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Research Article |
Department of Animal Science, the Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
ABSTRACT
This study investigated the development of bovine oocytes following intracytoplasmic injection of sperm heads from spermatozoa dried by heating. When sperm suspension was heated in a dry oven at 50, 56, 90, and 120°C, the mean amounts of residual water were about 0.3 g water/g dry weight within 8 h, 6 h, 1.5 h, and 20 min of heating, respectively. Oocyte activation, cleavage of oocytes, and development of cleaved embryos to the morula stage were better in oocytes injected with spermatozoa stored at 25°C for 7–10 days following drying at 50 and 56°C than at 90 and 120°C; however, only a small proportion of oocytes developed to the blastocyst stage. When spermatozoa were dried at 50°C for 16 h, activation, male pronucleus (MPN) formation, cleavage, and development to the morula stage were less good than when spermatozoa were dried for 8 and 10 h and no blastocysts were obtained. The development of oocytes was significantly better when spermatozoa were stored for 7–10 days at 4°C than 25°C after drying at 50°C for 8 h. Longer storage (7 days–12 mo) of heat-dried spermatozoa at 4°C did not affect MPN formation in activated oocytes, but blastocyst development was significantly lower when spermatozoa were stored for 3 mo or more. These results demonstrate that bovine oocytes can be fertilized with heat-dried spermatozoa and that the fertilized oocytes can develop at least to the blastocyst stage.
assisted reproductive technology, bovine, fertilization, gamete biology, heat drying, ICSI, oocyte activation, ovum, sperm
INTRODUCTION
When mammalian spermatozoa lose their motility, they lose their natural ability to penetrate oocytes both in vivo and in vitro, but their ability to contribute to embryonic development can be investigated using the technique of intracytoplasmic sperm injection (ICSI). The first successful use of ICSI demonstrated that freeze-dried human spermatozoa could develop into pronuclei when injected into hamster oocytes [1]. In later studies, normal calves were born after transfer of blastocysts obtained following ICSI using bovine spermatozoa that had been immobilized and killed by freeze-thawing without any cryoprotectants [2].
Using freeze-dried spermatozoa, Katayose et al. [3] confirmed that hamster and human sperm nuclei remained able, even after 12 mo of storage at 4°C, to form male pronuclei (MPN) following injection into hamster oocytes. Similarly, Hoshi et al. [4] obtained MPN development with freeze-dried hamster and human spermatozoa injected into hamster oocytes and with freeze-dried rabbit spermatozoa injected into rabbit oocytes. In additional studies, more advanced stages of embryonic development (up to the blastocyst stage in some instances) were obtained when rabbit [4], cattle [5], and pig [6] oocytes were injected with freeze-dried spermatozoa from the same species. Since then, it has been demonstrated that freeze-dried mouse [7–11], rabbit [12], and rat [13] spermatozoa are able to produce live offspring when injected into oocytes. Recently, Bhowmick et al. [14] and McGinnis et al. [15] investigated the use of convective drying as a possible alternative to freeze-drying of mouse spermatozoa. They found that mouse oocytes injected with convectively dried spermatozoa could develop to the blastocyst stage in vitro and to 15-day live fetuses or live young after transfer of two- to four-cell embryos. Furthermore, it has been reported that mouse spermatozoa can support full-term development after being dehydrated in a high-osmolarity medium [16]. Thus, mammalian spermatozoa that have lost their motility and possibly their membrane integrity during desiccation would appear to have retained their developmental potential.
Similarly, heat treatment does not appear to irrevocably damage spermatozoa. Sperm nuclei isolated from hamster, mouse, and human spermatozoa heated to 90°C for 30 min were able to form pronuclei when injected into hamster oocytes [17], and rabbit spermatozoa heated to 60°C for 30 min and then injected into rabbit oocytes could support development of the six- to eight-cell stage [18]. More recently, mouse spermatozoa heated at 56°C for 30 min were shown to support full embryonic development [19]. Thus, mammalian spermatozoa appear to be highly resistant to nonphysiologically high temperatures.
This ability of sperm nuclei to withstand drying and high temperatures led us to investigate whether spermatozoa could withstand drying by heating. This is much simpler and less expensive than either freeze-drying or convective drying and would have useful applications in the preservation of male genomes of both laboratory and farm animals. Thus far, no reports have been published on the developmental potential of heat-dried mammalian spermatozoa following ICSI, so this study was undertaken to evaluate the ability of heat-dried bovine sperm to support early development following injection into bovine oocytes.
MATERIALS AND METHODS
All experiments were conducted in accordance with the International Guiding Principles for Biomedical Research Involving Animals as published by the Society for the Study of Reproduction.
Media
Unless otherwise stated, all chemicals used in the present study were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO). The medium used for collection of oocytes was Dulbecco phosphate-buffered saline (DPBS) supplemented with 0.1% polyvinylalcohol (PVA). The medium used for maturation of oocytes was medium 199 (M-199) with Earles salts (Gibco, Grand Island, NY) buffered with 25 mM Hepes and supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Gibco), 60 µg/ml potassium penicillin G, 100 µg/ml streptomycin sulfate, 5 mM hemicalcium lactate, and 0.4 mM sodium pyruvate. The medium used for sperm treatment and ICSI was a low-bicarbonate Tyrodes albumin lactate pyruvate (TALP) composed of 114 mM NaCl, 3.2 mM KCl, 2 mM CaCl2, 0.5 mM MgCl2, 2 mM NaHCO3, 10 mM Hepes, 10 mM sodium lactate, 0.25 mM sodium pyruvate, 0.4 mM NaH2PO4, 75 µg/ml potassium penicillin G, 50 µg/ml gentamycin sulfate, and 1 mg/ml PVA instead of 3 mg/ml BSA (TALP-PVA; pH 7.4). The medium used for culture of ICSI oocytes was CR1aa [20] supplemented with 3 mg/ml BSA (Sigma A-6003; fatty acid free).
Preparation of Oocytes
Ovaries were collected from Holstein and Japanese black cows or heifers at a local abattoir within 30 min of slaughter and then transported to the laboratory within 6 h in 0.9% (w/v) NaCl solution containing 50 µg/ml streptomycin sulfate and 75 µg/ml potassium penicillin G at about 25°C in a vacuum flask. Cumulus-oocyte complexes (COCs) were aspirated from small antral follicles of 2–5 mm in diameter with an 18-gauge needle attached to a 10-ml disposable syringe and washed three times with DPBS. A group of 10–15 COCs was introduced into a 100-µl drop of maturation medium covered with paraffin oil (No. 261–17; Nacalai Tesque, Kyoto, Japan), which had previously been equilibrated in an atmosphere of 5% CO2 in air at 39°C for at least 3 h. The COCs were then cultured for 20–24 h at 39°C under the same atmospheric conditions. After culture, oocytes were freed from cumulus cells by vortexing for 1 min in TALP-PVA containing 0.1% hyaluronidase (Sigma H3506) and washed three times in the same medium. Fifteen to 20 denuded oocytes with the first polar body were transferred into 5 µl of TALP-PVA covered with paraffin oil in the cover of a Petri dish (50 x 4 mm; Falcon No. 1006; Becton Dickinson Co., Franklin Lakes, NJ) and kept in a CO2 incubator (39°C) until used for ICSI.
Heat Drying of Spermatozoa
Two 0.5-ml straws of frozen semen obtained from a Japanese black bull were thawed in a water bath at 37°C for 20 sec. Spermatozoa were washed twice by centrifugation at 250 x g for 10 min each after dilution with 10 ml TALP-PVA in 15-ml conical tubes (Greiner Bio-One; Frickenhausen, Germany). The final sperm pellet was covered with 3 ml TALP-PVA, and spermatozoa were allowed to swim up for about 1 h at 39°C in 5% CO2 in air. Spermatozoa present in the top of 1 ml of medium were collected in a 15-ml conical tube and washed once by centrifugation at 250 x g for 5 min after dilution with 5 ml TALP-PVA. Approximately 4 ml of the supernatant was removed, and spermatozoa (0.5–1 x 105 spermatozoa/ml) were resuspended in the remaining 
1 ml of medium; 100-µl aliquots of the suspension were transferred into 2-ml vial bottles (Maruemu Corporation, Ltd, Osaka, Japan). The bottles then were heated in a dry oven (MOV-212; Sanyo Electric Co., Ltd, Osaka, Japan) at 50, 56, 90, and 120°C for various times. After heating, the bottles were closed quickly with rubber caps without removing oxygen, firmly sealed with parafilm, and stored at 4°C for 7 days to 12 mo or 25°C for 7–10 days.
Water Content Measurement of Heat-Dried Spermatozoa
The water content of four heated sperm samples for each temperature and each length of heating was determined by gravimetric analysis using an analytical balance (BP210S; Sartorius AG, Goettingen, Germany) as reported by McGinnis et al. [15]. The residual water content was expressed as g water/g dry weight according to the formula moisture = (dried weight – baked weight)/baked weight. The dried weight is the weight of the sample after drying, and the baked weight is the weight of the sample after heating at 90°C for a further 24 h. The weight of both samples was determined by subtracting initial bottle weight from the weight of the bottle containing the sample after drying or baking. The baked weight is assumed to be entirely free of water.
Rehydration of Dried Spermatozoa
Dried sperm samples were rehydrated by adding 100 µl of sterile distilled water to the bottles and then transferred into 15-ml conical tubes, and about 1 ml of TALP-PVA was added. The tubes were placed in ice water, and the spermatozoa were sonicated for 20 sec (0.3-sec bursts at 0.7-sec interval/sec) using a 20% power output of a Branson Sonifier Model 250 (Branson Ultrasonics Co., Danbury, CT). The separation of heads from tails was successful in more than 80% of spermatozoa regardless of heating temperatures, storage periods, and storage temperatures. After diluting the 100-µl aliquot of sonicated sperm suspension by adding 100 µl of TALP-PVA supplemented with 10% (w/v) polyvinylpyrrolidone (PVP-360), a 5-µl drop was transferred to a Petri dish cover, adjacent to the drop containing previously prepared oocytes.
Intracytoplasmic Sperm Injection
Microinjection of isolated sperm heads into oocytes was performed on a microwarm plate (MPF-10-N; Kitazato Supply Co., Ltd, Sizuoka, Japan) at 37°C at 200x magnification using a piezomicromanipulator controller (PMAS-CT150; Prime Tech, Tsukuba, Japan). The injection and holding pipettes were prepared from borosilicate glass capillary tubes (Sutter Instrument Co., Novato, CA) using a micropipette puller (P-97/IVF; Sutter Instrument) and a microforge (MF-9; Narishige Co., Ltd, Tokyo, Japan). The external and internal diameters of the tip of the injection pipette were 10–11 µm and 8–9 µm, respectively. Mercury was not used in the injection pipette. After a sperm head had been aspirated into the injection pipette in a minimal amount of medium, the tip of the pipette was brought in contact with the zona pellucida of the oocyte, which was held by a holding pipette with an external diameter of 100–110 µm and an internal opening of 15–20 µm. The zona was drilled by applying two to three pulses (intensity 5, speed 3). Once the tip had reached the perivitelline space, it was forced onto the oolemma and then into the cytoplasm. After a small amount of cytoplasm had been withdrawn to confirm that the tip was in the oocyte, a sperm head in a minimal amount of medium was expelled into the oocyte; the pipette was then gently withdrawn from the oocyte. The injection procedure was completed within 50 min after the preparation of oocytes. A portion of sperm suspension without heating was used for ICSI as the control in one experiment. In that case, the final sonicated sperm suspension for ICSI was prepared with the same procedures as employed for obtaining dried samples. Sham injection was performed in a similar manner, with a minimum volume of medium expelled into the oocyte.
Culture of ICSI Oocytes
Prior to culture, the ICSI oocytes were treated with ionomycin in combination with 6-dimethylaminopurine (DMAP) to induce oocyte activation, as reported by Roh et al. [21]. Briefly, all oocytes used for ICSI were washed three times with TALP containing 3 mg/ml BSA (Sigma A-7638; fraction V) and cultured for 1 h in the same medium (50 µl) covered with paraffin oil in a culture dish (35 x 10 mm; Falcon No. 1008) in a CO2 incubator (5% CO2 in air at 39°C). After culture, oocytes were treated with 10 µM ionomycin in TALP containing 1 mg/ml BSA for 5 min at room temperature and then with ionomycin-free TALP containing 30 mg/ml BSA for 5 min to stop the activation process. The treated oocytes were washed three times with culture medium (CR1aa containing 3 mg/ml fatty-acid-free BSA), and cultured for 3 h in the same medium (50 µl) at 39°C under 5% CO2 in air. Then oocytes were transferred to culture medium (50 µl) containing 1.9 mM DMAP and cultured for a further 3 h. Finally, after washing three times with culture medium, 15–20 treated oocytes were cultured for various times in the same medium (50 µl) at 39°C under 5% CO2 in air.
Examination of Activation and In Vitro Development of ICSI Oocytes
At 18–21 h after ICSI, oocytes were fixed for 48–72 h in 25% (v/v) acetic acid in ethanol at room temperature, stained with 1% (w/v) orcein in 45% (v/v) acetic acid in water, and examined for evidence of activation of oocytes and male pronucleus (MPN) formation using a phase-contrast microscope at a magnification of 200x or 400x . The oocytes with an intact sperm head, an enlarged sperm head, or an MPN were considered to have been successfully injected. Since it was difficult to distinguish the MPN from the female pronucleus, the oocytes with either two pronuclei and a second polar body or three pronuclei without a second polar body were considered to have an MPN. The oocytes with either one female pronucleus and a second polar body or two female pronuclei without a second polar body were considered to be activated. For evaluation of cleavage potential, injected oocytes were cultured for 48, 144, and 192 h after ICSI to assess cleavage, development to the morula stage, and development to the blastocyst stage, respectively. At 72 h after ICSI, these oocytes were transferred into culture medium supplemented with 10% (v/v) heat-inactivated FBS instead of BSA and cultured further.
Statistical Analysis
All proportional data on activation and cleavage of ICSI oocytes and development of embryos obtained from four replicated experiments were treated using one-way ANOVA. When ANOVA revealed a significant effect, the treatments were compared by the Fisher protected least significant difference test.
RESULTS
Change of Residual Water Content in Sperm Suspensions Dried at Various Temperatures
Heating the bottles containing sperm suspension aliquots at 50, 56, 90, and 120°C resulted in a water content of about 0.3 g H2O/g dry weight within 8 h (0.31 g H2O/g dry weight), 6 h (0.27 g H2O/g dry weight), 1.5 h (0.29 g H2O/g dry weight), and 20 min (0.29 g H2O/g dry weight), respectively, of heating (Fig. 1).
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Appearance of Rehydrated Spermatozoa after Drying
More than 80% of spermatozoa dried at 50°C for 8 h and at 56°C for 6 h and stored at 25°C for 7–10 days appeared to be morphologically normal after rehydration (Fig. 2, A and C). However, when dried at 90°C for 90 min and at 120°C for 20 min, about 60%–75% of rehydrated spermatozoa had morphologically abnormal heads or bent tails (Fig. 2, B and D). When dried at 50°C for 8, 10, 12, and 16 h, the proportion of spermatozoa with bent tails was about 20%, 22%, 25%, and 30%, respectively. When dried at 50°C for 8 h, no difference in the proportions (about 80%–85%) of spermatozoa showing normal appearance after rehydration was observed either between different storage temperatures (4 and 25°C) or among different storage periods (7–10 days and 1, 3, 6 and 12 mo) at 4°C.
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Development of Oocytes Injected with Spermatozoa Dried at Various Temperatures and Stored at 25°C for 7–10 Days
Injection of spermatozoa dried at 50 and 56°C resulted in significantly more (P < 0.05) activated oocytes than obtained following injection of cells dried at 90 and 120°C or sham injection; however, these values were significantly lower (P < 0.05) than those obtained with unheated spermatozoa (Fig. 3A). The incidence of MPN formation in activated oocytes decreased significantly (P < 0.05) as the temperature of drying was raised, and no MPN formation was observed when spermatozoa were dried at 120°C. Significantly more (P < 0.05) oocytes cleaved and developed to the morula stage when spermatozoa dried at 50 and 56°C, compared with 90 and 120°C, were used (Fig. 3B). Results obtained with sperm heads dried at 90 and 120°C were similar to those obtained following sham injection. Few injected oocytes developed to the blastocyst stage regardless of the temperature used for drying; the values were similar to those obtained with sham injection.
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Development of Oocytes Injected with Spermatozoa Dried at 50°C for Various Times and Stored at 25°C for 7–10 Days
There were no significant differences in developmental parameters following ICSI with spermatozoa dried at 50°C for either 8 or 10 h (Fig. 4, A and B), but significantly lower values (P < 0.05) were obtained using spermatozoa dried for 16 h compared with 8–10 h. Only a small proportion of oocytes developed to the blastocyst stage regardless of length of drying time at 50°C.
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Development of Oocytes Injected with Spermatozoa Dried at 50°C for 8 h and Stored at Different Temperatures for 7–10 Days
The proportion of activated oocytes and the incidence of MPN formation in activated oocytes were significantly higher (P < 0.05) when dried spermatozoa were stored at 4°C than at 25°C (Fig. 5A). Although there was no significant difference in the proportion of cleaved oocytes between the two storage temperatures, significantly more (P < 0.05) developed to the morula and blastocyst stages when spermatozoa stored at 4°C after drying were used (Fig. 5B).
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Development of Oocytes Injected with Spermatozoa Dried at 50°C for 8 h and Stored at 4°C for Various Periods
Significantly fewer (P < 0.05) oocytes activated when injected with spermatozoa stored for 6 and 12 mo than 7–10 days and 1 mo after drying (Fig. 6A), but the incidence of MPN formation was similar in all the treatment groups. Embryonic development was significantly poorer (P < 0.05) when dried spermatozoa stored for 
3 mo were used (Fig. 6B). A typical blastocyst obtained in these experiments is shown in Figure 7.
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DISCUSSION
The present study has demonstrated for the first time that bovine oocytes can be activated following cytoplasmic injection of sperm heads isolated from heat-dried spermatozoa and that male pronuclei can be formed in the activated oocytes. Although injected oocytes were generally difficult to activate and develop to the blastocyst stage when spermatozoa were dried at 90 and 120°C, about 15% of oocytes developed to the blastocyst stage when they were injected with spermatozoa dried at 50°C for 8 h and stored at 4°C for 1 mo. The development to the blastocyst stage observed in the injected oocytes does not appear to be parthenogenetic because only about 2% of sham-injected oocytes developed to blastocysts, although we did not examine directly by genetic tests to confirm that blastocysts from the sperm-injected oocytes were not parthenotes.
In the present study, the ovaries from which the COCs were eventually collected were kept at 25°C in a vacuum flask for around 6 h after removal from slaughtered cows. This is because new regulations in Japan do not allow bovine material to be removed from the slaughterhouse until the test for bovine spongiform encephalopathy has proved to be negative. This takes about 5.5 h at the slaughterhouse in our district, and then transport to the laboratory takes 0.5 h. This lengthy storage of ovaries does not appear to affect the developmental potential of oocytes because a majority of oocytes injected with frozen-thawed spermatozoa were activated and contained an MPN; of these, about 23% developed to the blastocyst stage in the present study. This rate of blastocyst formation is very similar to the values reported in earlier bovine ICSI studies using frozen-thawed spermatozoa [5, 21–23], even though experimental conditions varied among the studies. Furthermore, Yang et al. [24] reported that storage of isolated bovine ovaries at 25°C for 8 h did not adversely affect development of oocytes after in vitro fertilization.
In the bovine, neither the injection process nor the spermatozoon/sperm head is sufficient to activate oocytes following ICSI [5, 21, 25]. The artificial activation procedure used in the present study was able to activate about 80% of bovine oocytes injected with frozen-thawed sperm heads, but activation of oocytes injected with heat-dried sperm heads was less successful, and there was variation depending on the temperature used for drying. This difference in activation success may indicate that a heat-sensitive factor(s) present in sperm heads, such as sperm-born oocyte activating factor [26], contributes to oocyte activation in bovine ICSI; in heat-dried spermatozoa, such a factor may be damaged or inactivated. The mouse sperm oocyte-activating factor is reported to be heat sensitive [27].
Mammalian spermatozoa appear to be resistant to nonphysiologically high temperatures [17–19]. However, in the present study, MPN formation in activated oocytes and subsequent embryonic development were less successful when heat-dried sperm heads were used for ICSI, especially if dried at high temperatures such as 90 and 120°C. At those temperatures, the amount of residual water in sperm samples was reduced to less than 0.3 g H2O/g dry weight within 90 min, indicating very rapid drying. However, this may not be the only reason for impaired oocyte development following ICSI with sperm heads dried at high temperatures. In a study using convection drying of mouse spermatozoa, a slow drying rate appeared to be more detrimental to the DNA integrity of spermatozoa than a rapid drying rate [14]. Mouse spermatozoa heated at 56°C for 30 min had a disrupted plasma membrane and acrosome but had apparently normal chromatin in the nucleus [19]. Although we did not examine the microstructure of heat-dried spermatozoa in the present study, phase-contrast micrographs showed that high proportions of spermatozoa dried at 90 or 120°C had heads showing a morphologically abnormal appearance. This may indicate that not only the plasma membrane and acrosome but also the chromatin of the nucleus has been damaged when bovine spermatozoa were dried at high temperatures. Mechanical damage of the chromatin induced by both heating and drying might be responsible for impairment of oocyte activation and subsequent development. Since it has been reported that inclusion of a calcium chelator such as EGTA [8] and a protective agent such as trehalose [15] can prevent chromosomal damage during freeze-drying and convective drying, respectively, of mouse spermatozoa, resulting in improved development of injected oocytes, a similar approach could be included in future experiments using heat-dried bovine spermatozoa.
The present study, the first to examine the effect of heating time at a fixed temperature on the ability of spermatozoa to fertilize oocytes, has demonstrated that oocyte activation and development were significantly reduced when spermatozoa were dried at 50°C for 16 h compared with 8 and 10 h. Since about 30% of rehydrated spermatozoa had bent tails when dried at 50°C for 16 h, a long time of heating may affect not only morphology or structure but also some substances responsible for oocyte activation such as a sperm-born oocyte activating factor of sperm heads. The amount of residual water in sperm samples dried at 50°C for 8, 10, and 16 h was 0.31, 0.25, and 0.19 g H2O/g dry weight, respectively, in the present study. Therefore, the most likely reason for impairment is that samples were overdried, even though Bhowmick et al. [14] reported that the final water content (0.5%–7%) in convectively dried mouse spermatozoa did not affect the rate of blastocyst formation of ICSI oocytes.
In addition to the temperature used for heat drying, the temperature used for storage and the length of storage of dried spermatozoa also affected the success of ICSI. We have demonstrated that spermatozoa dried at 50°C and then stored at 4°C for a short time were more successful than those stored at 25°C; longer storage at 4°C resulted in poorer development. These results suggest that temperature- and time-dependent damage, possibly to DNA, occurs during storage, with more detrimental effects occurring at 25°C than at 4°C. In the present study, sample bottles containing the dried sperm samples were closed in the presence of air, and this may account, at least in part, for the damage in the present study; it is known that exposure of cells with damaged membranes to radical oxygen species can result in loss of DNA integrity [28].
In the bovine, it has been reported that about 30% of oocytes developed to the blastocyst stage when they were injected with freeze-dried spermatozoa and stored at 4°C for 1–3 mo [5]. In the present study using heat-dried spermatozoa, the frequency of blastocyst development observed under the optimal conditions was somewhat lower (about 15%). However, the fact that heat-dried spermatozoa can support embryonic development indicates that heat drying is an effective technique for storage of spermatozoa. Although the heat-dried spermatozoa retained their developmental competence for only a month under the present experimental conditions, the technique would be useful for the animal industry; for example, it would allow long-distance shipping of spermatozoa without a need to keep them frozen in liquid nitrogen. It is also possible that further refinements of this technique in the future might provide maintenance of developmental competence over longer storage times.
In conclusion, we have demonstrated that bovine spermatozoa, dried by heating and used for ICSI, can induce oocyte activation and subsequent embryonic development at least as far as the blastocyst stage. However, further experiments are needed to optimize the protocols in terms of temperatures used for heating, subsequent storage conditions of dried spermatozoa, and procedures for rehydration to improve the fertility of heat-dried bovine spermatozoa following long-term storage. Finally, it is vital to determine whether pregnancies can be obtained following embryo transfer. Our results thus far suggest that heat drying might provide a relatively simple method of preserving mammalian spermatozoa for reasonable lengths of time.
ACKNOWLEDGMENTS
The authors are grateful to Professor Lynn R. Fraser, King's College London, for critical reading and valuable suggestions for the manuscript.
FOOTNOTES
1 Correspondence: FAX: 81 86 251 8388; kniwa{at}cc.okayama-u.ac.jp 
Received: 18 June 2005.
First decision: 21 July 2005.
Accepted: 23 September 2005.
REFERENCES
This article has been cited by other articles:
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  M. W Li, J. D Biggers, H. Y Elmoazzen, M. Toner, L. McGinnis, and K C K. Lloyd Long-term storage of mouse spermatozoa after evaporative drying Reproduction, May 1, 2007; 133(5): 919 - 929. [Abstract] [Full Text] [PDF]
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