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Effects of various concentrations of glycerol on post-thaw motility and velocity of human spermatozoa
CRYOBIOLOGY
Effects
19, 147-
153 (1982)
of Various Concentrations of Glycerol on Post-thaw and Velocity of Human Spermatozoa S. PILIKIAN, Laboratoire
de Biologie
J. C. CZYBA,
March
6, 198 1; accepted
J. F. GUERIN
de la Reproduction, H6pital Edouard-Herriot, 69373 Lyon, France
MATERIALS
Since the introduction of glycerol as a cryoprotective agent (19) for conservation of spermatozoa, considerable work has been done to test different cryoprotective agents, complex cryoprotective media, and various freezing and thawing methods, with the objective of improving the post-thaw survival, motility recovery, and fertilizing capacity of frozen semen for artificial insemination. Different concentrations of glycerol-5% (.8), 7 to 7.7% (1, 10, 18), 8.3% (21), 8 to 12% (3), and 10% (13, 15, 24, 25)-were used under different experimental conditions for freezing human semen. In all frozen-thawed samples spermatozoa undergo a marked decrease in motility and lose this motility much more rapidly than in fresh samples. These changes in time of the post-thaw motility characteristics have already been reported (11, 13, 14, 22, 23). However, there has not been much work on the behavior of spermatozoa during the first hour interval following thawing. This study investigates post-thaw motility and velocity of semen cryopreserved in 6, 8, 12, 16, and 24% glycerol and frozen in liquid nitrogen vapor. The goal is to compare both the percentage motility recovery and kinetic changes of spermatozoa in the first hour interval after thawing, and to make a comparison of the motility measures obtained with the optical method and the microphotographic technique.
Received
AND
September
Motility
Place
d’Arsonva1,
AND
METHODS
Normal semen specimens were obtained at the laboratory after 3 days of sexual abstinence and within 1 hr of production, and the percentage motility and velocity of each sample were assessed at room temperature. Each sample was split into five equal portions and diluted with equal volumes of glycerol-egg yolk-citrate (GEYC) medium. The cryoprotecting medium prepared after that proposed by Behrman and Ackerman (1) has the following composition for 100 ml: egg yolk, 23.25 ml; 5% glucose 30.23 ml; 3% sodium citrate, 45.34 ml; glycine, 17.44 mg; and 1.18 ml of a mixture of penicillin, 50,000 IU/lOO ml, and streptomycin, 25 mg/lOO ml. The medium was divided into five portions, and a desired volume of phosphate-buffered glycerol (KHBPOI, 0.136 g; Na2HP0,. 12H20, 3.220 g; distilled water, 20 ml; glycerol, 80 ml) was added to each portion to give tinal concentrations of 6, 8, 12, 16, and 24% glycerol. The mixture was heated for 30 min at 56”C, the pH was adjusted to 7.2-7.4 with 13% NaHCO,, and the mixture was then stored in a refrigerator. Before each experiment, the needed volumes of cryoprotecting medium were brought to room temperature and carefully added to semen samples, drop by drop, in order to avoid sudden changes in osmotic pressure and to minimize the harmful effect of glycerol on the spermatozoa. Addition of the medium was carried out at room temperature since human spermatozoa appear independent of the effect on permeability of prefreeze
4, 198 1. 147
001 l-2240/82/020147-07$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
148
PILIKIAN,
CZYBA,
glycerolation temperature with respect to freeze-thaw survival (24). The mixture was equilibrated for 15 min. A small sample was used for the percentage motility and velocity measurements. The rest was put in 0.25 ml color-coded straws for each concentration of glycerol and sealed with sealing powder and was ready to be frozen in liquid nitrogen. The rapid freezing in nitrogen vapors with two intermediate steps was a modification of Emperaire and Riviere’s technique (7). The straws, horizontally on a rack, were rapidly lowered into a container of liquid nitrogen at 18 cm above the liquid surface (-60 to -70°C) for 10 min, then lowered 3 to 4 cm above the liquid surface (-150 to -160°C) for another 10 min, and then dipped in liquid nitrogen for storage at - 196°C. Thawing was performed by simple exposure to laboratory temperature for a few minutes. Two sets of experiments were performed. (a) The post-thaw percentage motility and mean velocity of sperm frozen in 6, 8, 12, and 16% glycerol were measured with Laser Doppler velocimetry (SKM 100 SORO optics) immediately after thawing (To), and successively at 15min intervals for 1 hr (T,, T2, T,, TJ. Laser Doppler velocimetry utilizes a He-Ne laser of low power as the monochromatic light source (6). The Semen mixture is placed in a chamber of optical glass and measurements are performed at 37°C and rapidly given with a high precision.
The Effect
of Cryoprotecting
Medium
AND
GUERIN
(b) The post-thaw percentage motility of sperm frozen in 8, 12, and 24% glycerol was measured at T, with two different methods: Laser Doppler velocimetry (as described above) and the micrographic technique using dark-field illumination (16), with a 2-set exposure time; motile spermatozoa leave a typical white track contrasting with the bright spots left by immotile spermatozoa. In all our experiments the percentage motility recovery was calculated as ..
% mot&y after thawmg initial motility after addition of cryoprotecting medium
Data were analyzed statistically: Wilcoxon’s test (nonparametric test) was used for the analysis of motility results and the Student t test for that of velocity (4). RESULTS
All the specimens we worked with had a percentage motility of 45 to 65% with a velocity of 35 to 60 prnlsec. The addition of the cryoprotecting medium to fresh semen caused a consistent decrease in the percentage motility, whatever the glycerol concentration used, but no apreciable change in velocity (Table 1). There were no significant differences between 6, 8, 12, and 16% glycerol, but a significant decrease (P < 0.01) appeared for the highest concentration of glycerol (24%). Figure 1 represents the temperature curve during freezing of a semen sample.
TABLE 1 on Percentage Motility
and Velocity
Fresh
Fresh Percentage motility Velocity (pm/se@
semen 50 48
Note. Results are the means for 14 samples. a Significant difference (P < 0.01) compared b Significant difference (P i 0.01) compared
6% Glycerol 38” 47
x loo.
semen
8% Glycerol 37” 49
to fresh semen. to the other glycerol
of Human
+ Ackerman’s 12% Glycerol 33” 46
concentrations.
Sperm
before
Freezing
medium 16% Glycerol 32” 45
24% Glycerol 27a.b 45
EFFECTS 20
OF GLYCEROL
I
0 c\ -50
Cl
VI
-150 E : m
.k -200 B z
Time in minutes FIG. 1. Freezmg curve in liquid nitrogen with two intermediate steps.
vapor
The freeze-thawing process was responsible for an important drop in both motility and velocity (as recorded at time T,, right after thawing in all four concentrations of glycerol (Table 2). However, post-thaw motility recovery improved with increasing concentrations of glycerol. The registered increase, though not significant between the intervals of 6 to 8% and 12 to 16%, was
ON HUMAN
149
SPERMATOZOA
very significant (P < 0.01) when the glycerol concentration was raised from 8 to 12%, for all time intervals studied. A rapid decrease in post-thaw motility followed in the next hour. The nonparametric pairs test showed that this drop in percentage motility was significant (P < 0.05) 15 min after thawing for 16% glycerol and 30 min after thawing for 6, 8, and 12% glycerol. The post-thaw motility results are represented graphically in Fig. 2. While post-thaw motility increased with glycerol concentration, velocity remained rather constant. Moreover post-thaw velocity dropped less rapidly than motility since the decrease reached significance (P < 0.01) only after 1 hr (Fig. 3). In the second set of experiments with 14 specimens, the post-thaw motility at time To was estimated with two different methods: the Doppler technique and dark-field illumination photography. (Table 3). The photographic results support the previous observations, obtained with the Doppler technique, of an improvement in post-thaw motility when glycerol concentration is increased from 8 to 16%. The two methods compare well as far as the relative increase
TABLE 2 Percentage Motility Recovery and Velocity at Different Time Intervals for a Population of Spermatozoa Frozen in Different Glycerol Concentrations Concentration Time
of glycerol in Ackerman’s medium
6%
8%
12%
16%
T,: at thawinlg
% motility Velocity (pm/set)
27 36
34 35
46” 36
51 37
T,: 15 min
% Motility Velocity (pm/set)
22 33
26 35
43 34
450 36
T,: 30 min
% Motility Velocity (pm/set)
20* 33
2lb 34
36b 34
37 35
T,: 4.5 min
% Motility Velocity (ymlsec)
18 31
19 32
28 34
34 34
60 min
% Motility Velocity (ymlsec)
16 2t3*
19 30”
27 32b
33 32”
T,:
Note. Results are the means for 22 samples. n Significant inc:rease (P < 0.05) compared to 8% glycerol. b Significant decrease (P < 0.01) compared to T,.
150
PILIKIAN,
CZYBA,
AND GUERIN
52
48
16
L 0
I 15
45
30
60
NINUTES
FIG. 2. Evolution in time of the percentage motility recovery of spermatozoa frozen in complex cryoprotecting medium containing 6% (A), 8% (O), 12% (O), 16% (a) glycerol.
in motility is concerned although the values given with the photomicrographs are smaller than those of the Doppler technique. However, there is a large discrep-
38
ancy in the values for 24% glycerol: whereas a drop in motility is registered with the photomicrographic method, the Doppler technique shows no such difference.
c
36
32 30 28
0
30
15
45
60
MINUTES FIG. 3. Evolution in time of the velocity of spermatozoa frozen in complex cryoprotecting medium containing 6% (A), 8% (0), 12% (O), 16% (a) glycerol.
EFFECTS
OF GLYCEROL
Percentage Motility
ON HUMAN
151
SPERMATOZOA
TABLE 3 Recovery Measured with Two Different Techniques Concentration 8%
Doppler Velocimetry Microphotographic technique
of glycerol in Ackerman’s medium 12% % Motility 52” 32”
40 24
24% 54 206
Note. Results are the means for 13 samples. a Significant difference (P < 0.05) compared to 8% glycerol. b Significant difference (P < 0.05) compared to 12% glycerol,
DISCUSSION
The significant decrease in post-thaw motility and velocity agrees with the findings of recent investigations (14, 23). However, results vary (1, 2, 13, 14, 24), due to differences in semen quality, processes of cryopreservation, i.e., cryoprotecting media, glycerol concentrations, equilibration time, freezing and thawing methods, as well as general laboratory conditions. No systematic comparative studies had been done with 6,8, 12, and 16% glycerol in Ackerman’s medium. Under our experimental conditions, no significant difference was noticed in the mean prefreeze motility and velocity for the different concentrations of glycerol used in spite of the high osmotic pressure in 12 and 16% glycerol (815 and 1.086 mosm, respectively). The toxic effect of glycerol became obvious, however, after addition of 24% glycerol and when samples with lower concentrations were kept unfrozen for longer than 1 hr. Alterations in motility have been ascribed to glycerol “osmotically inducing plasmalemma distention” (5). While other mammalian spermatozoa are greatly affected by the hypertonicity of the expanding medium (20), human spermatozoa seem to have a better resistance when motility is considered. Human spermatozoa seemed to be less affected by hypertonic diluters than hypotonic diluters as reported by Guerin and Czyba (12), though these authors used media less hypertonic than those used in the present work.
Ten percent glycerol or less is considered optimum for freeze-preservation of human semen. In our present work, where only percentage motility recovery after freezing was considered, we found that 12 and 16% glycerol gave better motility results than 8% glycerol. Possibly the increased motility recovery in high-osmotic-pressure media was due to the prevention of osmotic swelling during thawing. Wooley and Richardson (25) suggested that, during thawing, cells are exposed to the lower osmotic strength of the melting diluent and are damaged by osmotic swelling. Therefore, it may be possible to prevent osmotic damage by thawing semen in hypertonic media. The high osmotic pressure resulting from 12 and 16% glycerol probably prevents this osmotic swelling. The difference in motility of spermatozoa frozen in 12 and 16% glycerol was not statistically significant. The increase in 16% glycerol was not systematically observed and was mainly due to some samples which continued to give better motility in 16% glycerol. This, once more, shows the importance of individual differences in ability to withstand freezing and cryoprotecting media of different osmotic pressure values. The motility results obtained with the two different methods (Table 3) show: (a) an increase in percentage motility with both methods as the concentration of glycerol is raised from 8 to 16%. The values recorded for the micrographs are smaller than those of the Doppler velocimeter probably because only spermatozoa mov-
152
PILIKIAN,
CZYBA,
ing on the surface are viewed on the microscopic field. Also, friction forces created by the slide and coverslip may slow down movement of spermatozoa moving alongside the glass and immobilize initially slowmoving spermatozoa. Moreover in the Doppler velocimeter, the temperature is constant which is not the case with the micrographic technique. (b) No difference is shown in percentage motility recovery of spermatozoa frozen in 16 and 24% glycerol according to the optical measurements, whereas the photographic method showed a sharp decrease in 24% glycerol. This discrepancy is due to the increased number of moving but nonprogressing spermatozoa after freezing in 24% glycerol. On the dark-field illumination microphotographs, only progressing spermatozoa leaving a typical white track are counted whereas the blurred images of moving but nonprogressing spermatozoa are not taken into consideration. On the contrary, the Doppler method does not discriminate the progressing and nonprogressing but motile sperm, and considers all of them as “motile.” Nevertheless, both methods showed that the velocity of sperm exhibiting forward motility in 24% glycerol was comparable to that of sperm in 8 or 16% glycerol. The rapid loss in motility and velocity of freeze-thawed semen samples is important when artificial insemination with frozen semen is to be performed. Although postthaw motility and fertilizing capacity are not always correlated, they are the only factors which significantly predict the penetration in cervical mucus (9). Insemination in the first 15 min following thawing becomes imperative if any further loss in motility and velocity is to be prevented. SUMMARY
The percentage post-thaw motility and velocity of semen samples mixed one to one by volume with Ackerman protective
AND
GUERIN
medium, with final buffered glycerol concentrations of 6, 8, 12, 16, and 24%, and frozen in liquid nitrogen vapor were studied. Semen frozen in 12 and 16% glycerol gave better motility recovery than those frozen in the other concentrations considered. The changes in motility and kinetics of thawed samples, recorded at 15 min intervals, showed a significant drop 30 min after thawing for motility and after 1 hr for velocity. Results obtained with the photomicrographic method confirm the increase in percentage motility given with the SKM measures when the concentration of glycerol is raised from 8 to 12%. The physical method did not discrimate between progressing and nonprogressing spermatozoa motilities while recording the percentage motility of a population of spermatozoa. REFERENCES
1. Behrman, S. J., and Ackerman, D. R. Freeze preservation of human sperm. Amer. J. Obstet. Gynecol. 103, 654-658 (1969). 2. Beck, W. W., and Silverstein, I. Variable motility recovery of spermatozoa following freeze preservation. Ferf. Steril. 26, 863-867 (1975). 3. Czyba, J. C., Pinatel, M. C., and Guerin, J. F. Preservation and storage of human sperm. Acta Med. Pal. 19, 133-146 (1978). 4. Dagnelie, P. “Theorie et methodes statistiques” (J. Ducolot, Ed.), Vol. 2, pp. 35-40. Grembloux, 1970. 5. Drevius, L. 0. The permeability of bull spermatozoa to water polyhydric alcohols and univalent anions and the effects of the anions upon the kinetic activity of spermatozoa and sperm models. J. Reprod. Fert. 28, 41-54 (1972). 6. Dubois, P., Jouannet, P., Berge, P., Volochine, B., Serre, C., and David, G. Methode et appareillage de mesure objective de la mobilitk des spermatozo’ides humains. Ann. Physiol. Biol. Med. 9, 19-41 (1975). 7. Emperaire, J. C., and Riviere, J. La congelation du sperme humain normal. J. Gynecol. O&ret. Biol. Reprod. 3, 215-222 (1974). 8. Femandez-Cano, L. F., Menkin, M. F., Garcia, G. R., and Rock, J. Refrigerant preservation of human spermatozoa. Fert. Steril. 15, 390-405 (1964). 9. Fjallbrant, B., and Ackerman, D. R. Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimens. J. Reprod. Ferr. 20, 515-521 (1%9).
EFFECTS
OF GLYCEROL
10. Freund, M., and Wiederman, J. Factors affecting the dilution, freezing and storage of human semen. J. Reprod. Fer?. 11, 1-17 (1966). 11. Friberg, J., and Gemzell, C. Insemination of sperm after freezing in liquid nitrogen vapors with glycerol or glycerol-egg yolk-citrate as protective media. Amer. J. Obstet. Gynecol. 116, 330-334 (1975). 12. Guerin J. F., and Czyba, J. C. Effects de la pression osmotique sur la mobilite et metabolisme des spermatozdides humains. In “Comptes rendus des seances de la Societe de Biologie 1977,” pp. 822-825. 13. Harrison, R. IF., and Sheppard, B. L. A comparative study in methods of cryoprotection for human semen. Cryobiology 17, 25-32 (1980). 14. Keel, B. A., and Black, J. B. Reduced motility longevity in thawed human spermatozoa. Arch. Androl. 4, 213-215 (1980). 15. Keel, B. A., and Karow, A. A., Jr. Motility characteristics of human sperm, nonfrozen and cryopreserved. Arch. Androl. 4, 205-212 (1980). 16. Makler, A. A new multiple exposure photography method for objective human spermatozoa1 motility determination. Fert. Steril. 30, 192-199, (1978). 17. Overstreet, J., Katz, D., Hanson, F. W., and Fonseca, J. R. A simple inexpensive method for objective assessment of human sperm movement characteristics Fertil. Steril. 31, 162- 172 (1979).
ON HUMAN
SPERMATOZOA
153
18. Pedersen, H., and Lebech, P. E. Ultrastructural changes in the human spermatozoa after freezing for artificial insemination. Fert. Steril. 22, 125-133 (1971). 19. Polge, G., Smith, A. U., and Parkes, A. S. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature (London) 164, 666-669 (1949). 20. Rao, P. S., Sikes, J. D., and Merilan, C. P. Freezing point depression and viability of bovine sperm during freezing and storage. Fert. Steril. 19, 129-136 (1968). 21. Rubin, S. O., Anderson, L., and Bostrom, K. Deep-freeze preservation of normal and pathologic human semen. &and. J. Ural. Nephrol. 3, 144- 150 (1%9). 22. Sawada, Y., Ackerman, D. R., and Behrman, S. J. Motility and respiration of human spermatozoa after cooling to various low temperatures. Fert. Steril. 18, 775-781 (1967). 23. Serres C., Jouannet, P., Czylik, F., and David, G. Effects of freezing on spermatozoa motility. In “Human Artificial Insemination and Semen Preservation” (G. David and S. P. Wendel, Eds.), p. 147. Plenum, New York, 1980. 24. Sherman, J. K. Improved methods of preservation of human spermatozoa by freezing and freezedrying. Fert. Steril. 14, 49-64 (1963). 25. Woolley, D. M., and Richardson, D. W. Ultrastructural injury to human spermatozoa after freezing and thawing. .I. Reprod. Fert. 53, 389-394 (1978).
Effects
19, 147-
153 (1982)
of Various Concentrations of Glycerol on Post-thaw and Velocity of Human Spermatozoa S. PILIKIAN, Laboratoire
de Biologie
J. C. CZYBA,
March
6, 198 1; accepted
J. F. GUERIN
de la Reproduction, H6pital Edouard-Herriot, 69373 Lyon, France
MATERIALS
Since the introduction of glycerol as a cryoprotective agent (19) for conservation of spermatozoa, considerable work has been done to test different cryoprotective agents, complex cryoprotective media, and various freezing and thawing methods, with the objective of improving the post-thaw survival, motility recovery, and fertilizing capacity of frozen semen for artificial insemination. Different concentrations of glycerol-5% (.8), 7 to 7.7% (1, 10, 18), 8.3% (21), 8 to 12% (3), and 10% (13, 15, 24, 25)-were used under different experimental conditions for freezing human semen. In all frozen-thawed samples spermatozoa undergo a marked decrease in motility and lose this motility much more rapidly than in fresh samples. These changes in time of the post-thaw motility characteristics have already been reported (11, 13, 14, 22, 23). However, there has not been much work on the behavior of spermatozoa during the first hour interval following thawing. This study investigates post-thaw motility and velocity of semen cryopreserved in 6, 8, 12, 16, and 24% glycerol and frozen in liquid nitrogen vapor. The goal is to compare both the percentage motility recovery and kinetic changes of spermatozoa in the first hour interval after thawing, and to make a comparison of the motility measures obtained with the optical method and the microphotographic technique.
Received
AND
September
Motility
Place
d’Arsonva1,
AND
METHODS
Normal semen specimens were obtained at the laboratory after 3 days of sexual abstinence and within 1 hr of production, and the percentage motility and velocity of each sample were assessed at room temperature. Each sample was split into five equal portions and diluted with equal volumes of glycerol-egg yolk-citrate (GEYC) medium. The cryoprotecting medium prepared after that proposed by Behrman and Ackerman (1) has the following composition for 100 ml: egg yolk, 23.25 ml; 5% glucose 30.23 ml; 3% sodium citrate, 45.34 ml; glycine, 17.44 mg; and 1.18 ml of a mixture of penicillin, 50,000 IU/lOO ml, and streptomycin, 25 mg/lOO ml. The medium was divided into five portions, and a desired volume of phosphate-buffered glycerol (KHBPOI, 0.136 g; Na2HP0,. 12H20, 3.220 g; distilled water, 20 ml; glycerol, 80 ml) was added to each portion to give tinal concentrations of 6, 8, 12, 16, and 24% glycerol. The mixture was heated for 30 min at 56”C, the pH was adjusted to 7.2-7.4 with 13% NaHCO,, and the mixture was then stored in a refrigerator. Before each experiment, the needed volumes of cryoprotecting medium were brought to room temperature and carefully added to semen samples, drop by drop, in order to avoid sudden changes in osmotic pressure and to minimize the harmful effect of glycerol on the spermatozoa. Addition of the medium was carried out at room temperature since human spermatozoa appear independent of the effect on permeability of prefreeze
4, 198 1. 147
001 l-2240/82/020147-07$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
148
PILIKIAN,
CZYBA,
glycerolation temperature with respect to freeze-thaw survival (24). The mixture was equilibrated for 15 min. A small sample was used for the percentage motility and velocity measurements. The rest was put in 0.25 ml color-coded straws for each concentration of glycerol and sealed with sealing powder and was ready to be frozen in liquid nitrogen. The rapid freezing in nitrogen vapors with two intermediate steps was a modification of Emperaire and Riviere’s technique (7). The straws, horizontally on a rack, were rapidly lowered into a container of liquid nitrogen at 18 cm above the liquid surface (-60 to -70°C) for 10 min, then lowered 3 to 4 cm above the liquid surface (-150 to -160°C) for another 10 min, and then dipped in liquid nitrogen for storage at - 196°C. Thawing was performed by simple exposure to laboratory temperature for a few minutes. Two sets of experiments were performed. (a) The post-thaw percentage motility and mean velocity of sperm frozen in 6, 8, 12, and 16% glycerol were measured with Laser Doppler velocimetry (SKM 100 SORO optics) immediately after thawing (To), and successively at 15min intervals for 1 hr (T,, T2, T,, TJ. Laser Doppler velocimetry utilizes a He-Ne laser of low power as the monochromatic light source (6). The Semen mixture is placed in a chamber of optical glass and measurements are performed at 37°C and rapidly given with a high precision.
The Effect
of Cryoprotecting
Medium
AND
GUERIN
(b) The post-thaw percentage motility of sperm frozen in 8, 12, and 24% glycerol was measured at T, with two different methods: Laser Doppler velocimetry (as described above) and the micrographic technique using dark-field illumination (16), with a 2-set exposure time; motile spermatozoa leave a typical white track contrasting with the bright spots left by immotile spermatozoa. In all our experiments the percentage motility recovery was calculated as ..
% mot&y after thawmg initial motility after addition of cryoprotecting medium
Data were analyzed statistically: Wilcoxon’s test (nonparametric test) was used for the analysis of motility results and the Student t test for that of velocity (4). RESULTS
All the specimens we worked with had a percentage motility of 45 to 65% with a velocity of 35 to 60 prnlsec. The addition of the cryoprotecting medium to fresh semen caused a consistent decrease in the percentage motility, whatever the glycerol concentration used, but no apreciable change in velocity (Table 1). There were no significant differences between 6, 8, 12, and 16% glycerol, but a significant decrease (P < 0.01) appeared for the highest concentration of glycerol (24%). Figure 1 represents the temperature curve during freezing of a semen sample.
TABLE 1 on Percentage Motility
and Velocity
Fresh
Fresh Percentage motility Velocity (pm/se@
semen 50 48
Note. Results are the means for 14 samples. a Significant difference (P < 0.01) compared b Significant difference (P i 0.01) compared
6% Glycerol 38” 47
x loo.
semen
8% Glycerol 37” 49
to fresh semen. to the other glycerol
of Human
+ Ackerman’s 12% Glycerol 33” 46
concentrations.
Sperm
before
Freezing
medium 16% Glycerol 32” 45
24% Glycerol 27a.b 45
EFFECTS 20
OF GLYCEROL
I
0 c\ -50
Cl
VI
-150 E : m
.k -200 B z
Time in minutes FIG. 1. Freezmg curve in liquid nitrogen with two intermediate steps.
vapor
The freeze-thawing process was responsible for an important drop in both motility and velocity (as recorded at time T,, right after thawing in all four concentrations of glycerol (Table 2). However, post-thaw motility recovery improved with increasing concentrations of glycerol. The registered increase, though not significant between the intervals of 6 to 8% and 12 to 16%, was
ON HUMAN
149
SPERMATOZOA
very significant (P < 0.01) when the glycerol concentration was raised from 8 to 12%, for all time intervals studied. A rapid decrease in post-thaw motility followed in the next hour. The nonparametric pairs test showed that this drop in percentage motility was significant (P < 0.05) 15 min after thawing for 16% glycerol and 30 min after thawing for 6, 8, and 12% glycerol. The post-thaw motility results are represented graphically in Fig. 2. While post-thaw motility increased with glycerol concentration, velocity remained rather constant. Moreover post-thaw velocity dropped less rapidly than motility since the decrease reached significance (P < 0.01) only after 1 hr (Fig. 3). In the second set of experiments with 14 specimens, the post-thaw motility at time To was estimated with two different methods: the Doppler technique and dark-field illumination photography. (Table 3). The photographic results support the previous observations, obtained with the Doppler technique, of an improvement in post-thaw motility when glycerol concentration is increased from 8 to 16%. The two methods compare well as far as the relative increase
TABLE 2 Percentage Motility Recovery and Velocity at Different Time Intervals for a Population of Spermatozoa Frozen in Different Glycerol Concentrations Concentration Time
of glycerol in Ackerman’s medium
6%
8%
12%
16%
T,: at thawinlg
% motility Velocity (pm/set)
27 36
34 35
46” 36
51 37
T,: 15 min
% Motility Velocity (pm/set)
22 33
26 35
43 34
450 36
T,: 30 min
% Motility Velocity (pm/set)
20* 33
2lb 34
36b 34
37 35
T,: 4.5 min
% Motility Velocity (ymlsec)
18 31
19 32
28 34
34 34
60 min
% Motility Velocity (ymlsec)
16 2t3*
19 30”
27 32b
33 32”
T,:
Note. Results are the means for 22 samples. n Significant inc:rease (P < 0.05) compared to 8% glycerol. b Significant decrease (P < 0.01) compared to T,.
150
PILIKIAN,
CZYBA,
AND GUERIN
52
48
16
L 0
I 15
45
30
60
NINUTES
FIG. 2. Evolution in time of the percentage motility recovery of spermatozoa frozen in complex cryoprotecting medium containing 6% (A), 8% (O), 12% (O), 16% (a) glycerol.
in motility is concerned although the values given with the photomicrographs are smaller than those of the Doppler technique. However, there is a large discrep-
38
ancy in the values for 24% glycerol: whereas a drop in motility is registered with the photomicrographic method, the Doppler technique shows no such difference.
c
36
32 30 28
0
30
15
45
60
MINUTES FIG. 3. Evolution in time of the velocity of spermatozoa frozen in complex cryoprotecting medium containing 6% (A), 8% (0), 12% (O), 16% (a) glycerol.
EFFECTS
OF GLYCEROL
Percentage Motility
ON HUMAN
151
SPERMATOZOA
TABLE 3 Recovery Measured with Two Different Techniques Concentration 8%
Doppler Velocimetry Microphotographic technique
of glycerol in Ackerman’s medium 12% % Motility 52” 32”
40 24
24% 54 206
Note. Results are the means for 13 samples. a Significant difference (P < 0.05) compared to 8% glycerol. b Significant difference (P < 0.05) compared to 12% glycerol,
DISCUSSION
The significant decrease in post-thaw motility and velocity agrees with the findings of recent investigations (14, 23). However, results vary (1, 2, 13, 14, 24), due to differences in semen quality, processes of cryopreservation, i.e., cryoprotecting media, glycerol concentrations, equilibration time, freezing and thawing methods, as well as general laboratory conditions. No systematic comparative studies had been done with 6,8, 12, and 16% glycerol in Ackerman’s medium. Under our experimental conditions, no significant difference was noticed in the mean prefreeze motility and velocity for the different concentrations of glycerol used in spite of the high osmotic pressure in 12 and 16% glycerol (815 and 1.086 mosm, respectively). The toxic effect of glycerol became obvious, however, after addition of 24% glycerol and when samples with lower concentrations were kept unfrozen for longer than 1 hr. Alterations in motility have been ascribed to glycerol “osmotically inducing plasmalemma distention” (5). While other mammalian spermatozoa are greatly affected by the hypertonicity of the expanding medium (20), human spermatozoa seem to have a better resistance when motility is considered. Human spermatozoa seemed to be less affected by hypertonic diluters than hypotonic diluters as reported by Guerin and Czyba (12), though these authors used media less hypertonic than those used in the present work.
Ten percent glycerol or less is considered optimum for freeze-preservation of human semen. In our present work, where only percentage motility recovery after freezing was considered, we found that 12 and 16% glycerol gave better motility results than 8% glycerol. Possibly the increased motility recovery in high-osmotic-pressure media was due to the prevention of osmotic swelling during thawing. Wooley and Richardson (25) suggested that, during thawing, cells are exposed to the lower osmotic strength of the melting diluent and are damaged by osmotic swelling. Therefore, it may be possible to prevent osmotic damage by thawing semen in hypertonic media. The high osmotic pressure resulting from 12 and 16% glycerol probably prevents this osmotic swelling. The difference in motility of spermatozoa frozen in 12 and 16% glycerol was not statistically significant. The increase in 16% glycerol was not systematically observed and was mainly due to some samples which continued to give better motility in 16% glycerol. This, once more, shows the importance of individual differences in ability to withstand freezing and cryoprotecting media of different osmotic pressure values. The motility results obtained with the two different methods (Table 3) show: (a) an increase in percentage motility with both methods as the concentration of glycerol is raised from 8 to 16%. The values recorded for the micrographs are smaller than those of the Doppler velocimeter probably because only spermatozoa mov-
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ing on the surface are viewed on the microscopic field. Also, friction forces created by the slide and coverslip may slow down movement of spermatozoa moving alongside the glass and immobilize initially slowmoving spermatozoa. Moreover in the Doppler velocimeter, the temperature is constant which is not the case with the micrographic technique. (b) No difference is shown in percentage motility recovery of spermatozoa frozen in 16 and 24% glycerol according to the optical measurements, whereas the photographic method showed a sharp decrease in 24% glycerol. This discrepancy is due to the increased number of moving but nonprogressing spermatozoa after freezing in 24% glycerol. On the dark-field illumination microphotographs, only progressing spermatozoa leaving a typical white track are counted whereas the blurred images of moving but nonprogressing spermatozoa are not taken into consideration. On the contrary, the Doppler method does not discriminate the progressing and nonprogressing but motile sperm, and considers all of them as “motile.” Nevertheless, both methods showed that the velocity of sperm exhibiting forward motility in 24% glycerol was comparable to that of sperm in 8 or 16% glycerol. The rapid loss in motility and velocity of freeze-thawed semen samples is important when artificial insemination with frozen semen is to be performed. Although postthaw motility and fertilizing capacity are not always correlated, they are the only factors which significantly predict the penetration in cervical mucus (9). Insemination in the first 15 min following thawing becomes imperative if any further loss in motility and velocity is to be prevented. SUMMARY
The percentage post-thaw motility and velocity of semen samples mixed one to one by volume with Ackerman protective
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GUERIN
medium, with final buffered glycerol concentrations of 6, 8, 12, 16, and 24%, and frozen in liquid nitrogen vapor were studied. Semen frozen in 12 and 16% glycerol gave better motility recovery than those frozen in the other concentrations considered. The changes in motility and kinetics of thawed samples, recorded at 15 min intervals, showed a significant drop 30 min after thawing for motility and after 1 hr for velocity. Results obtained with the photomicrographic method confirm the increase in percentage motility given with the SKM measures when the concentration of glycerol is raised from 8 to 12%. The physical method did not discrimate between progressing and nonprogressing spermatozoa motilities while recording the percentage motility of a population of spermatozoa. REFERENCES
1. Behrman, S. J., and Ackerman, D. R. Freeze preservation of human sperm. Amer. J. Obstet. Gynecol. 103, 654-658 (1969). 2. Beck, W. W., and Silverstein, I. Variable motility recovery of spermatozoa following freeze preservation. Ferf. Steril. 26, 863-867 (1975). 3. Czyba, J. C., Pinatel, M. C., and Guerin, J. F. Preservation and storage of human sperm. Acta Med. Pal. 19, 133-146 (1978). 4. Dagnelie, P. “Theorie et methodes statistiques” (J. Ducolot, Ed.), Vol. 2, pp. 35-40. Grembloux, 1970. 5. Drevius, L. 0. The permeability of bull spermatozoa to water polyhydric alcohols and univalent anions and the effects of the anions upon the kinetic activity of spermatozoa and sperm models. J. Reprod. Fert. 28, 41-54 (1972). 6. Dubois, P., Jouannet, P., Berge, P., Volochine, B., Serre, C., and David, G. Methode et appareillage de mesure objective de la mobilitk des spermatozo’ides humains. Ann. Physiol. Biol. Med. 9, 19-41 (1975). 7. Emperaire, J. C., and Riviere, J. La congelation du sperme humain normal. J. Gynecol. O&ret. Biol. Reprod. 3, 215-222 (1974). 8. Femandez-Cano, L. F., Menkin, M. F., Garcia, G. R., and Rock, J. Refrigerant preservation of human spermatozoa. Fert. Steril. 15, 390-405 (1964). 9. Fjallbrant, B., and Ackerman, D. R. Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimens. J. Reprod. Ferr. 20, 515-521 (1%9).
EFFECTS
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10. Freund, M., and Wiederman, J. Factors affecting the dilution, freezing and storage of human semen. J. Reprod. Fer?. 11, 1-17 (1966). 11. Friberg, J., and Gemzell, C. Insemination of sperm after freezing in liquid nitrogen vapors with glycerol or glycerol-egg yolk-citrate as protective media. Amer. J. Obstet. Gynecol. 116, 330-334 (1975). 12. Guerin J. F., and Czyba, J. C. Effects de la pression osmotique sur la mobilite et metabolisme des spermatozdides humains. In “Comptes rendus des seances de la Societe de Biologie 1977,” pp. 822-825. 13. Harrison, R. IF., and Sheppard, B. L. A comparative study in methods of cryoprotection for human semen. Cryobiology 17, 25-32 (1980). 14. Keel, B. A., and Black, J. B. Reduced motility longevity in thawed human spermatozoa. Arch. Androl. 4, 213-215 (1980). 15. Keel, B. A., and Karow, A. A., Jr. Motility characteristics of human sperm, nonfrozen and cryopreserved. Arch. Androl. 4, 205-212 (1980). 16. Makler, A. A new multiple exposure photography method for objective human spermatozoa1 motility determination. Fert. Steril. 30, 192-199, (1978). 17. Overstreet, J., Katz, D., Hanson, F. W., and Fonseca, J. R. A simple inexpensive method for objective assessment of human sperm movement characteristics Fertil. Steril. 31, 162- 172 (1979).
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18. Pedersen, H., and Lebech, P. E. Ultrastructural changes in the human spermatozoa after freezing for artificial insemination. Fert. Steril. 22, 125-133 (1971). 19. Polge, G., Smith, A. U., and Parkes, A. S. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature (London) 164, 666-669 (1949). 20. Rao, P. S., Sikes, J. D., and Merilan, C. P. Freezing point depression and viability of bovine sperm during freezing and storage. Fert. Steril. 19, 129-136 (1968). 21. Rubin, S. O., Anderson, L., and Bostrom, K. Deep-freeze preservation of normal and pathologic human semen. &and. J. Ural. Nephrol. 3, 144- 150 (1%9). 22. Sawada, Y., Ackerman, D. R., and Behrman, S. J. Motility and respiration of human spermatozoa after cooling to various low temperatures. Fert. Steril. 18, 775-781 (1967). 23. Serres C., Jouannet, P., Czylik, F., and David, G. Effects of freezing on spermatozoa motility. In “Human Artificial Insemination and Semen Preservation” (G. David and S. P. Wendel, Eds.), p. 147. Plenum, New York, 1980. 24. Sherman, J. K. Improved methods of preservation of human spermatozoa by freezing and freezedrying. Fert. Steril. 14, 49-64 (1963). 25. Woolley, D. M., and Richardson, D. W. Ultrastructural injury to human spermatozoa after freezing and thawing. .I. Reprod. Fert. 53, 389-394 (1978).