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Effect of sugar type on the survival of frozen-thawed rhesus monkey (Macaca mulatta) sperm.

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American Journal of Primatology 68:103–108 (2006)
BRIEF REPORT
Effect of Sugar Type on the Survival of Frozen-Thawed
Rhesus Monkey (Macaca mulatta) Sperm
WEI SI1,2, HONG WANG1,2, CATHERINE REID3, THOMAS B. HILDEBRANDT3,
and WEIZHI JI1,2n
1
Kunming Primate Research Center, the Chinese Academy of Sciences, Yunnan, China
2
Kunming Institute of Zoology, the Chinese Academy of Sciences, Yunnan, China
3
Institute for Zoo Biology and Wildlife Research, Berlin, Germany
Sperm-freezing extenders supplemented with sugar or a combination of
different sugars are widely used for the cryopreservation of nonhuman
primate spermatozoa. Understanding which sugar or combination of
sugars offers the highest level of cryoprotection would be beneficial for
the development of sperm-freezing extenders. In the present study we
aimed to investigate the effect of glucose, lactose, and raffinose separately
or in combination on the cryosurvival of rhesus monkey spermatozoa.
Toward that end, we prepared eight extenders by adding various types of
sugars to a basic medium (BM): G-BM (0.3 M glucose), L-BM (0.3 M
lactose), R-BM (0.3 M raffinose), LG-BM (0.15 M lactose+0.15 M glucose),
RG-BM (0.15 M raffinose+0.15 M glucose), LR-BM (0.15 M lactose+0.15
M raffinose), and LRG-BM (0.1 M lactose+0.1 M raffinsoe+0.1 M
glucose). A saline control (0.157 M sodium chloride) was also used. The
results showed no significant difference in post-thaw motility when
spermatozoa were frozen with G-BM, L-BM, LG-BM, RG-BM, and LRGBM, but the post-thaw motility was significantly lower when it was frozen
with R-BM, LR-BM, and the saline control. The highest plasma
membrane integrity was achieved when spermatozoa were frozen with
G-BM, L-BM, LG-BM, RG-BM, and LRG-BM, and the highest acrosome
integrity was achieved with G-BM, L-BM, LG-BM, RG-BM, LRG-BM, and
the saline control. The results indicate that the various sugars offered
different protective effects. For the cryopreservation of rhesus monkey
spermatozoa, glucose (monosaccharide) and lactose (disaccharide) were
shown to be more suitable than raffinose (trisaccharide) for preserving
spermatozoal motility, plasma membrane, and acrosome. Specifically,
Contract grant sponsor: Chinese Academy of Sciences; Contract grant number: KXCX1-05;
Contract grant sponsor: Ministry of Science and Technology of China; Contract grant number:
2001DEA10009-09; Contract grant number: China National Science Foundation; Contract grant
number: 30370166.
n
Correspondence to: Weizhi Ji, Kunming Institute of Zoology, Chinese Academy of Sciences,
Jiaochang Donglu 32, Kunming, Yunnan 650223, China. E-mail: wji@mail.kiz.ac.cn
Received 12 December 2004; revised 4 April 2005; revision accepted 7 April 2005
DOI 10.1002/ajp.20209
Published online in Wiley InterScience (www.interscience.wiley.com).
r
2006 Wiley-Liss, Inc.
104 / Si et al.
raffinose was detrimental to sperm acrosome integrity. Am. J. Primatol.
r 2006 Wiley-Liss, Inc.
68:103–108, 2006.
Key words: rhesus monkey; sperm; cryopreservation; sugar
INTRODUCTION
Supplementing semen-freezing extenders with sugar as a nonpenetrating
cryoprotectant has demonstrated beneficial effects on the cryosurvival of
spermatozoa. The protective effects of sugar have been reported to include cell
dehydration, which reduces intracellular ice crystal formation [Chen et al., 1993],
and the prevention of liposome fusion and leakage during dehydration and
rehydration [Crowe et al., 1986]. Recently, it was found that the various types of
sugars have different cryoprotective effects [Yildiz et al., 2000]. For the
cryopreservation of nonhuman primate spermatozoa, the most extensively used
sugars are glucose, lactose, and raffinose [Morrell & Hodges, 1998]. However, to
our knowledge, no reports have been published on nonhuman primate
spermatozoa cryopreservation comparing the effect of various types of sugars as
cryoprotectants in one species, and the influence of these different sugars on
nonhuman primate spermatozoa during freezing and thawing is not clear.
Determining which sugar or combinations of sugars provide the highest postthaw parameters would be beneficial for the development of freezing extenders
and the cryosurvival of spermatozoa. Thus, the present study was designed to
examine the effect of adding glucose (monosaccharide), lactose (disaccharide), and
raffinose (trisaccharide) separately or in combination on sperm motility and
plasma membrane/acrosome integrity of rhesus monkey spermatozoa during
freezing and thawing. Eight sperm-freezing extenders containing various types of
sugars in different concentrations and combinations were tested.
MATERIALS AND METHODS
Sperm-Freezing Extender Preparation
We prepared the freezing extenders by dissolving various types of sugars in
different concentrations and combinations in a basic medium (BM), which was
prepared by the addition of 10% (v/v) fresh egg yolk to Milli-Q water. The
compositions of the extenders are listed in Table I.
Table I. The Composition of the Sperm-Freezing Extendersn
Concentration (M)
Components
Glucose
Lactose
Raffinose
NaCl
Egg yolk
Glycerol
G-BM
L-BM
R-BM
LG-BM
RG-BM
LR-BM
LRG-BM
Saline
control
0.3
–
–
–
10%
10%
–
0.3
–
–
10%
10%
–
–
0.3
–
10%
10%
0.15
0.15
–
–
10%
10%
0.15
–
0.15
–
10%
10%
–
0.15
0.15
–
10%
10%
0.1
0.1
0.1
–
10%
10%
–
–
–
0.157
10%
10%
n
Note. All chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) unless indicated otherwise. The
extenders were centrifuged at 7000g for 1 hr to separate egg yolk droplets. Before use, glycerol was added to the
concentration of 10%.
Am. J. Primatol. DOI 10.1002/ajp
Effect of Sugar Type on Cryopreservation / 105
Semen Collection and Freezing
Ten semen samples were collected by penile electro-ejaculation from four
male rhesus macaques (8–10 years old) provided by the Kunming Primate
Research Center, Chinese Academy of Sciences. To avoid disturbing sugar from
the seminal plasma, the semen samples were washed with TALP-Hepes
containing 3 mg/mL BSA by centrifugation at 150 g for 10 min. Each sample
was divided into eight aliquots and diluted with nine volumes of the eight
experimental freezing extenders without glycerol at room temperature. The
extended samples were equilibrated at 41C for 2 hr, and further diluted with an
equal volume of the same freezing extender variant containing 10% glycerol to
reach a final concentration of 5% glycerol. The samples were loaded in 0.25-ml
cryostraws (IMV, L’Aigle, France) and sealed. To freeze the specimens, the straws
were held horizontally 5 cm above liquid nitrogen for 10 min and then submerged
directly into the liquid nitrogen for storage [Sankai et al., 1994]. For thawing, the
straws were plunged directly into a 371C water bath for 2 min. The spermatozoa
sample was then diluted with 5 volumes of TALP-Hepes containing 3 mg/mL
BSA, for 5 times at 30-sec intervals.
Sperm Motility
With the use of a prewarmed hemocytometer counting chamber, we assessed
the fresh unwashed sperm samples and thawed sperm samples for percentage of
progressive motility by counting 200 in duplicate. The sperm motility recovery
rate was calculated by the following formula: (post-thaw motility % 100)/fresh
sperm motility %.
Assessment of Sperm Membrane and Acrosome Integrity
Nuclei stain Hoechst 33258 and fluorescein isothiocyanate-conjugated peanut
agglutinin (FITC-PNA) was used to determine the integrity of the sperm plasma
membrane and acrosome, as described by Cross et al. [1986] and Esteves et al.
[2000]. At least 200 spermatozoa were scored for each sample.
Statistical Analysis
All data were expressed as the means7SD. The percentage data of sperm
motility and membrane/acrosome integrity were subjected to arcsine square root
transformation before a statistical analysis was performed. An analysis of
variance (ANOVA), followed by Tukey’s test, was used to analyze the differences
among different groups. Values with Po0.05 were considered statistically
different.
RESULTS
Sperm Motility After Freezing and Thawing
As shown in Table II, similar post-thaw spermatozoa motility and motility
recovery rates were found in G-BM, L-BM, LG-BM, RG-BM, and LRG-BM groups
(P40.05). These rates were significantly higher than those of the R-BM, LR-BM,
and saline control groups (Po0.05). The extender containing only raffinose
(R-BM) and the saline control group provided similar cryoprotection for sperm
motility (P40.05). This was significant lower than that obtained from the
medium containing lactose and raffinose together (LR-BM) (Po0.05).
Am. J. Primatol. DOI 10.1002/ajp
106 / Si et al.
Table II. Motility of Rhesus Monkey Spermatoza After Freezing and Thawing With Various
Types of Sugarsn
Sperm-freezing
extender
Saline control
G-BM
L-BM
R-BM
LG-BM
RG-BM
LR-BM
LRG-BM
Fresh sperm
motility (%)
Post-thaw
sperm motility (%)
Motility
recovery rate (%)
72.976.5
18.573.9a
41.274.6b
38.176.0b
16.073.1a
39.775.6b
38.173.6b
28.175.9c
42.378.1b
25.776.6a
55.878.5b
52.678.8b
22.174.6a
54.576.1b
52.574.9b
39.179.8c
58.4712.1b
n
Different superscripts in a column indicate significant difference (Po0.05).
Table III. Plasma Membrane Integrity and Acrosoem Integrity of Rhesus Monkey Spermatozoa after Freezing and Thawing with various Types of Sugarsn
Integrity (%)
Sperm-freezing extender
Fresh sperm
Saline control
G-BM
L-BM
R-BM
LG-BM
RG-BM
LR-BM
LRG-BM
Plasma membrane
Acrosome
77.475.3a
18.975.5b
48.772.4c
45.474.0c,d
23.776.7b
48.874.4c
45.374.8c,d
38.976.2d
47.275.4c
89.771.9a
76.474.1b
77.874.4b
78.173.2b
62.9710.6c
80.376.2b
77.378.9b
68.975.7c
77.574.5b
n
Different superscripts in a column indicate significant different (Po0.05).
Sperm Plasma Membrane and Acrosome Integrity of Fresh and
Cryopreserved Sperm
The status of the plasma membrane and acrosome of the rhesus spermatozoa
cryopreserved with various types of sugars is shown in Table III. Compared to
fresh sperm, cryopreservation damaged the plasma membrane significantly
(Po0.05). The plasma membrane integrity obtained with G-BM, L-BM, LG-BM,
RG-BM, and LRG-BM was significantly higher than that obtained with R-BM and
the saline control after cryopreservation (Po0.05). No statistical difference was
found among LR-BM, L-BM, and RG-BM (P40.05). R-BM and the saline control
provided the worst cryoprotection of the plasma membrane among the eight
groups (Po0.05), but no difference was found between these two groups
(P40.05).
Compared to fresh sperm, the percentage of cryopreserved spermatozoa with
intact acrosome decreased significantly (Po0.05). G-BM, L-BM, LG-BM, RG-BM,
LRG-BM, and the saline control showed similar protection values for sperm
acrosome (P40.05), but they were significantly higher than those obtained with
R-BM and LR-BM (Po0.05).
Am. J. Primatol. DOI 10.1002/ajp
Effect of Sugar Type on Cryopreservation / 107
DISCUSSION
Sugars have been widely used in animal spermatozoa cryopreservation.
Previous studies indicated that monosaccharide is more effective than disaccharide for freezing ram sperm [Molinia et al., 1994], and trisaccharide is a less
effective cryoprotective agent than monosaccharide for bull spermatozoa [Garcia
& Graham, 1989]. In our study, the individual sugars glucose (G-BM), and lactose
(L-BM) were more effective in protecting sperm motility and plasma membrane/
acrosome integrity compared to raffinose (R-BM) for rhesus monkey spermatozoa
cryopreservation. No difference was found between monosaccharide and
disaccharide for the rhesus monkey. However, both monosaccharides and
disaccharides were more effective than trisaccharide. On the other hand, it has
been reported that some types of sugars damage the spermatozoa acrosome
during freezing and thawing [Yildiz et al., 2000]. In dogs, glucose, lactose, and
raffinose increased the percentage of damaged acrosomes after freezing and
thawing, but fructose, galactose, xylose, trehalose, malthose, and sucrose
decreased the percentage of damaged acrosomes. However, in the rhesus monkey,
of the three sugars examined (G-BM, L-BM, and R-BM), only raffinose (R-BM)
increased the percentage of spermatozoa with damaged acrosome compared to the
saline control (without sugar). The results indicate that the cryoprotective effects
of various types of sugars may be species dependent.
Sugars usually do not penetrate the sperm membrane, and they provide
cryoprotection outside of the membrane by stabilizing cell membranes via
the loose, electrostatic binding of saccharide hydroxyl groups to phosphate groups
on the membrane lipid head [Chen et al., 1993]. In our study, compared to the
saline control group (without sugar), the post-thaw sperm motility and plasma
membrane integrity of spermatozoa cryopreserved by G-BM, L-BM, LG-BM, RGBM, and LRG-BM were significant higher. However, no statistical difference in
sperm acrosome integrity was found between these groups and the saline
control. The results indicate that during freezing and thawing, the motility and
plasma membrane of rhesus monkey spermatozoa was mostly damaged when
extender without sugar was used, and compared to sodium chloride, the sugars
provided effective protection outside of the membrane to the sperm plasma
membrane. It has been suggested that disaccharide is more effective than
monosaccharide because the former has a lower carbon : hydrogen (C:H) ratio
and a greater number of hydroxyl groups per molecule [Bakas & Disalvo,
1991]. However, our results revealed no significant difference between
glucose and lactose (G-BM vs. L-BM). The discrepancy may due to the fact that
in addition to the effect of stabilizing the cell membrane, sugars may also affect
the pattern of crystallization, the shape and width of the channels in the
unfrozen solution, and the mechanical properties of the solidified medium, which
could relieve or prevent damage to spermatozoa [Nicolajsen & Hvidt, 1994].
The sugar-type-dependent properties and the different mechanisms of cryoprotection could contribute to the different cryoprotective properties of various
types of sugars.
Because of the different cryoprotective mechanisms found in various types of
sugars, it may be hypothesized that the combined use of monosaccharide and
disaccharide in proper concentrations could provide better protection than that
achieved with the use of monosaccharide or disaccharide alone [Yildiz et al.,
2000]. In the present study, the results indicate that the combined use of
monosaccharide, disaccharide, and trisaccharide could provide better protection compared to the use of trisaccharide alone (LG-BM, RG-BM, LRG-BM, and
Am. J. Primatol. DOI 10.1002/ajp
108 / Si et al.
LR-BM vs. R-BM), and in a low concentration (0.15 M), glucose combined with
raffinose could provide better cryoprotection than lactose combined with raffinose
(RG-BM vs. LR-BM).
In conclusion, this study shows that glucose and lactose have better
protective effects than raffinose for preserving sperm motility, plasma membrane
function, and acrosome integrity. Raffinose was detrimental to the acrosome
integrity of rhesus monkey spermatozoa. These results can help researchers
design more effective sperm-freezing extenders and improve the cryopreservation
of nonhuman primate spermatozoa.
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sugars and sugar alcohols on post thaw
motility. Theriogenology 31:1029–1037.
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Morrell JM, Hodges JK. 1998. Cryopreservation of non-human primate sperm: priorities for future research. Anim Reprod Sci
53:43–63.
Nicolajsen H, Hvidt A. 1994. Phase behavior
of the system trehalose-NaCl-water. Cryobiology 31:199–205.
Sankai T, Terao K, Yanagimachi R, Cho F,
Yoshikawa Y. 1994. Cryopreservation of
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