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Effect of amino acids on cryopreservation of cynomolgus monkey (macaca fascicularis) sperm.

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American Journal of Primatology 59:159–165 (2003)
BRIEF REPORT
Effect of Amino Acids on Cryopreservation of Cynomolgus
Monkey (Macaca fascicularis) Sperm
YAHUI LI1–3, WEI SI1,2, XIUZHEN ZHANG1,2, ANDRAS DINNYES4, and WEIZHI JI1n
1
Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan, People’s
Republic of China
2
Graduate School, Chinese Academy of Sciences, Beijing, People’s Republic of China
3
College of Food Science and Technology, Yunnan Agricultural University, Kunming
Yunnan, People’s Republic of China
4
Research Group for Applied Animal Genetics and Biotechnology, Hungarian Academy of
Sciences and Szent Istvan University, Godollo, Hungary
The effects of three amino acids (proline, glutamine, and glycine) added
to the freezing medium Tes-Tris-egg yolk (TTE) for cryopreservation of
cynomolgus monkey (Macaca fascicularis) spermatozoa were studied.
This is the first report on the effects of amino acids on nonhuman primate
sperm cryopreservation. The addition of 5 mM proline, 10 mM glutamine,
and 10 or 20 mM glycine each significantly improved post-thaw sperm
motility and membrane and acrosome integrity compared with the
control (TTE alone). However, a significant decrease in motility and
membrane/acrosome integrity was observed when amino acid concentrations increased to 60 mM for proline and glutamine, and 80 mM for
glycine. The results suggest that adding a limited amount of amino acids
to the freezing media is beneficial for freezing cynomolgus monkey
sperm. Am. J. Primatol. 59:159–165, 2003. r 2003 Wiley-Liss, Inc.
Key words: amino acid; sperm; cryopreservation; Macaca fascicularis
INTRODUCTION
Sperm cryopreservation is an important element in preserving rare or
endangered animals, treating human infertility, and further developing reproductive techniques, such as artificial insemination (AI) and in vitro fertilization
(IVF). Quite a few spermatozoa are damaged during the freeze-thaw process.
Many factors, such as the cryoprotectant used, influence this cryodamage.
Glycerol has been widely used as a cryoprotectant during sperm cryopreservation.
Various other compounds have also been demonstrated to have the ability to
Contract grant sponsor: China National Science Foundation; Contract grant number: 39970117;
Contract grant sponsor: Chinese Academy of Sciences; Contract grant number: KZ952-J1-109.
n
Correspondence to: Weizhi Ji, Kunming Institute of Zoology, Chinese Academy of Sciences, 32
Estern Jiaochang Road, Kunming, Yunnan 650223, People’s Republic of China.
E-mail: wji@mail.kiz.ac.cn(W.Ji)
Received 3 December 2002; revision accepted 14 February 2003
DOI 10.1002/ajp.10073
Published online in Wiley InterScience (www.interscience.wiley.com).
r
2003 Wiley-Liss, Inc.
160 / Li et al.
protect sperm during freezing [Chen et al., 1993; De Leeuw et al., 1993; Sztein
et al., 2001; Woelders et al., 1997], and an intensive search for new cryoprotectants continues. Certain plants are known to accumulate the amino acid
proline to resist cold temperatures [Stewart & Lee, 1974], and it has also been
reported that some amino acids protect animal cells against hypothermia [Kruuv
& Glofcheski, 1992]. Protective effects of the amino acids glutamine, proline,
glycine, alanine, taurine, and hypotaurine during cryopreservation of spermatozoa in rams [Kundu et al., 2001], stallions [Lindeberg et al., 1999; Trimeche et al.,
1999], bulls [Chen et al., 1993], and humans [Renard et al., 1996] have been
described. However, no reports have been published on nonhuman primate sperm
preservation using amino acids as cryoprotectants.
In the present study, we investigated the cryoprotective potential of three
amino acids for spermatozoa of the cynomolgus monkey, one of the most widely
used nonhuman primate models.
METHODS
Media Preparation
All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless
otherwise indicated.
The base for the cryoprotectant solutions was Tes-Tris-egg yolk (TTE), as
described by Sankai et al. [1994]. Before the experiments were conducted, Lproline was added to the TTE at concentrations of 1, 3, 5, 10, 20, 40, or 60 mM.
Both L-glutamine and L-glycine were added separately to the medium at 3, 5, 10,
20, 40, 60, and 80 mM. All freezing media were adjusted to pH 7.0–7.2. The
osmotic pressures of the media were measured with an automatic osmometer
(Osmette A, 5002; Precision Systems, Inc., Natick, MA). Finally, 10% glycerol was
added to prepare the freezing medium (the final concentration for sperm freezing
was 5% glycerol).
Semen Collection and Cryopreservation of Spermatozoa
Fifteen sperm samples were collected by penile electroejaculation [Si et al.,
2000] from four anesthetized cynomolgus monkeys (5–11 years old) provided by
the Laboratory Animal Center of the Kunming Institute of Zoology, Chinese
Academy of Sciences. Semen was collected into a disposable plastic test tube
containing 2 ml of TALP-Hepes [Bavister et al., 1983], which was kept at 371C in a
water bath for 30 min to allow the clot to liquefy. After the clot liquified, a small
sample was examined for sperm motility and membrane and acrosome integrity;
the rest was washed twice with 9 volumes of TALP-Hepes and centrifuged at
200 g for 10 min. The supernatant was then aspirated, and the sperm pellet was
dispersed and mixed with a Pasteur pipette before it was frozen.
The sperm freezing procedure was identical to that described by Sankai et al.
[1994]. After they were stored in liquid nitrogen for more than 7 days, 0.25-ml
plastic straws (IMV, L’Aigle, France) containing frozen spermatozoa were thawed
in a 371C water bath for 2 min. Thawed sperm suspension was diluted with 5
volumes of TALP-Hepes, 5 times at 30-sec intervals, and then washed twice with
TALP-Hepes by centrifugation at 200 g for 10 min. The sperm pellet was
dispersed immediately.
Amino Acids and Sperm Cryopreservation / 161
Examination of Sperm Motility and Membrane/Acrosome Integrity
Sperm motility.
With the use of a hemocytometer counting chamber, fresh and thawed sperm
samples were assessed for percentage of progressive motility by counting 200
spermatozoa, in duplicate.
Membrane integrity.
Sperm membranes were stained with Hoechst 33258 according to the
method of Mortimer et al. [1990]. At least 200 spermatozoa were scored for each
sample.
Acrosome integrity.
Acrosome staining was performed as described by Valcarcel et al. [1997], with
the exception that in the present study only FITC-PNA was used. A minimum of
200 spermatozoa per sample were counted.
Statistical Analysis
All data are expressed as mean 7 STD. The sperm motility and membrane/
acrosome integrity data were subjected to arcsine (square root) transformation
and analyzed by analysis of variance (ANOVA) and Tukey’s test. Po0.05 was
considered statistically significant.
RESULTS
Effect of L-Proline
The effect of L-proline on cynomolgus monkey sperm cryopreservation is
shown in Table I. When compared with the control, sperm motility and
membrane/acrosome integrity were significantly increased by the addition of
5 mM proline. However, the addition of 60 mM proline significantly decreased
sperm motility and membrane/acrosome integrity.
Effect of L-Glutamine
The effect of L-glutamine on cynomolgus monkey sperm cryopreservation
is presented in Table II. The addition of 10 mM glutamine improved sperm
motility, and 5–40 mM glutamine increased membrane integrity significantly.
Furthermore, 3–10 mM glutamine significantly improved acrosome integrity.
Overall, 10 mM glutamine provided the best cryoprotection for sperm. It was
found that 80 mM glutamine had negative effects for all three parameters
examined.
Effect of L-Glycine
The results of L-glycine addition are shown in Table III. The addition of
10–40 mM glycine significantly improved post-thaw motility. Membrane and
162 / Li et al.
acrosome protection was provided by both 10 and 20 mM glycine. At a
concentration of 80 mM, the three values decreased significantly. Overall,
10 mM glycine produced the highest values of sperm motility and membrane/
acrosome integrity.
TABLE I. Effect of Proline Addition on Cynomolgus Monkey Sperm Cryopreservation
Proline
addition
Osmotic
pressure
(mOsm/kg)
Fresh sperm
0 (TTE control)
1 mM
400
402
3 mM
403
5 mM
406
10 mM
408
20 mM
418
40 mM
442
60 mM
465
F(7,
32)
a,b,c,d,e
values
Motility
(%)
80.672.2
55.071.0ae
48.373.4b
(P=0.022)
51.571.3ab
(P=0.576)
64.073.2c
(P=0.001)
60.671.9ce
(P=0.071)
60.770.8ce
(P=0.065)
55.173.7ae
(P=1.000)
38.074.8d
(P=0.000)
41.159
Membrane
intact (%)
76.572.0
45.673.2ae
43.971.8e
(P=0.915)
47.271.6ce
(P=0.918)
54.573.1b
(P=0.000)
49.971.6ac
(P=0.55)
49.371.1ac
(P=0.126)
44.771.9e
(P=0.998)
36.471.7d
(P=0.000)
31.606
Acrosomal
integrity (%)
91.770.5
70.671.3ac
68.473.6c
(P=0.955)
69.172.1c
(P=0.993)
77.971.4be
(P=0.007)
75.274.2ae
(P=0.211)
75.172.8ae
(P=0.246)
67.074.8c
(P=0.631)
58.871.7d
(P=0.000)
19.607
Groups with different superscripts in the same column are significantly different (Po 0.05).
TABLE II. Effect of Glutamine Addition on Cynomolgus Monkey Sperm Cryopreservation
Glutamine
addition
Osmotic
pressure
(mOsm/kg)
Fresh sperm
0 (TTE control)
3 mM
400
402
5 mM
403
10 mM
408
20 mM
420
40 mM
440
60 mM
460
80 mM
482
F(7,
32)
a,b,c,d,e
values
Motility
(%)
81.471.4
55.174.7a
59.471.4ac
(P=0.298)
60.171.0abc
(P=0.145)
65.372.7b
(P=0.000)
57.573.3ac
(P=0.893)
54.371.0ac
(P=1.000)
45.873.2d
(P=0.000)
46.273.5d
(P=0.001)
27.087
Membrane
intact (%)
78.272.4
45.470.9ac
49.172.4ce
(P=0.092)
50.372.4e
(P=0.008)
58.872.4b
(P=0.000)
49.871.7e
(P=0.027)
49.672.2e
(P=0.036)
41.671.4ad
(P=0.076)
38.871.7d
(P=0.000)
48.299
Acrosomal
integrity (%)
92.970.8
69.573.7ae
74.772.7c
(P=0.042)
75.471.2c
(P=0.014)
80.571.6b
(P=0.000)
74.572.5ce
(P=0.055)
73.573.7ce
(P=0.216)
64.871.4ad
(P=0.101)
60.071.2d
(P=0.000)
34.202
Groups with different superscripts in the same column are significantly different (Po 0.05).
Amino Acids and Sperm Cryopreservation / 163
TABLE III. Effect of Glycine Addition on Cynomolgus Monkey Sperm Cryopreservation
Glycine
addition
Osmotic
pressure
(mOsm/kg)
Fresh sperm
0 (TTE control)
3 mM
400
402
5 mM
403
10 mM
410
20 mM
421
40 mM
440
60 mM
462
80 mM
482
F(7,
32)
a,b,c,d,e,f
values
Motility (%)
81.672.7
54.571.0ac
53.471.5c
(P=0.992)
53.973.0c
(P=1.000)
64.672.4b
(P=0.000)
64.271.7be
(P=0.000)
59.871.2de
(P=0.022)
56.773.2acd
(P=0.829)
40.273.1f
(P=0.000)
55.885
Membrane
intact (%)
78.771.2
50.172.9acde
47.271.4c
(P=0.598)
49.571.4cd
(P=1.000)
59.972.6b
(P=0.000)
59.473.0b
(P=0.000)
53.771.2de
(P=0.316)
51.374.0cd
(P=0.994)
41.971.6f
(P=0.000)
30.001
Acrosomal
integrity (%)
93.170.7
70.570.9a
63.675.7bd
(P=0.018)
70.572.9ae
(P=1.000)
78.771.5c
(P=0.001)
77.473.2cf
(P=0.007)
73.170.5aef
(P=0.808)
71.572.9ae
(P=0.999)
60.971.5d
(P=0.000)
23.259
Groups with different superscripts in the same column are significantly different (Po 0.05).
DISCUSSION
In this study we have shown that the addition of amino acids to spermfreezing media can be beneficial in the cryopreservation of cynomolgus monkey
sperm.
A few previous reports demonstrated that amino acids had certain
cryoprotective effects on sperm [Chen et al., 1993; Kundu et al., 2001; Lindeberg
et al., 1999; Renard et al., 1996; Trimeche et al., 1999]; however, none of these
studies involved nonhuman primates. Our study showed that proline, glutamine,
and glycine have a positive effect on sperm cryopreservation. Different amino
acids had different optimal concentrations (B5–20 mM), which were lower
than those reported in previous studies for other species (40–80 mM). Aside
from species differences, these contrasting findings can be explained by
differences in the extenders and freezing procedures used. In most previous
studies, the sperm was cooled by a programmed freezing machine at a rate of
10–201C/min, whereas in our study it was cooled much faster (at a rate of
approximately 601C/min). In the present study, the concentration of egg yolk in
the TTE was 20%; in Trimeche et al.’s [1999] study (in which semen was cooled at
a similarly fast rate) it was 2%.
The reasons for the cryoprotection provided by the three amino acids
tested in this study remain unclear. Kundu et al. [2001] suggested that
the protective effects of amino acids may stem from their ability to form a layer
on the sperm surface, as these positively charged molecules can combine with
the phosphate groups of the sperm plasma membrane phospholipids. In the
present study, however, at pH 7.0–7.2 of freezing media, proline, glutamine, and
glycine are negatively charged. According to Alvarez and Storey [1983], some
amino acids can prevent lipid peroxidation of sperm membrane during
164 / Li et al.
cryopreservation; however, whether this occurred in the present study remains to
be proved.
It is interesting that the addition of 1 mM proline significantly decreased
sperm motility while 3 mM glycine decreased acrosome integrity significantly.
There is currently no explanation for this, and it merits further study.
Trimeche et al. [1999] observed that high concentrations of glutamine are
detrimental to the freezing of sperm, and suggested that this may be the result of
high osmotic pressure when the concentration of glutamine rises. In our study,
sperm damage also occurred at high amino acid concentrations, in accordance
with the findings of Trimeche et al. [1999].
In conclusion, we have shown that the three amino acids studied had positive
effects on cynomolgus monkey sperm cryopreservation. The optimal concentrations for proline, glutamine, and glycine were 5, 10, and 10 mM, respectively.
However, at high concentrations all three amino acids had negative effects on
sperm after freezing. Further studies are needed to demonstrate whether other
amino acids have beneficial effects as well, and whether any positive additive
effects can be derived from combining several amino acids.
ACKNOWLEDGMENTS
We thank Dr. Lei Su, Hong Wang, and our colleague Yijing Pei for their
assistance.
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