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,
From * Kunming Institute of Zoology, Chinese
Academy of Sciences, Kunming, Yunnan, China;
Graduate School of the Chinese Academy of
Sciences, Beijing, China; College of Food
Science and Technology, Yunnan Agricultural University, Kunming, Yunnan,
China; and Research Institute for Animal
Breeding and Nutrition, Herceghalom, Gesztenyes, Hungary.
| Correspondence to: Prof Weizhi Ji, Kunming Institute of Zoology, Chinese Academy of Sciences, 32 Jiaochang Donglu, Kunming, Yunnan, 650223, China (e-mail: wji{at}mail.kiz.ac.cn). |
| Received for publication September 17, 2004; accepted for publication January 4, 2005. |
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Abstract |
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Key words: Sperm, freezing, motility, plasma membrane, acrosome, permeability coefficient
Cryoprotectant is another factor that influences sperm survival during sperm cryopreservation. Although glycerol is a commonly used permeating cryoprotectant in NHP sperm freezing, its toxicity can cause loss in sperm motility and fertility. This encourages searching for other less toxic, effective cryoprotectants for sperm freezing. Recently, other permeating cryoprotectants have been employed in sperm cryopreservation for nonprimate mammalian species. For instance, dimethyl sulfoxide (DMSO) has been successfully used in sperm freezing for rabbit (Vicente and Viudes-de-Castro, 1996), rhinoceros (O'Brien and Roth, 2000), and mouse (Sztein et al, 2001); similar success have been obtained in ram (Molinia et al, 1994) and stallion (Mantovani et al, 2002) with ethylene glycol, as well as in rabbit with acetamide (Arriola and Foote, 2001). As for NHP, dimethyl sulfoxide, propylene glycol, and ethylene glycol have been tested in sperm cryopreservation for gorillas (Sadleir, 1966; Gould and Styperek, 1989), cynomolgus monkeys (Feradis et al, 2001), and rhesus monkeys (Si et al 2004). Even with one such certain cryoprotectant, the results are different between laboratories and species. Since different extenders were used to freeze spermatozoa in those studies, whether extender type affects the efficiencies of these permeating cryoprotectants is yet to be proved. However, no studies have compared the effect of cryoprotectants other than glycerol on sperm freezing using various extenders at the same time. In addition, no reports have been published on NHP sperm cryopreservation using acetamide as a permeating cryoprotectant.
In the present study, the comparison of 11 extenders for sperm cryopreservation has been made using cynomolgus monkey as a NHP model and several optimal extenders for cryopreservation have been sifted. On the basis of this, effects of 5 commonly used permeating cryoprotectants (ie, glycerol, ethylene glycol, dimethyl sulfoxide, propylene glycol, and acetamide) on sperm cryopreservation for this species have been examined.
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Materials and Methods |
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Media Preparation
The composition and osmolality of extenders as well as the concentrations
of glycerol are presented in Table
1. Penicillin G (6.32 mg) and Streptomycin (sulfate) (5.0 mg) were
added separately to each extender (100 mL), and the pH was adjusted to
7.0–7.2. The osmolality of extenders was measured using a freezing-point
depression osmometer (Osmette A, 5002, Precision System Inc, Natick,
Mass).
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The extender was prepared as follows: fresh chicken eggs laid within 8 hours were purchased from a hennery for preparation of extender. Egg yolks were obtained with the procedure of Si et al (2000). For TCG preparation, egg yolks were mixed with a portion of Milli-Q water first, and then the mixture was centrifuged at 7000 x g for 1 hour at 4°C to sediment yolk granules. The supernatants were extracted, and the remaining components were dissolved in water complemented accordingly. For other extenders, after dissolving all the ingredients in Milli-Q water, the egg yolk was added and mixed thoroughly and then the yolk granules were separated by centrifugation at 7000 x g for 1 hour. The supernatants were adjusted to pH 7.0–7.2 with 1 N NaOH or HCl when needed and used as extenders. The extenders were divided into 4-mL aliquots and stored at -30°C for no more than 2 weeks. Before an experiment, extenders were thawed in a 37°C water bath. The freezing extenders were made by adding cryoprotectants (glycerol, ethylene glycol, dimethyl sulfoxide, acetamide, propylene glycol) to extenders to obtain extenders with certain cryoprotectant concentrations.
Semen Collection
Four sexually mature male cynomolgus monkeys, aged 5–11 years,
provided by the Laboratory Animal Center of the Kunming Institute of Zoology,
were used for collecting semen. The animals were individually caged and kept
with lights on from 0600 to 1800 hours at a temperature of
20°C–25°C. Each animal was anaesthetized with ketamine
hydrochloride (Xingang Co, Shanghai, China) using 5 mg/kg body weight
intramuscularly and stimulated with penile electro-ejaculation procedure
elaborated in our laboratory (Yang et al,
1994). Semen was collected into a disposable plastic test tube
containing 2 mL of prewarmed TALP-Hepes
(Bavister et al, 1983). The
diluted semen was kept at 37°C water bath for 30 minutes to allow the clot
to liquefy. After liquefaction, the semen was transferred into a 15-mL
disposable plastic tube. A small sample was taken to examine sperm motility,
plasma membrane, and acrosomal integrity (see below), the rest was washed
twice with 9 mL TALP-Hepes and centrifuged at 200 x g for 10
minutes. The supernatant was aspirated and the sperm pellet was dispersed and
mixed with a Pasteur pipette before freezing.
Experimental Design
Two experiments were performed within this study. In experiment 1, a total
of 10 ejaculates (2–3 ejaculates per male) were studied to compare the
efficiency of 11 extenders on sperm cryopreservation in which glycerol was
used as cryoprotectant. In experiment 2, a total of 8 ejaculates (2 ejaculates
per male) were used in a 6 x 5 factor experiment (6 extenders x 5
cryoprotectants) to investigate effects of permeating cryoprotectant on sperm
cryopreservation in which the final concentration of each cryoprotectant was
5% (vol/vol).
Sperm Freezing and Thawing
Before freezing, washed spermatozoa were divided into several aliquots
according to experiment design. The procedure of sperm freezing was that
described by Sankai et al
(1994). Briefly, each portion
of spermatozoa was diluted with 1 of the extenders without cryoprotectant and
kept at 4°C (water bath) for 2 hours. An equal volume of precooled
(4°C) corresponding extender containing cryoprotectant was added stepwise
(5 times) at intervals of 6–7 minutes within 30 minutes. Spermatozoa
were equilibrated in extenders containing cryoprotectant at 4°C for
another 30 minutes. Just before the end of equilibration, spermatozoa were
drawn into 0.25-mL plastic straws (IMV, L'Aigle, France) with a syringe,
sealed with straw heater (Tew Impulse Sealer, Tish-200, Tew Electric Heating
Equipment Co Ltd, Taiwan, ROC). Straws were frozen in liquid nitrogen vapor
within 10 minutes and plunged into liquid nitrogen and stored for at least 7
days before thawing. For thawing, straws were placed in a 37°C water bath
for 2 minutes.
Examination of Sperm Motility and Motility Recovery
Sperm Motility—
With a prewarmed hemocytometer counting chamber, sperm samples were
assessed for percentage of progressive forward motility by counting 200
spermatozoa, in duplicates. All samples were evaluated by the same operator,
who did not know the identity of the sperm samples offered.
Sperm Motility Recovery Rate— The sperm motility recovery rate was calculated by comparing the motility of prefreeze (Mpr) and postthaw (Mps) spermatozoa. If Mpr and Mps are the sperm motility percentages before and after freezing, then the recovery rate would be Mps/Mpr x 100%.
Examination of Sperm Plasma Membrane and Acrosomal Integrity
Sperm Plasma Membrane Integrity—
Sperm membrane integrity was measured by means of a dual DNA staining
technique using Hoechst 33342 (H342) and propidium iodide (PI), through use of
flow cytometry and excited simultaneously by a single ultraviolet laser.
Briefly, 6 µL H342 (1 mg/mL) and 8 µL PI (1 mg/mL) were added to each
sperm sample (1 mL). Each sperm suspension was mixed 3 times by gently
pipetting and incubated in a 37°C water bath for 15 minutes. After
incubation, all samples were processed for flow cytometric analysis.
Sperm Acrosomal Integrity— Acrosome status was determined by fluorescein isothiocyanate-conjugated peanut agglutinin (FITC-PNA), and the acrosome staining process was that described by Esteves et al (2000). Under a fluorescence microscope, spermatozoa with intact acrosome showed uniform apple-green fluorescence in the acrosomal region of the sperm head, while acrosome-reacted spermatozoa showed little or no green fluorescence in the anterior part of the head. A minimum of 200 spermatozoa were counted for each sample.
Statistical Analysis
All data are expressed as mean ± SD. Data of sperm motility,
membrane integrity, and acrosomal integrity were subjected to arcsine square
root transformation and analyzed by ANOVA and Fisher protected least
significant difference (LSD) test. P < .05 was considered as
statistically significant.
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Results |
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Plasma membrane integrity of frozen-thawed sperm for all treatments is given in Figure 1. The percentages of spermatozoa with intact membrane for TTE, DM, mDM, LG-DM, G-DM, and TCG were significantly higher than other extenders (P < .05) and no significant difference was found among them (P > .05). Among the lower membrane integrity groups of TEST, TSM, Test-M, Test-H, and LM, the ability of cryoprotection on sperm membrane for these extenders is as follows: Test-H provides greater cryprotection than TEST and Test-M, which provide greater cryprotection than TSM and LM (P < .05).
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Acrosomal integrity of frozen-thawed sperm for 11 extenders is presented in Figure 2. Spermatozoa cryopreserved with TTE, DM, mDM, LG-DM, and G-DM showed similar and highest acrosomal integrity in all groups. Unlike sperm membrane integrity, cryopreservation in TCG yielded lower percentage of acrosomal intact spermatozoa than in TTE, DM, mDM, LG-DM, and G-DM (P < .05), but this acrosome value of TCG was significantly higher than those of TEST, TSM, Test-M, Test-H, and LM (P < .05). The TEST, TSM, Test-M, Test-H, and LM resulted in lower percentage of acrosome integrity as compared with TTE, DM, mDM, LG-DM, G-DM, and TCG (P < .05). Additionally, there was no statistical difference for acrosomal integrity among TSM, Test-M, and Test-H, neither was there difference between TEST and Test-M (P > .05). LM yielded the lowest acrosomal integrity in all treatments (P < .05).
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Experiment 2: Effects of Permeating Cryoprotectant on Cryopreservation
Postthaw sperm motility, membrane integrity, and acrosomal integrity for
spermatozoa cryopreserved in different cryoprotective solutions are shown in
Table 3.
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The results demonstrated that in general, sperm motility, membrane integrity, and acrosomal integrity for glycerol or ethylene glycol were highest among all groups (P < .05). Sperm parameters for dimethyl sulfoxide or acetamide were similar and lower than glycerol or ethylene glycol but higher than that of propylene glycol (P < .05). The percentages of the 3 sperm parameters for propylene glycol were lowest in all treatments (P < .05). In addition, the percentage of acrosomal integrity for TCG-glycerol was statistically lower than those of TCG-ethylene glycol and other extender groups of glycerol or ethylene glycol (P < .05).
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Discussion |
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Sanchez-Partida et al (2000) obtained 4 offspring of rhesus monkey with spermatozoa cryopreserved in TCG extender and glycerol. They reported as high as 85% of postthaw motility, which was not significantly different compared with fresh sperm. However, the acrosomal integrity and its recovery in frozen-thawed spermatozoa in that study was only 75% and 79.94%, respectively. In our study, the corresponding data for acrosome with TCG were 76% and 81.1%, respectively, consistent with that of Sanchez-Partida et al (2000), but postthaw sperm motility in this study was much lower than that. According to Sanchez-Partida et al (2000), different freezing rates (liquid nitrogen vapor vs dry ice) might be a major reason. There were no significant differences in postthaw motility and membrane integrity for spermatozoa cryopreserved with TCG, TTE, DM, mDM, LG-DM, and G-DM (P > .05), but the acrosomal integrity for TCG differed, slightly but significantly, from the other treatments (P < .05). In addition, rapid motility, somewhat like hyperactivity, could be observed for spermatozoa frozen with TCG but not with other extenders in this study. Therefore, we infer that a proportion of sperm population undergo capacitation or acrosome reaction after thawing when spermatozoa are frozen with TCG, which will in turn reduce the rate of acrosomal integrity.
Tollner et al (1990) used TEST and TSM to freeze cynomolgus monkey spermatozoa, and postthaw sperm motilities were 56% ± 3% and 67% ± 2%, respectively. Afterward, a baby monkey was born after artificial insemination with spermatozoa frozen in TSM. In our study, the postthaw motility of spermatozoa frozen with these 2 extenders was extremely low (16.1% ± 2.9% vs 8.8% ± 3.2%). The time and temperature of equilibration with glycerol are the 2 main differences between our study and that of Tollner et al (1990). In the present study, spermatozoa were equilibrated in glycerol extender for 0.5 hours, while in the study of Tollner et al (1990) the duration was 2 hours. The equilibration temperatures in 2 studies were 4°C vs room temperature, respectively. It seems that these 2 differences may partially account for the conflicting results. What needs to be noted is that the osmolalities of TEST and TSM before addition of glycerol were as high as 731 mOsm and 825 mOsm when prepared according to the reported formula. These considerably high osmolalities might exceed the osmolality limit that monkey sperm cells can tolerate, and this might cause an irreversible damage to these cells. Sperm viability parameters increased dramatically when the osmolalities of these 2 extenders were reduced to around 400 mOsm (data not shown). Therefore, we maintain that hyperosmolality of TEST and TSM may be the major reason for the poor results in the present study.
Using LM extender and programmed freezing procedure, Leverage et al (1972) reported 68% postthaw motility recovery of rhesus monkey spermatozoa. However, the corresponding rate was only 7.22% in this study. Different concentrated salt ions were included in LM, and this may be deleterious to freezing of cynomolgus monkey spermatozoa. In the freezing of bull (Yassen and Foote, 1967), mouse (An et al, 2000), and rhesus monkey (Si, 2004) spermatozoa, postthaw sperm motility decreased dramatically if spermatozoa were exposed to salt ions. Our study confirms these findings, but the mechanisms of this remain unclear, maybe Mazur's 2 factor hypothesis would explain this (Mazur et al 1972). Additionally, the converse result may be partially due to different freezing procedures and primate species used in the 2 studies.
Marmoset and human spermatozoa were diluted and frozen with Test-M and Test-H in previous studies (Weidel and Prins, 1987; Morrell 1997). The results given by Test-M and Test-H in the present study were poorer than those studies. Test-M and Test-H also possessed high osmolalities of 728 and 665 mOsm prepared according to the published formulas. We suggest the hyperosmolality might be a major reason for our poor data; however, differences in species and freezing procedure may also exist.
Although glycerol is most widely used in sperm cryopreservation for primate animals, its cytotoxicity should not be ignored. Recently, some researchers have reported the success of sperm cryopreservation in a few NHP animals with other permeating cryoprotectants, stimulating the study of these for NHP. However, limited information indicated that the efficiencies of nonglycerol cryoprotectants varied in studies of NHP sperm cryopreservation. Using glycerol, dimethyl sulfoxide, and propylene glycol, Feradis et al (2001) cryopreserved epididymal spermatozoa from cynomolgus monkeys and discovered that glycerol had similar effectiveness to that of dimethyl sulfoxide, while the cryoprotection provided by propylene glycol was considerably poorer. On the contrary, Sadleir (1966) found glycerol to be better than dimethyl sulfoxide for cryopreserving chimpanzee spermatozoa. Besides, Gould and Styperek (1989) reported a failure of in vitro fertilization with spermatozoa cryopreserved with dimethyl sulfoxide.
Comparing glycerol, ethylene glycol, dimethyl sulfoxide, and propylene glycol for rhesus monkey sperm cryopreservation, Si et al (2004) found that both glycerol and ethylene glycol could offer good protection for this species spermatozoa and there was no significant difference between them, but sperm parameters in case of dimethyl sulfoxide and propylene glycol were significantly worse.
A lot of variability, which is supposed to be attributed to the different results, has been found in these reports, and the type of the extender is probably the main difference that can be readily controlled. To investigate whether extender composition influences the role of permeating cryoprotectant, cynomolgus monkey spermatozoa have been cryopreserved in TTE, DM, mDM, LG-DM, G-DM, and TCG, with glycerol, ethylene glycol, dimethyl sulfoxide, acetamide, and propylene glycol as permeating cryoprotectants.
The results of this study indicate that glycerol and ethylene glycol yield the best and similar cryoprotection for cynomolgus monkey spermatozoa. Dimethyl sulfoxide and acetamide are significantly worse in efficiency than glycerol and ethylene glycol but better than propylene glycol. In addition, no significant difference was found between dimethyl sulfoxide and acetamide. Propylene glycol resulted in the poorest protection for spermatozoa among these 5 cryoprotectants. These findings are basically consistent with those of Si et al (2004). Besides, the efficacy of dimethyl sulfoxide is similar to that described by Sadleir (1966) and Gould and Styperek (1989), but disagrees with the result of Feradis et al (2001). This may be due to different sperm source and equilibration procedures in the 2 studies. Furthermore, our study demonstrates that the effects of permeating cryoprotectants are independent of extender type but probably only related to their own nature, such as their chemical structure and/or physical characters. Hence, spermatozoa from different species and sources may have different responses to these permeating cryoprotectants. Additionally, different freezing techniques may interfere with different cryoprotectants.
From these points, we can see that the results of sperm cryopreservation in monkeys might greatly vary because of variation among semen donor individuals and different semen collection methods. This might be one of the reasons why it is not easy to repeat the results of other teams.
The mechanisms of permeation of cryoprotectants into sperm are still not known. According to Gilmore et al (1997), the optimal cryoprotectant for human sperm is one that can permeate the cell in the shortest period of time, causing the least amount of volume excursion during its addition and removal. Therefore, the cryoprotective action of a cryoprotectant depends on its permeability coefficient. According to the study of Gao et al (1995), approximately 60% of human spermatozoa exposed to glycerol will lose their motility when their volume decreases to 0.68 times or exceeds 1.38 times their iso-osmotic volume.
From the report of Gilmore et al (1997) we can see a great disparity in values of solute and water permeability for dimethyl sulfoxide and propylene glycol. When spermatozoa are exposed to the 2 solutions, great sperm volume excursion, exceeding the limits that human sperm can tolerate, will occur. In this way, the rate of sperm survival reduces considerably. On the contrary, the solute and water permeability for glycerol or ethylene glycol match well enough to avoid great volume excursion, so they can protect sperm cells effectively. Combining our experiment, we suggest that the permeability coefficients of glycerol, ethylene glycol, dimethyl sulfoxide, acetamide, and propylene glycol are similar to cynomolgus monkey and human spermatozoa, so the survival percentage of spermatozoa cryopreserved with glycerol or ethylene glycol is significantly higher than those with dimethyl sulfoxide or propylene glycol. On the other hand, there was no significant difference in motility, membrane integrity, and acrosomal integrity for spermatozoa cryopreserved with glycerol or ethylene glycol. In addition, as a permeating cryoprotectant, acetamide may take its action in the same way as the others, and probably its permeability coefficient is an unmatched value that resulted in poor cryopreservation in our study.
In the present study, plasma membrane was damaged dramatically in spermatozoa cryopreserved with propylene glycol, but this damage was less compared with the damage of motility. This finding implies that apart from head plasma membrane, other compartments of sperm regarding sperm motility, such as tail membrane and mitochondria, may be damaged more severely. Another surprising finding is that there was no significant difference in acrosomal integrity for spermatozoa cryopreserved either with propylene glycol, dimethyl sulfoxide, or acetamide. This finding indicates that the sperm acrosome is the most resistant to freezing compared with motility or plasma membrane; meanwhile, it indicates indirectly that sperm motility is the most convincing parameter for evaluation of sperm function.
In conclusion, the present study confirms the following: 1) TTE, DM, mDM, LG-DM, G-DM, and TCG are excellent extenders and suitable for sperm cryopreservation of cynomolgus monkey. 2) The finding that TEST, TSM, Test-M, and Test-H are unable to protect cynomolgus monkey spermatozoa during freezing may be due to the hyperosmolality these extenders possess, which exceeds the sperm cell toleration limit. Few spermatozoa can survive the freezing with LM; this may result from its concentrated salt ions. 3) Ethylene glycol and glycerol exhibited the best cryoprotective ability for cynomolgus monkey spermatozoa. Dimethyl sulfoxide and acetamide resulted in worse effectiveness than ethylene glycol or glycerol, and propylene glycol yielded the worst results. This is probably because dimethyl sulfoxide, acetamide, and propylene glycol have unmatched coefficients that make sperm cells shrink or expand so greatly that they are irreversibly damaged.
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Acknowledgments |
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Footnotes |
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  Q. Dong, S. E. Rodenburg, C. Huang, and C. A. Vandevoort Cryopreservation of Rhesus Monkey (Macaca mulatta) Epididymal Spermatozoa Before and After Refrigerated Storage J Androl, May 1, 2008; 29(3): 283 - 292. [Abstract] [Full Text] [PDF]
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