Development of a Simple Sperm Cryopreservation Model Using a Chemically Defined Medium and Goat Cauda Epididymal Spermatozoa

Cryobiology 40, 117–125 (2000) doi:10.1006/cryo.2000.2230, available online at http://www.idealibrary.com on

Development of a Simple Sperm Cryopreservation Model Using a Chemically Defined Medium and Goat Cauda Epididymal Spermatozoa C. N. Kundu,* J. Chakraborty,* P. Dutta,* D. Bhattacharyya,† A. Ghosh,‡ and G. C. Majumder* *Indian Institute of Chemical Biology, 4, Raja S. C. Mullick Road, Jadavpur, Calcutta 700032, India; †Advanced Research for Cryogenic Center, Jadavpur University, Calcutta 700032, India; and ‡Bose Institute, 93 A. P. C. Roy Road, Calcutta 700009, India This investigation was carried out to develop a simple sperm cryopreservation model using a chemically defined synthetic medium (modified Ringer’s solution) and mature goat cauda epididymal sperm as the model system. Rates of cooling, freezing, and maximum freezing temperature were manipulated with the help of a computer-controlled programmable biofreezer. Highly motile goat cauda sperm dispersed in a modified Ringer’s solution was subjected to the freezing protocol: cooling 0.25°C min ⫺1 to 5°C, 5°C min ⫺1 to ⫺20°C, 20°C min ⫺1 to ⫺100°C, prior to plunging into liquid nitrogen. In the absence of any cryoprotective agent, all of the spermatozoa lost their motility. Addition of glycerol (0.22 to 0.87 M) caused a dose-dependent increase of sperm motility recovery. The highest recovery of forward and total motility was (32 and 35%, respectively) at 0.87 M. Further increase of the glycerol concentration caused a marked decrease in motility. Changes in the cooling rate particularly before and during freezing had a notable effect on the sperm motility recovery. There was no or low recovery (0 –18%) of sperm motility when the cells were transferred directly to liquid nitrogen from the initial two cooling stages. The data demonstrate the importance of all of the cooling stages in the cryopreservation of the cells. Like glycerol, dimethyl sulfoxide (Me 2SO) and ethylene glycol also showed a dose-dependent increase in motility recovery as well as a biphasic curve of cryoprotection. At optimal concentrations, dimethyl sulfoxide (1.00 M) and ethylene glycol (1.29 M) were effective in recovering sperm motility to the extent of 20 and 13%, respectively. Thus these reagents have markedly lower cryoprotection potential than glycerol. © 2000 Academic Press

Key Words: cryopreservation; goat; spermatozoa; glycerol; dimethyl sulfoxide; ethylene glycol.

of frozen semen for artificial insemination (AI) in cattle breeding. Semen cryopreservation also has importance for solving some of the problems of human male infertility, a global social problem. Human semen banks have been established in many countries (22). Although current cryopreservation methods are useful, some limitations exist because the recovery of motile spermatozoa is too low for some applications (1, 4, 14, 21, 24, 25, 27, 31). In most mammalian species, the recovery rate does not exceed 50%. During cryopreservation a substantial portion of sperm cells undergo damage from thermal, mechanical, chemical, and osmotic stresses (32, 34, 37). Generally, cell damage during freezing and thawing procedures is believed to be due to extensive dehydration and intracellular ice crystallization (32). Ice-crystal-mediated damage of the sperm plasma membrane is believed to be a major

Since the successful cryopreservation of spermatozoa reported in 1949 by Polge et al. (26), spermatozoa from many mammalian species have been successfully frozen (36). The method is based on careful manipulation of cooling, freezing, and thawing processes. The semen is usually diluted with a suitable diluent containing an extender (e.g., egg yolk citrate) and a cryoprotectant (e.g., glycerol) prior to preservation at ⫺196°C in liquid nitrogen. Cryopreservation is a potentially useful way of banking cells until needed for experimentation or insemination. Long-term preservation of spermatozoa in liquid nitrogen is a subject of paramount interest because of the extensive use Received June 9, 1999, accepted with revision January 10, 2000. This work was supported by a research grant from the Department of Science & Technology, New Delhi, India. 117

0011-2240/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

118

KUNDU ET AL.

cause of cell death during cryopreservation. A high proportion of cell destruction has been noted when the transition period from the liquid to solid phase or vice versa is prolonged. Thus, an optimal cooling and warming rate is usually needed for the successful cryopreservation of each specific type of cell. Cooling from 37 to 5°C causes a specific type of alteration that is related to membrane lipid phase transitions and is very different from those caused by freezing and thawing (e.g., osmotic, mechanical stresses, Refs. 9, 13, 34). However, the biochemical mechanism of cell cryodamage is largely unknown. Media containing the complex extenders such as egg yolk, milk, skim milk, and milk whey are routinely used for cryopreservation of sperm of different species (3, 5, 10, 35). Glycerol is the most widely used reagent for cryoprotection of spermatozoa. Despite the use of complex media and cryoprotectants, a substantial portion of the cells die during freezing and thawing. As the complex media contain large numbers of undefined biomolecules (proteins, lipids, carbohydrates), it is rather difficult to analyze the beneficial effects of a particular compound on sperm cryopreservation. A synthetic medium will be most appropriate for investigating the biochemical basis of sperm cryodamage as well as cryoprotection offered by the cryoprotectants and extenders. In the present study, efforts have been made to develop a synthetic medium for cryopreservation of cells using the mature goat cauda– epididymal sperm as the model. MATERIALS AND METHODS

Reagents. Inorganic salts, glucose, glycerol, ethylene glycol, and dimethyl sulfoxide used were of reagent grade and were purchased from the local market. Penicillin G and other biochemical reagents used in the experiments were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Collection of goat epididymides. Goat epididymides were collected from the nearby slaughterhouses. The tissue samples were taken to the research laboratory in a plastic container at at-

mospheric temperature. Spermatozoa were extracted from the epididymides within 2–3 h of slaughtering (17, 20). Preparation of spermatozoa and epididymal plasma. Mature goat epididymal spermatozoa were isolated at room temperature (30 ⫾ 2°C) from the cauda segments of the epididymis by the procedure described earlier (20). Cauda epididymides were cut into four to five pieces with a sharp razor blade and were suspended in modified Ringer’s solution (RPS medium) to get the sperm for experimentation. The composition of the medium was 119 mM NaCl, 5 mM KCl, 1.2 mM MgSO 4, 10 mM glucose, penicillin G (50 unit/ml), and 16.3 mM potassium phosphate, pH 6.9. After 5 min, the sperm suspension was filtered through four layers of cotton cheese cloth. Spermatozoa were sedimented by centrifugation at 800g for 5 min at room temperature and the pellet was washed twice with RPS medium. The spermatozoal pellet was dispersed in RPS medium, and this spermatozoa preparation was used for the studies. The sperm preparation was highly purified as judged by phase contrast microscopy, and it contained less than 2% broken or damaged cells. The number of spermatozoa in the sample was determined with a hemocytometer and the spermatozoa preparation was left at room temperature for subsequent use. Goat cauda epididymal plasma (EP) was prepared by the procedure described earlier (20). Freshly extracted sperm preparations were centrifuged at 800g for 5 min when most of the spermatozoa were removed as a pellet. The resulting supernatant, which appeared to be slightly turbid, were again spun at 14,000g for 10 min yielding the cell free EP. A pooled sample of EP was concentrated (20) at 4 – 6°C with polyethylene glycol compound (mol wt 20,000) and preserved at ⫺10°C for subsequent use. The concentration of EP was expressed as its protein content. Estimation of protein. The protein content of EP was estimated according to Lowry et al. (16) using bovine serum albumin as standard. Motility assay method. The percentages of forward motility (FM) and total motility (TM) were assayed prefreeze (initial sperm motility in

119

SPERM CRYOPRESERVATION MODEL

RPS medium prior to the addition of cryoprotectant) and postthaw (i.e., after removing cryoprotectant) by the conventional microscopic method using the hemocytometer as the counter chamber. The motility assay was carried out in the presence of EP (1.2 mg protein/ml) to prevent the adhesion of sperm to glass (33). Spermatozoa (0.5 ⫻ 10 6 cells) were incubated with EP (0.6 mg protein) at room temperature (30 ⫾ 2°C) for 1 min in a total volume of 0.5 ml of RPS medium. A portion of the cell suspension was then injected into the hemocytometer. Immediately spermatozoa that showed well-defined FM (FM cells) (cells that moved in small or large circles were excluded) and TM (including all types of motile cells) and total cell numbers were counted under a phase contrast microscope at 400⫻ magnification. The percentages of FM and TM cells were then calculated. Cryopreservation procedure. Specified concentrations of glycerol, ethylene glycol, and Me 2SO were prepared by dissolving them in RPS medium. Sperm preparations were then added to the cryopreservation medium in such a way that the concentration of the sperm cells was 60 – 80 ⫻ 10 6 cells/ml. For proper mixing, sperm cells were gently stirred with the medium and kept at room temperature for 10 min. In each experiment the sperm preparation contained about 30 – 40% forward motility and 50 – 60% total motility. Addition of cryoprotectants before cooling had no appreciable effect on sperm motility. Aliquots of the sperm suspension were loaded with and without cryoprotectant (i.e., control) into 0.5-ml straws by aspirating the suspension until it reached a measured mark. The open ends of the straws were sealed by polyvinyl chloride powder and were then dipped in double-distilled water for complete sealing. Then the straws were put into the Teflon-made straw holder and placed inside the computer-controlled programmable biofreezer (PTC-1000 C, Apex Instruments, Calcutta, India) for freezing. The processed sperm preparations were frozen by a four-step cooling technique, from room

temperature (30 ⫾ 2°C) to 5°C at the rate of 0.25°C min ⫺1, then from 5 to ⫺20°C at the rate of 5°C min ⫺1 and again from ⫺20 to ⫺100°C at the rate of 20°C min ⫺1. When the run of the programmed protocol was completed, the straws were removed from the freezing chamber and immediately plunged into liquid nitrogen (⫺196°C) for storage. Thawing methods. After 24 h, the straws were removed from storage and rapidly thawed in a 37°C constant water bath for 2 min. Both ends of the sealed straws were cut with a sharp razor blade and the spermatozoa were suspended in RPS medium. The cryoprotectants were removed from the sperm suspension by centrifugation at 800g for 5 min at room temperature and the pellet was washed twice with RPS medium. Finally the pellet was suspended in the same medium. The motility was determined by the methods described earlier. Calculation of motility recovery (%). The motility recovery was calculated by comparing the prefreeze and postthaw motilities. If M b and M f are the percentages of motility of spermatozoa before and after freezing, then recovery (%) would be M f/M b ⫻ 100. Statistical analysis. Results were expressed as means (⫾) standard deviation. Significance was tested using Students’ t test and compared with standard tables. A P value of P ⬍ 0.02 was considered statistically significant. RESULTS

Standardization of Freezing Protocol Using 0.87 M Glycerol as Cryoprotectant The total cooling process consisted of four stages of cooling: (a) from room temperature 30 ⫾ 2 to 5°C, (b) 5 to ⫺20°C, (c) ⫺20 to ⫺100°C (or ⫺50°C), and (d) immersion in liquid nitrogen. The cooling rates of individual steps were optimized by a computer-controlled programmable biofreezer using 0.87 M glycerol in modified Ringer’s solution as cryopreservation medium. When the chamber was cooled at the rate of 5 and 1°C min ⫺1 from room temperature (30 ⫾ 2°C) to 5°C, all of the spermatozoa lost their motility (Table 1). With the lowering

120

KUNDU ET AL. TABLE 1 Effect of Different Cooling Rates Using 0.87 M Glycerol as Cryoprotectant Motility recovery (%)* Protocol 5°C/m

5°C/m

20°C/m

RT** ™™™3 5°C ™™™3 ⫺20°C ™™™3 ⫺100°C 1°C/m

5°C/m

20°C/m

RT ™™™3 5°C ™™™3 ⫺20°C ™™™3 ⫺100°C

Forward

Total

0

0

0

0

0

0

0.5°C/m

10°C/m

20°C/m

0.5°C/m

5°C/m

20°C/m

15 ⫾ 1.2

23 ⫾ 2

0.2°C/m

5°C/m

20°C/m

18 ⫾ 1.5

35 ⫾ 2.5

0.2°C/m

5°C/m

20°C/m

16 ⫾ 2

34 ⫾ 2

32 ⫾ 3

35 ⫾ 2

RT ™™™™3 5°C ™™™3 ⫺20°C ™™™3 ⫺100°C RT ™™™™3 5°C ™™™3 ⫺20°C ™™™3 ⫺100°C RT ™™™™3 5°C ™™™3 ⫺20°C ™™™3 ⫺100°C RT ™™™™3 5°C ™™™3 ⫺20°C ™™™3 ⫺50°C 0.25°C/m

5°C/m

20°C/m

RT ™™™™3 5°C ™™™3 ⫺20°C ™™™3 ⫺100°C

Note. Values indicate the mean ⫾ standard deviation of five experiments. * The prefreeze motility (control) was 35 ⫾ 5% for FM and 60 ⫾ 5% for TM. ** RT represents room temperature (30 ⫾ 2°C).

of the rate, the motility recovery first increased up to 0.25°C min ⫺1. On further lowering of the rate, the recovery of FM decreased but TM remained same (P ⬍ 0.01) (Fig. 1). The percentage of motility recovered was 15 ⫾ 1.2% (FM) and 23 ⫾ 2% (TM) when the cooling rate was 0.5°C min ⫺1 (Table 1). The optimum recovery was found to be 32 ⫾ 3% (FM) and

35 ⫾ 2% (TM) at the rate of 0.25°C min ⫺1 (P ⬍ 0.01) (Table 1). But motility recovery was 18 ⫾ 1.5% (FM) and 35 ⫾ 2.5% (TM) at the cooling rate 0.2°C min ⫺1. So the initial cooling rate of 0.25°C min ⫺1 was better than 0.2°C min ⫺1. The optimum recovery of motility was not only dependent on the initial cooling rate but also on the initial freezing rate (5 to ⫺20°C).

FIG. 1. Effect of initial cooling rate on motility recovery of goat spermatozoa. —E— represents forward motility recovery and —䊐— represents total motility recovery. The values indicate the mean ⫾ standard deviation of five experiments. The prefreeze motility (control) was 36 ⫾ 4% for FM and 65 ⫾ 4% for TM.

121

SPERM CRYOPRESERVATION MODEL TABLE 2 Cryorecovery of Sperm Motility after Each Stage of Cooling Motility recovery (%)* Protocol RT** 3 LN 2 0.25°C/m

RT ™™™™3 5°C 3 LN 2 0.25°C/m

5°C/m

0.25°C/m

5°C/m

RT ™™™™3 5°C ™™™3 ⫺20°C 3 LN 2 20°C/m

RT ™™™™3 5°C ™™™3 ⫺20°C ™™™3 ⫺100°C 3 LN 2

Forward

Total

0

0

0

0

15 ⫾ 2

18 ⫾ 3

32 ⫾ 2

35 ⫾ 3

Note. Values indicate the mean ⫾ standard deviation of five experiments. * The prefreeze motility (control) was 37 ⫾ 3% for FM and 62 ⫾ 2% for TM. ** RT represents room temperature (30 ⫾ 2°C).

Freezing at 5°C min ⫺1 was better than 10°C min ⫺1. When the straws were cooled at 10°C min ⫺1, all of the sperm cells lost their motility. But at 5°C min ⫺1 the motility recovery was 15 ⫾ 1.2% (FM) and 23 ⫾ 2% (TM) (Table 1).

In the third step, the cooling and freezing rates were kept constant but different maximum freezing temperatures were used to assess the motility recovery. When the straws were frozen to ⫺50°C prior to transfer into liquid nitrogen,

FIG. 2. Effects of glycerol concentration on recovery of goat spermatozoa after freezing and thawing. —E— represents the percentage of forward motility recovery. —䊐— represents the percentage of total motility recovery. The values indicate the mean ⫾ standard deviation of five experiments. The prefreeze motility (control) was 35 ⫾ 5% for FM and 60 ⫾ 5% for TM.

122

KUNDU ET AL.

the optimum motility recovery was 16 ⫾ 2% (FM), and 34 ⫾ 2% (TM). But when they were cooled to ⫺100°C before transfer into liquid nitrogen, the recovery was 18 ⫾ 1.5% (FM) and 35 ⫾ 2.5% (TM) (Table 1). This result suggests that cooling to ⫺100°C appeared to be superior to that of ⫺50°C. An experiment was designed to elucidate the importance of the various stages of cooling on sperm cryopreservation (Table 2). Spermatozoa lost their motility completely when the straws were directly plunged into liquid nitrogen either from room temperature or 5°C. But the recovery of FM and TM was 15 ⫾ 2 and 18 ⫾ 2% when the straws were transferred to liquid nitrogen from ⫺20°C. However, maximal recovery (FM, 32 ⫾ 2%; TM, 35 ⫾ 3%) was obtained when the straws were plunged into liquid nitrogen from ⫺100°C. The data show that all the three stages of cooling are important in the optimal cryopreservation of the cells. Effects of Cryoprotectants on Motility Recovery After freezing and thawing all of the spermatozoa lost their FM and TM in the straws containing no cryoprotecting agent. Glycerol showed a dose-dependent increase in the recovery of both FM and TM of frozen sperm from 0.22 to 0.87 M concentration (P ⬍ 0.001). The motility recovery of FM and TM at 0.22 M concentration was 3 ⫾ 1 and 5 ⫾ 1%, respectively. The optimum recovery of FM and TM was 32 ⫾ 3 and 35 ⫾ 2%, respectively, at 0.87 M concentration. On further increase in concentration the potentiality decreased sharply (P ⬍ 0.01) (Fig. 2). The recovery of FM and TM was 15 ⫾ 3 and 21 ⫾ 3%, respectively, at 1.63 M. Me 2SO caused a dose-dependent increase in FM and TM up to 1.0 M. It did not show any significant recovery below 0.25 M. The optimum recovery of FM and TM was 16 ⫾ 1 and 20 ⫾ 2%, respectively, at 1.0 M (P ⬍ 0.02). Beyond this range the motility sharply decreased (P ⬍ 0.02) (Fig. 3). Ethylene glycol also served as a cryoprotectant for sperm cells. It showed a dose-dependent increase in the FM and TM from 0.32 to 1.29 M (P ⬍ 0.01). The highest recovery was found (recovery: FM 10 ⫾

FIG. 3. Effects of dimethyl sulfoxide (Me 2SO) concentration on recovery of goat spermatozoa after freezing and thawing. —E— represents the percentage of forward motility recovery. —䊐— represents the percentage of total motility recovery. The values indicate the mean ⫾ standard deviation of five experiments. The prefreeze motility (control) was 37 ⫾ 2% for FM and 60 ⫾ 5% for TM.

1%, TM 13 ⫾ 1%) at 1.29 M. Further increase in concentration decreased motility recovery sharply (P ⬍ 0.01) (Fig. 4). At 1.93 M concentration the recovery of FM and TM was 4 ⫾ 1 and 8 ⫾ 2%, respectively. DISCUSSION

Earlier investigators have used complex media containing egg yolk, milk, milk whey, etc., as extenders for cryopreservation of ejaculated spermatozoa (3, 5, 10, 35). Such media were also used for investigating the effects of multiple cryoprotectants such as glycerol, Me 2SO, and ethylene glycol (11, 12). Because of the complexity of the media, it was difficult to analyze the biochemical basis of sperm cryodamage and cryoprotection offered by the various reagents. For such an investigation it would be ideal to develop a simple sperm model for cryopreservation in a synthetic medium. Mature cauda– epididymal sperm is a better model than ejaculated sperm because the latter cells are exposed to a variety of undefined constituents from the secretions of seminal vesicles and

SPERM CRYOPRESERVATION MODEL

123

FIG. 4. Effects of ethylene glycol concentration on recovery of goat spermatozoa after freezing and thawing. —E— represents the percentage of forward motility recovery. —䊐— represents the percentage of total motility recovery. The values indicate the mean ⫾ standard deviation of five experiments. The prefreeze motility (control) was 38 ⫾ 2% for FM and 62 ⫾ 2% for TM.

prostate that may complicate the interpretation of the data. Studies from our laboratory during the past 2 decades have shown that goat cauda– sperm is a suitable model for investigating the biochemical regulation of sperm motility (for reviews see Refs. 18, 19). Methodologies have been developed for measuring intactness and forward motility of these cells (7). A method has also been evolved for the isolation of highly purified sperm plasma membranes (28). We have investigated the functional characteristics of the cell membrane with special reference to cell adhesion, cell-surface antigens (2), protein phosphorylation/dephosphorylation mechanisms (8, 23), lipid phase fluidity, lipid constituents, and phospholipid asymmetry (29, 30). An important advantage of the cauda–sperm model

is that spermatozoa can be extracted from the epididymides, which can be procured rather easily from the slaughterhouses. Studies on the cryoprotection mechanism using a cell model will require a large amount of spermatozoa that will be rather difficult to procure from ejaculated mammalian semen samples. We have therefore studied goat cauda–sperm for developing a simple sperm cyropreservation model with a view to analyzing the biochemical mechanism of cryopreservation of these cells. The present study has developed, for the first time, a synthetic medium for sperm cryopreservation using goat cauda–sperm as the model. This has been achieved by manipulating the rates of cooling of the sperm suspension (in a modified Ringer’s solution) before freezing,

124

KUNDU ET AL.

during freezing, and after freezing and by analyzing the profiles of cryoprotectants such as glycerol, Me 2SO, and ethylene glycol. The best cryoprotection was offered by glycerol at the 0.87 M level, when the freezing protocol was cooling 0.25°C min ⫺1 to 5°C, 5°C min ⫺1 to ⫺20°C, 20°C min ⫺1 to ⫺100°C. The sperm cells are highly sensitive to cooling rates particularly during cooling before and during freezing. Under the optimal cryopreservation condition nearly 35% of the motile cells were recovered. It is well known that elevated levels of extracellular reagents will increase osmotic pressure of the medium, thereby inflicting greater damage to the cells (6, 15). Consequently each cryoprotectant is expected to have a characteristic optimal concentration for cryosurvival of the cells. Further increase of the level of cryoprotectant will damage the cells primarily due to elevated osmotic pressure of the medium and as a result there will be lower cryosurvival of the cells. The biphasic curve of the action of the cryoprotectants (Figs. 2– 4) can thus be explained. Although glycerol is widely used as a cryoprotectant for a variety of mammalian cells (1, 9, 11, 21, 31), its mechanism of action is still not clear. The main cause of cell damage is the formation of intra- and extracellular ice crystals during freezing. Glycerol is believed to protect the cells by minimizing ice-crystal formation (32). Glycerol (CH 2OH–CHOH–CH 2OH) has three OH groups and hydrogen atoms of these OH groups are likely to form H-bonding with the oxygen atoms of the phosphate groups of the membrane phospholipids. Ethylene glycol (CH 2OH–CH 2OH) by virtue of having two –OH groups may as well bind to the sperm membrane by H-bonding. In aqueous solutions Me 2SO occurs as a charged molecule ((CH 3) 2S ⫹–O ⫺) that may interact electrostatically with phosphate head groups of the sperm inner/outer membranes. The synthetic model has great promise as its medium contents can be modulated for elucidating the mechanism of sperm cryodamage and cryoprotection offered by cryoprotectants. Understanding this mechanism will form the

basis for formulating improved cryopreservation technology that may be extremely beneficial for more efficacious cattle breeding and for rectifying some of the problems of human infertility. ACKNOWLEDGMENT The authors are grateful to Dr. D. K. Ganguli, Director of the Indian Institute of Chemical Biology, for his interest in this study. REFERENCES 1. Berndfson, W. E., and Foote, R. H. The freezability of spermatozoa after minimal pre-freezing exposure to glycerol or lactose. Cryobiology 9, 57– 60 (1972). 2. Chatterjee, T., and Majumder, G. C. Identification of membrane antigens of goat epididymal spermatozoa. Biochem. Biophys. Res. Commun. 162, 550 – 556 (1989). 3. Chen, Y., Foote, R. H., Tobback, C., Zhang, L., and Hough, S. Survival of bull spermatozoa seeded and frozen at different rates in egg-tris and whole milk extenders. J. Dairy Sci. 76, 1028 –1034 (1993). 4. Chen, Y., Foote, R. H., and Brockett, C. C. Effect of sucrose, trehalose, taurine, and blood serum on survival of frozen bull sperm. Cryobiology 30, 423– 431 (1993). 5. Colas, G. Semen technology in the ram. In “The Male in Farm Animal Production” (M. Courof, Ed.), pp. 219 –236, Amsterdam, Holland, Martinus, Nijhoff (1984). 6. Curry, M. R., and Watson, P. F. Osmotic effects on ram and human sperm membranes in relation to thawing injury. Cryobiology 31, 39 – 46 (1994). 7. Dey, C. S., and Majumder, G. C. A simple quantitative method of estimation of cell-intactness based on ethidium bromide fluorescence. Biochem. Int. 17, 367–374 (1988). 8. Dey, C. S., and Majumder, G. C. Maturation specific type II cyclic AMP- dependent protein kinase in goat sperm plasma membrane. Biochem. Int. 21, 659 – 665 (1990). 9. Fiser, P. S., and Fairful, R. W. The effect of glycerol related osmotic changes on post thaw motility and acrosomal integrity of ram spermatozoa. Cryobiology 26, 64 – 69 (1989). 10. Ganguli, N. C., Bhosrekar, M., and Stephan, J. Milk whey as a diluent for buffalo semen. J. Reprod. Fertil. 35, 355–358 (1973). 11. Gilmore, J. A., Liu, J., Gao, D. Y., and Crister, J. K. Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa. Hum. Reprod. 12(1), 112–118 (1997). 12. Gilmore, J. A., Liu, J., Gao, D. Y., and Crister, J. K. Determination of plasma membrane characteristics

SPERM CRYOPRESERVATION MODEL

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

of boar spermatozoa and their relevance to cryopreservation. Biol. Reprod. 58(1), 28 –36 (1998). Hammerstedt, R. H., Graham, J. K., and Nolan, J. P. Cryopreservation of mammalian sperm: What we ask them to survive. J. Androl. 11, 73– 88 (1990). Kennedy, J. J., Hoyt, R. S., Foote, R. H., and Bratton, R. W. Survival of rapidly frozen bovine spermatozoa. J. Dairy Sci. 43, 1140 –1146 (1960). Liu, Z., and Foote, R. H. Osmotic effects on volume and motility of bull sperm exposed to membrane permeable and nonpermeable agents. Cryobiology 37, 207–218 (1998). Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randal, R. J. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951). Majumder, G. C., and Chakrabarti, C. K. A simple spectrophotometric method of assay of forward motility of goat spermatozoa. J. Reprod. Fertil. 70, 235–241 (1984). Majumder, G. C., Dey, C. S., Haldar, S., and Barua, M. Biochemical parameters of initiation and regulation of sperm motility. Arch. Androl. 24, 287–303 (1990). Majumder, G. C., Jaiswal, B. S., Nath, D., Banerjee, S., Barua, M., Sarkar, M., Rana, A. P. S., Mitra, S., Dutta, P., Chatterjee, T., Misra, S., and Ghosh, A. Biochemistry of sperm motility initiation during epididymal maturation. In “Comparative Endocrinology and Reproduction” (K. P. Ray, A. Krishna, and C. Halder, Eds.), Narosa, New Delhi, India (1999). Mandal, M., Banerjee, S., and Majumder, G. C. Stimulation of forward motility of goat cauda epididymal spermatozoa by a sperm glycoprotein factor. Biol. Reprod. 41, 983–989 (1989). Mortimer, R. G., Berndtson, W. E., Ennen, B. D., and Pickett, B. W. Influence of glycerol concentration and freezing rate on post-thaw motility of bovine spermatozoa in continental straws. J. Dairy Sci. 59, 2134 –2137 (1976). Mossad, H., Morshedi, M., Tonner, J. P., and Oehninger, S. Impact of cryopreservation on spermatozoa from infertile men: implications for artificial insemination. Arch. Androl. 33, 51–57 (1994). Nath, D., and Majumder, G. C. Maturation dependent modification of protein phosphorylation profile of isolated goat sperm plasma membrane. J. Reprod. Fertil. 115, 29 –37 (1999). O’Dell, W. T., Almquist, J. O., and Marsh, L. A. Freezing bovine semen. III. Effect of freezing rate on bovine spermatozoa frozen and stored at ⫺79°C. J. Dairy Sci. 41, 79 – 89 (1958).

125

25. Parkinson, T. J., and Whitfield, C. H. Optimization of freezing conditions for bovine spermatozoa. Theriogenology 27, 781–798 (1987). 26. Polge, C., Smith, A. U., and Parkes, A. S. Revival of spermatozoa after vitrification and dehydration at low temperature. Nature 164, 666 – 668 (1949). 27. Polge, C. Low-temperature storage of mammalian spermatozoa. Proc. R.Soc. B (London) 147, 498 –508 (1957). 28. Rana, A. P. S., and Majumder, G. C. Factors influencing the yield and purity of goat sperm plasma membranes isolated by means of an aqueous two- phase polymer system. Prep. Biochem. 17(3), 261–281 (1987). 29. Rana, A. P. S., and Majumder, G. C. Changes in the fluidity of the goat sperm plasma membrane in the transit from caput to cauda epididymis. Biochem. Int. 21, 797– 803 (1990). 30. Rana, A. P. S., Mishra, S., Majumder, G. C., and Ghosh, A. Phospholipid asymmetry of goat sperm plasma membrane during epididymal maturation. Biochim. Biophys. Acta 120, 1–7 (1993). 31. Robbins, R. K., Saacke, R. G., and Chandler, P. T. Influence of freeze rate, thaw rate and glycerol level on acrosomal retention and survival of bovine spermatozoa frozen in French straws. J. Anim. Sci. 42, 145–154 (1976). 32. Rodojcic, L., Vukotic-Maletic, V., and Balint, B. Current knowledge on cryopreservation of spermatozoa, ovum cells and zygotes. Medicinski pregled. 51 (1– 2), 29 –36 (1998). 33. Roy, N., and Majumder, G. C. Purification and characterization of an antisticking factor from goat epididymal plasma that inhibits sperm– glass and sperm– sperm adhesion. Biochim. Biophys. Acta 991, 114 – 122 (1989). 34. Watson, P. F. The effects of cold shock on sperm cell membranes. In “Effect of Low Temperatures on Biological Membranes” (G. J. Morris and A. Clarke, Eds.), Academic Press, London (1981). 35. Watson, P. F. The role of lipids and proteins in the protection of ram spermatozoa at 5°C by egg yolk lipoprotein. J. Reprod. Fertil. 62, 483– 492 (1981). 36. Watson, P. F. Artificial insemination and the preservation of semen. In “Marshall’s Physiology of Reproduction” (G. E. Lamming, Ed.), Vol. 2, pp. 747– 869. Churchill Livingstone, New York (1990). 37. Watson, P. F. Recent developments and concepts in the cryopreservation of spermatozoa the assessment of their post-thawing function. Reprod. Fertil. Dev. 7, 871– 891 (1995).