were
exposed to glycerol for as short as 10 s. However, Almlid
and Johnson  discovered that there was no
difference in sperm cryosurvival when boar semen was ex
posed to glycerol for 0.5, 2, 5, 15, or 75 min.
Equilibrium time is also quite different in nonhuman primate
sperm cryopreservation. Leverage et al.  and Morrell
 found that the equilibration time for rhesus and
marmoset sperm should be within 10 min to get a maximal
sperm survival. Mahone and Dukelow  concluded
that there was no difference in total sperm motility for
1-, 25- or 45-min glycerol equilibration in cynomolgus
monkeys. In Roussel and Austin's study ,
ejaculates from five species of nonhuman primates were added
to the glycerol extenders and equilibrated for 30 min at
room temperature and an average of 50 % post-thaw survival in the sperm was obtained. However, the result
of our study showed that sperm motility and head
membrane integrity for 10-, 60- and 90-min equilibration in
the chemically defined extender was significantly lower
(P < 0.05) than that of 30 min, indicating that shorter or
longer equilibration time was directly related with the cell
survival of cryopreservation. The shorter equilibration
time might not allow enough glycerol to penetrate into
spermatozoa, but longer exposure to glycerol could be
toxic for them. The results suggested that the
equilibration time not only depended on the species, but also
related with the extenders as well as experimental
In this study, four male animals used as semen
donors were chosen randomly from the Laboratory Animal
Center of Kunming Institute of Zoology and it was
unclear whether the semen of each individual was suitable
for cryopreservation. Additionally, no significant
difference was found in susceptibility of spermatozoa to
cryodamage among the individual males or between the
ejaculates in any donor. Even so, whether there is sperm
freezing variability among cynomolgus monkeys and if
the protocol described here is suitable for any donor of
this species, further investigation is required. Moreover,
though the post-thaw motility of spermatozoa is good
with the present procedure, their capability of
fertilization has to be examined.
In conclusion, our study demonstrates that cynomolgus monkey spermatozoa can be successfully
cryopreserved in a chemically defined extender. An
extender consisting of 5 % glycerol and 30 min
equilibration yielded the highest post-thaw sperm motility and head
membrane integrity. The concentration and
equilibration time of glycerol proved to be the key factors to
The authors thank Mr Bence Baranyai for his critical
revision of the manuscript. This work was supported
by grants from the Chinese Academy of Science's KSCX
1-05-01, major state research development program G2000016108, Ministry of Science and Technology of
China (2001 DEA 10009-09) and National Nature Science Foundation of China (No. 30370166).
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