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Sublethal Damage during Cryopreservation of Rainbow Trout Sperm
37, 245–253 (1998) CY982121
CRYOBIOLOGY ARTICLE NO.
Sublethal Damage during Cryopreservation of Rainbow Trout Sperm E. Cabrita,* R. Alvarez,* L. Anel,† K. J. Rana,‡ and M. P. Herraez* *Department of Biologı´a Celular y Anatomı´a and †Department of Patologı´a Animal, University of Leo´n, Spain; and ‡Fisheries Department, Food and Agriculture Organization, Italy Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at 220°C/min in a programmable cooler and thawed in a 25°C water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures. © 1998 Academic Press
Cryopreservation of freshwater fish spermatozoa usually reduces the fertilizability of postthawed milt. Cellular damage to the sperm cells have been monitored in ultrastructural and biochemical studies (3, 11, 12, 14, 19), and permeability to different fluorescent probes has also been studied (12, 19, 20). However, the causal relationship between such damage and fertility has not been clearly demonstrated (12, 19), and electron microscopic studies suggest greater and varied damage during the processing of frozen–thawed cells which appear to be more liable to suffer artifacts. The fragility of the membrane of these cells could explain the low fertility rates achieved with frozen/thawed sperm, even when the live spermatozoa/egg ratio is theoretically sufficient to guarantee a high percentage of fertility (4, 5, 19). The loss of fertility could be attributed to additional undetected sublethal cryodamage, which could destabilize the sperm membrane and thereby af-
fect spermatozoa function and viability at later times (12). This loss of function is extremely important for freshwater fish spermatozoa whose motility, activation, and ability to fertilize the egg require their dilution in water or hypoosmotic solutions (3, 4). These media constitute a hazardous environment for the cells, which, even under ideal conditions, remain motile for only around 1 min. The aim of this study was to evaluate sublethal damage caused during cryopreservation by analyzing the resistance of live spermatozoa to hypoosmotic shock and examining the effect of egg yolk as a membrane stabilizer.
Received January 20, 1998; accepted July 30, 1998.
MATERIAL AND METHODS
Animals Three-year-old male and female rainbow trout (Oncorhynchus mykiss), provided by a commercial farm, were kept during the experimental period in 50,001 fiberglass tanks with recycled freshwater at 12°C under a natural photoperiod.
245 0011-2240/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
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Milt
Assessment of Sublethal Damage
The milt was obtained by catheterization and the quality from each individual male was checked using the same methods that were applied in previous studies (12). Only samples whose quality parameters ranged between the following values were used: pH, 8.08 – 8.42; osmolality, 310 –319 mOsm/kg; motility, 80 – 100% motile cells; cell density, 48 3 109– 114 3 109 cells/ml; live cells (nonpermeable to Hoechst 33285), 85–100%. All sperm manipulations were carried out at 4°C using individual males for every trial. Samples diluted in the cryoprotectant solutions were frozen after 30 min of equilibration in 0.5-ml plastic straws. Freezing was carried out at 220°C/min in a programmable biofreezer, and thawing was carried out in a water bath at 25°C for 30 s.
Effects on cell stability were studied by carrying out serial time course measurements of viability following dilution 1:100 in different hypoosmotic solutions. The solutions were prepared at the beginning of the experimental period, stored at 220°C, and thawed at room temperature immediately before use. Different solutions were used in the two experiments: fresh water (3 6 1 mOsm/kg); Tris–NaCl buffer, pH 7.4 (10, 50, 150, and 300 mOsm/kg); activation solutions from Steyn et al. (20), Wheeler and Thorgaard (22), and Billard (2) (DIA532). Cell permeability to Hoechst 33258 was checked at different times after dilution and related to the nonpermeable (live) cell count before the hypoosmotic shock. Experimental Design
Fertility Trials Eggs were obtained from four females, pooled and fertilized with the sperm, using the wet method. The activation medium was water. The sperm/egg ratio was always in excess of 10 3 106 live cells/egg in order to guarantee sufficient live spermatozoa in the frozen/thawed samples. Eggs were placed in a vertical incubator for salmonids at 11 6 1°C, in the dark, and fertility was expressed as the percentage of eyed embryos. Cell Viability Sperm samples were fixed in 4% glutaraldehyde in phosphate-buffered saline (PBS) (at pH 7.4) for 1 min and the fixation was stopped by dilution 1:500 in PBS. Cell permeability to Hoechst 33258 was analyzed after 5 min of incubation at room temperature in the dark with equal volume of a solution of 20 mg/ml of Hoechst in sodium citrate buffer. Two smears from each sample (100 –120 cells each) were analyzed using an epifluorescence microscope. Unlabeled cells were designated as live cells, but when they displayed an intense fluorescence all around the head or close to the middle piece, they were noted as nonviable cells.
Trial I Fresh sperm. Immediately after it was extracted from four males, milt was used to carry out the standard semen analysis and the time course in viability after hypoosmotic shock. Water and all the sodium chloride solutions were used and viability was assessed at 30 s and 2, 5, 10, and 30 min after dilution. Diluted sperm. Semen from each of three individual males was diluted 1:3 in the extender No. 6 from Erdhal and Graham (10) containing different cryoprotectants (CPTs) as follows: 7% Me2SO, 7% DMA, 5% methanol. After 30 min of equilibration, the viability of the cells and their stability in hypoosmotic solutions were assessed under the same conditions that were used for the undiluted sperm. Frozen/thawed sperm. After freezing and thawing, viability by the Hoechst dye, fertility, and viability in hypoosmotic solutions were assessed for semen from three individual males. Two hypoosmotic solutions usually used to activate the motility of the frozen/thawed sperm were also checked: one from Steyn et al. (20) and the other from Wheeler and Thorgaard (22). Viability was also checked 10 sec after the hypoosmotic shock.
SUBLETHAL DAMAGE IN CRYOPRESERVED FISH SPERM
Trial II After the standard parameters were checked, the milt from five individual males were separately diluted in four different media: two extenders (No. 6 from Erdhal and Graham (10) and Lahnsteiner (15)) containing 7% Me2SO, each with and without 10% egg yolk. Osmolality of the dilution media ranged between 1630 to 1770 mOms/kg without significant differences between extenders. The dilution ratio was 1:3 (sperm/extender) and freezing was carried out after equilibration for 30 min. After freezing and thawing the viability, fertility, and time course of viability in hypoosmotic solutions were measured. The hypoosmotic solutions tested were the 50 mOsm/kg, 150 mOsm/kg, and 300 mOsm/kg sodium chloride solutions and the activation solution DIA532 from Billard (2). Viability was assessed 10 and 30 s and 5, 10, and 30 min after hypoosmotic shock. All experiments were conducted in triplicate. Biometrics Results were examined using a one-way analysis of variance with the Student–Neuman– Keuls multiple range test. The significance level was 5%. RESULTS
Trial I—Evaluation of Cell Fragility during the Cryopreservation Procedure Unfrozen milt samples showed a high percentage of viable cells, ranging between 85 and 100%. Nevertheless, after dilution in hypoosmotic solutions between 30 s and 10 min the viability of these cells suffered a significant decrease (Fig. 1A). The cells exposed to water and the 10 mOsm/kg sodium chloride solution displayed a dramatic drop in viability at 5 min. Loss of viability took place later in 50 and 150 mOsm/kg solutions (10 min after dilution) and was not observed in the isoosmotic solution at 300 mOsm/kg. Dilution of sperm in the CPTs caused a slight but significant increase in fragility. The permeability of live cells to the probe increased more
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rapidly after the hypoosmotic shock and the viability in isoosmotic media was significantly reduced compared to untreated controls (Fig. 1B). Even in this solution (300 mOsm/kg), a decrease of viability with time was noticed. Sperm diluted in the extender containing Me2SO displayed significantly better results when compared with the other cryoprotectants that were tested. Freezing and thawing caused further significant losses in milt quality. The percentage of live cells suffered a significant decrease after the freeze/thaw process (Fig. 2), most pronounced when methanol was the cryoprotectant: with methanol only 3.9% of the postthawed cells were viable. Higher percentages of viable cells were observed when Me2SO or DMA were used (17.3 and 17.7%). Fertility was achieved only in samples frozen with Me2SO (10.4%): no fertility was observed in samples containing methanol or DMA, despite the samples containing live cells sufficient to fertilize the eggs. After thawing a dramatic increase in the fragility of the cells was also observed; live spermatozoa were dead in a few seconds after their contact with the hypoosmotic solutions (Fig. 3). Of the tested solutions, those formulated to activate cell motility gave significantly poorer results. Lower survival rates were registered after dilution with the Steyn solution, but with the medium from Wheeler and Thorgaard, the cells survived for a few minutes following freezing with Me2SO (Fig. 3A). These samples displayed significantly better results than those frozen with DMA (Fig. 3B) or methanol (Fig. 3C). Trial II—Effect of Egg Yolk on Cell Integrity Significant losses of semen quality were also noticed after thawing but the decrease was significantly smaller in samples frozen with media containing egg yolk: these gave 36 –38% of nonpermeable cells, whereas media without egg yolk reached only 21–23% live cells. No differences in viability were observed between extenders. Fertility dropped in all treatments in relation to the controls (98%) and the addition of egg yolk promoted a small but insignificant increase in eyed embryos when the extender
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FIG. 1. Permeability to Hoechst dye in fresh (A) and diluted sperm (B) (n 5 3). (A) Time course of permeability of fresh sperm in solutions of differing osmolality. (B) Time course of permeability of sperm after equilibration in different CPTs and dilution in the 300 mOsm/kg solution. *, no data available.
from Erdhal and Graham was used (6 and 4%, respectively). No fertilization occurred with the samples frozen with Lahnsteiner’s extender. The effect of egg yolk was more noticeable when resistance to hypoosmotic shock was analyzed (Fig. 4). Cells frozen in media without egg yolk did not show a significant proportion of live cells after 30 s in the solutions that were tested (Figs. 4A and 4C), but a significant resistance was observed when egg yolk was added to the extender (Figs. 4B and 4D). In these samples, viability decreased with the time, but a reasonable percentage of nonpermeable cells was observed even 5 min after dilution in the 10 mOsm/kg solution. The use of this stabilizer with the extender from Erdahl and Graham (Fig.
4B) provided significantly better results than the extender from Lahnsteiner (Fig. 4D). In relation to the hypoosmotically tested solutions, the cells displayed similar behavior in the 150 mOsm/kg solution and in DIA523, which was designed to activate trout sperm motility. DISCUSSION
The postthaw viability of cryopreserved spermatozoa has often given low fertility rates during insemination trials, despite the maintenance of adequate motility and structural integrity (viability) after thawing (12). The possibility exists that damage to the sperm membrane might occur despite preservation of other semen param-
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FIG. 2. Viability and fertility of fresh and frozen and thawed sperm (n 5 3). Statistically significant differences are noted by asterisks.
eters, but there is no published functional analysis that assesses cryopreserved fish sperm quality under physiological conditions. Motility in the spermatozoa of freshwater fishes with external fertilization is activated only after dilution in appropriate solutions that are usually hypoosmotic, and they then remain motile for very short periods—a few seconds in salmonids (4). The assessment of semen quality by biochemical studies, structural analysis, or permeability to fluorescence probes has always been carried out before activation, but this ignores the important stress to which the sperm is to be exposed in order to reach the egg. Functional tests should measure functionality under physiological conditions. Such measurements of membrane functional integrity performed in undiluted fresh semen, in semen diluted in CPTs, and in frozen/thawed semen could thus contribute to the assessment of functional damage during the cryopreservation process. Our results indicate that unfrozen, undiluted sperm show a progressive loss of viability with time of contact with hypoosmotic solutions. The cells display a behavior significantly different in water or 10 mOsm/kg solution, in which viability drops dramatically after 5 min. In 50 and 150 mOsm/kg solutions loss of viability takes place later. No loss is observed under isoosmotic conditions (300 mOsm/kg). Similar results were obtained with carp sperm (17), which showed a
high survival in solutions more concentrated than 250 mOsm/kg and an increase of dead cells in more hypoosmotic media. Nevertheless, the membrane is sufficiently resistant to allow cells to survive for the few minutes required to reach the egg after activation. Dilution in the CPTs causes a significant increase in fragility when the cells are subjected to hypoosmotic shock. Sperm diluted in extender containing Me2SO display significantly better results after 30 min equilibration than sperm exposed to the other cryoprotectants that were tested. In addition to direct effects of the CPTs on cells, the extender– cryoprotectant has a higher osmolality than the semen samples, so that subsequent dilution in hypoosmotic solutions will cause greater osmotic stress when the cells are first immersed in a hypertonic and then in a hypoosmotic medium. Curry and Watson (7) demonstrated that ram and human sperm show a high resistance to hyperosmotic stress, but their further dilution to isoosmotic conditions promotes loss of viability. The rapid influx of water into the cells under these conditions was proposed as the mechanism of cell damage. In the present study our results show that even when Me2SO was used as the CPT, the increase in sperm damage when exposed to hypoosmotic solutions was significant. The osmolality of the CPT solutions containing Me2SO used in the two experiments ranged from 1625 mOsm/kg to
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FIG. 3. Time course of permeability to Hoechst dye of frozen and thawed sperm after dilution in hypoosmotic solutions (n 5 3). (A) Samples frozen with Me2SO. (B) Samples frozen with DMA. (C) Samples frozen with methanol. Hypoosmotic solutions: A, Steyn et al. (20); B, Wheeler and Thorgaard (22); C, 50 mOsm/Kg; D, 150 mOsm/Kg; E, 300 mOsm/Kg.
1778 mOsm/kg, but cell viability did not appear to be affected by this exposure; loss of viability was observed only after hypoosmotic shock and could be explained by possible detrimental effects of the CPTs on the membranes. After freezing and thawing about 20% of the cells frozen with Me2SO or DMA were still nonpermeable to the Hoechst dye. A similar percentage of undamaged cells after thawing was reported by Ogier de Baulny et al. (19). In their study, Me2SO was also found to be the most favourable CPT as judged by membrane integrity after thawing. However, in the present study not only was the number of dead cells increased following cryopreservation, but there was also a dramatic increase in the fragility of the live cells. A few seconds after their contact with the hypoosmotic solutions they became permeable to the
probe. Samples frozen with Me2SO showed a higher resistance of the cell membrane than those frozen with DMA or methanol, which explains the superiority of this agent as a cryoprotectant for salmonid sperm. The fertility rates achieved in the present study are consistent with the observed increase in susceptibility to hypoosmotic shock, but not with the proportion of live cells measured before hypotonic exposure, demonstrating that sublethal cryodamage that is not detected by standard procedures had been caused. Donoghue et al. (9), working with turkey sperm, have also reported that there is a population of labile spermatozoa more sensitive to hypoosmotic stress, which is undetected if fluorescent probes are used alone. Membrane destabilization has been pointed out as the main factor causing damage to mammalian sperm during freezing and thawing (8).
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FIG. 4. Time course of permeability to Hoechst dye of frozen and thawed sperm after dilution in hypoosmotic solutions (n 5 5). Samples frozen with (A) No. 6 –Me2SO; (B) No. 6 –Me2SO– egg yolk; (C) Lahnsteiner– Me2SO; (D) Lahnsteiner–Me2SO– egg yolk. Hypoosmotic solutions: A, 10 mOsm/Kg; B, 150 mOsm/Kg; C, 300 mOsm/Kg; D, DIA532.
Alvarez and Storey (1) hypothesized two models to explain sublethal cryodamage in human sperm: peroxidation-related events due to lipid peroxidation of the plasma membrane; mechanical stress in the membrane, involving membrane embrittlement during phase transitions occurring during the freezing–thawing cycle. The use of lipid peroxidation inhibitors and membrane stabilizers, such as egg yolk or polyols, allowed them to confirm the stress-related proposal as the main contributor to sublethal cryodamage. Phase transitions were not detected during freezing and thawing of trout sperm (13) but our results show a significant increase of susceptibility to osmotic stress during the process, as well as a beneficial effect of egg yolk, suggesting that membrane embrittlement during the freezing/thawing process can
be significantly reduced by using membrane stabilizers. The sperm membrane of carp shows a significant lose of amino acids during cryopreservation and this could induce a destabilization of hydrophobic interactions between the proteins and lipids of the membrane during freezing (18). These results suggest that the decrease of sperm membrane stability in trout could also be due to changes in molecular interactions between membrane components. In the present study the use of egg yolk promoted significantly increased membrane resistance and sperm motility, but a similar increase in fertility was not observed. The cryoprotective action offered by the complex mixture that composes egg yolk is well known in mammals (8). The low density lipoprotein fraction is an extracellular cryoprotectant that
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renders the membrane more stable at low temperatures (21). The mode of action still remains unclear but the use of egg yolk in some species decreases the fertility rate, which has been explained by a possible interference between egg yolk lipids and other seminal plasma components during fertilization (6). In the present study, a similar interference by egg yolk could affect the sperm entry through the micropile or other unknown mechanisms involving the interaction of sperm and egg membrane. The low fertility rates achieved with postthawed semen in all trials could be exacerbated by the use of water as the sperm activator. As we have demonstrated, dilution of thawed sperm in such a medium causes cell death within a few seconds. All the tested solutions usually used to activate the cell motility, except DIA 532, were also hazardous for the stability of thawed cells. Our data suggest that new extenders containing different membrane stabilizers and motility activators with osmolalities exceeding 150 mOsm/kg are needed to minimize sublethal cryodamage and to improve fertility rates of trout sperm after freezing and thawing. CONCLUSION
The hypoosmotic solutions usually used to activate motility in artificial fertilization cause cell death within a few minutes in fresh sperm. Susceptibility to these solutions is slightly higher after dilution with cryoprotectants, but dramatically increases after freezing and thawing, showing sublethal cryodamage that is not demonstrated by ultrastructural or biochemical studies or by counting the live cells immediately after thawing. The results suggest that the addition of components that are able to increase the membrane stability and the use of higher osmolalities to activate motility should be analyzed in order to improve fertilizing ability of cryopreserved sperm. REFERENCES 1. Alvarez, J. G., and Storey, B. T. Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human
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sperm: Glycerol and other polyols as sole cryoprotectant. J. Androl. 14, 199 –209 (1993). Billard, R. Artificial insemination and gamete management in fish. Mar. Behav. Physiol. 14, 3–21 (1988). Billard, R. Ultrastructure of trout spermatozoa: Changes after dilution and deep-freezing. Cell. Tissue Res. 228, 205–218 (1983). Billard, R., Cosson, J., Crim, L. W., and Suquet, M. Sperm physiology and quality. In “Broodstock Management and Egg and Larval Quality” (Bromage and Roberts, Eds). Blackwell Sci. Oxford, 1995. Cabrita, E., Alvarez, R., Anel, L., Rana, K. J., and Herraez, P. Evidence of sublethal sperm membrane damages during cryopreservation of rainbow trout sperm. Proc. Cryo’97, 34th Annu. Meet. Soc. Cryobiol., Barcelona, Spain, June 1997, No. 150, 1997. Chauhan, M. S., and Anand, R. R. Effect of egg yolk lipids on the freezing of goat semen. Theriogenology 34, 1003–1013 (1990). Curry, M., and Watson, P. Osmotic effects on ram and human sperm membranes in relation to thawing. Cryobiology 31, 39 – 46 (1994). de Leeuw, F. E., de Leeuw, A. M., Den Daas, J. H. G., Colenbrander, B., and Verkleij, A. J. Effects of various cryoprotective agents and membrane-stabilizing compounds on bull sperm membrane integrity after cooling and freezing. Cryobiology 30, 32– 44 (1993). Donoghue, A. M., Garner, D. I., Donoghue, D. J., and Johnson, L. A. Assessment of the membrane integrity of fresh and stored turkey spermatozoa using a combination of hypo-osmotic stress, fluorescent staining and flow cytometry. Theriogenology 46, 153–163. Erdhal, D., and Graham, E. F. Cryopreservation of spermatozoa of the brook and rainbow trout. CryoLett. 1, 203–208 (1980). Gwo, J., and Arnold, C. R. Cryopreservation of Atlantic croaker spermatozoa: Evaluation of morphological changes. J. Exp. Zool. 264, 444 – 453 (1992). Herra´ez, P., Mediavilla, M., Alvarez, R., Manso, A., and de Paz, P. Cellular damages caused by cryopreservation in trout sperm. Proc. Int. Meet. Refrig. Aquacult., Bordeaux, France, 57– 64 (1996). Labbe´, C., Crowe, L. M., and Crowe, J. H. Stability of the lipid component of trout sperm plasma membrane during freeze thawing. Cryobiology 34, 176 – 182 (1997). Lahnsteiner, F., Weisman, T., and Patzner, R. A. Fine structural changes in spermatozoa of the grayling, Thymallus thymallus during routine cryopreservation. Aquaculture 103, 73– 84 (1992).
SUBLETHAL DAMAGE IN CRYOPRESERVED FISH SPERM 15. Lahnsteiner, F., Weismann, T., and Patzner, R. A. A uniform method for cryopreservation of semen of the salmonid fishes O. mykiss, S. trutta f. fario, S. trutta f. lacustris, Coregonus sp. Aquacult. Res. 26, 801– 807 (1995). 16. Malejac, M., Loir, M., and Maisse, G. Qualite´ de la membrane des spermatozoides de truite arc-en-ciel; relation avec l’aptitude du sperme a la congelation. Aquat. Living Resour. 3, 43–54 (1990). 17. Ma´ria´n, T., Krazsnai, Z., Balkay, L., Bala´zs, M., Emry, M., Bene, L., and Tro´n, L. Hypo-osmotic shock induces an osmolality-dependent permeabilization and structural changes in the membrane of carp sperm. J. Histochem. Cytochem. 41(2), 291–297 (1993). 18. Nauc, V., and Boronciuc, Gh. Cryogenic changes in carp spermatozoa content. Proc. Cryo ’97, 34th
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Annu. meet. Soc. Cryobiol. Barcelona, Spain, June 1997, No. 104 (1997). Ogier de Baulny, B., Le Vern, Y., Kerboeuf, D., and Maisse, G. Flow cytometric evaluation of mitochondrial activity and membrane integrity in fresh and cryopreserved rainbow trout spermatozoa. Cryobiology 34, 141–149 (1997). Steyn, G., Van Vuren, J., and Grobler, E. A new sperm diluent for the artificial insemination of rainbow trout. Aquaculture 83, 367–374 (1989). Watson, P. F., and Martin, I. C. A. The response of ram spermatozoa to preparation of egg yolk in semen diluents during storage at 5 or 2196°C. Aust. J. Biol. Sci. 26, 927–935 (1973). Wheeler, P. A., and Thorgaard, G. H. Cryopreservation of rainbow trout semen in large straws. Aquaculture 93, 95–100 (1991).
CRYOBIOLOGY ARTICLE NO.
Sublethal Damage during Cryopreservation of Rainbow Trout Sperm E. Cabrita,* R. Alvarez,* L. Anel,† K. J. Rana,‡ and M. P. Herraez* *Department of Biologı´a Celular y Anatomı´a and †Department of Patologı´a Animal, University of Leo´n, Spain; and ‡Fisheries Department, Food and Agriculture Organization, Italy Although cellular damage during cryopreservation of freshwater fish spermatozoa has been reported in several studies, there is a lack of correlation between this damage and the fertility rates of eggs using postthawed milt. The apparent lack of such correlation may be due to other undetected sublethal cryodamage, which could affect the cell functionality and viability. This may be extremely important for freshwater fish spermatozoa whose ability to fertilize the egg requires dilution in water or hypoosmotic solutions, an hazardous environment for the cells. This study tested the change in cell permeability during cryopreservation, using Hoechst 33258 to assess cell permeability. The permeability of spermatozoa at different times after dilution in several hypoosmotic media were investigated. In the first trial, fresh semen, sperm diluted in freezing media (CPT), and freeze/thawed semen were studied. Three CPT were tested (Me2SO, DMA, and methanol). In the second trial, the addition of egg yolk as a membrane stabilizer was investigated. Samples were frozen at 220°C/min in a programmable cooler and thawed in a 25°C water bath. Dilution in the CPTs slightly increased the susceptibility of cells to damage but freezing/thawing caused a dramatic increase in the fragility of cells, which were killed in a few seconds after their contact with the hypoosmotic solutions. Egg yolk provided a significant protection to the membrane, allowing the cells a greater and more prolonged survival in the fertilization media. Samples frozen with Me2SO displayed the best results. These results are consistent with the achieved fertility rates that demonstrated sublethal cryodamage in the function of the sperm membrane that was not detected by standard procedures. © 1998 Academic Press
Cryopreservation of freshwater fish spermatozoa usually reduces the fertilizability of postthawed milt. Cellular damage to the sperm cells have been monitored in ultrastructural and biochemical studies (3, 11, 12, 14, 19), and permeability to different fluorescent probes has also been studied (12, 19, 20). However, the causal relationship between such damage and fertility has not been clearly demonstrated (12, 19), and electron microscopic studies suggest greater and varied damage during the processing of frozen–thawed cells which appear to be more liable to suffer artifacts. The fragility of the membrane of these cells could explain the low fertility rates achieved with frozen/thawed sperm, even when the live spermatozoa/egg ratio is theoretically sufficient to guarantee a high percentage of fertility (4, 5, 19). The loss of fertility could be attributed to additional undetected sublethal cryodamage, which could destabilize the sperm membrane and thereby af-
fect spermatozoa function and viability at later times (12). This loss of function is extremely important for freshwater fish spermatozoa whose motility, activation, and ability to fertilize the egg require their dilution in water or hypoosmotic solutions (3, 4). These media constitute a hazardous environment for the cells, which, even under ideal conditions, remain motile for only around 1 min. The aim of this study was to evaluate sublethal damage caused during cryopreservation by analyzing the resistance of live spermatozoa to hypoosmotic shock and examining the effect of egg yolk as a membrane stabilizer.
Received January 20, 1998; accepted July 30, 1998.
MATERIAL AND METHODS
Animals Three-year-old male and female rainbow trout (Oncorhynchus mykiss), provided by a commercial farm, were kept during the experimental period in 50,001 fiberglass tanks with recycled freshwater at 12°C under a natural photoperiod.
245 0011-2240/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved.
246
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Milt
Assessment of Sublethal Damage
The milt was obtained by catheterization and the quality from each individual male was checked using the same methods that were applied in previous studies (12). Only samples whose quality parameters ranged between the following values were used: pH, 8.08 – 8.42; osmolality, 310 –319 mOsm/kg; motility, 80 – 100% motile cells; cell density, 48 3 109– 114 3 109 cells/ml; live cells (nonpermeable to Hoechst 33285), 85–100%. All sperm manipulations were carried out at 4°C using individual males for every trial. Samples diluted in the cryoprotectant solutions were frozen after 30 min of equilibration in 0.5-ml plastic straws. Freezing was carried out at 220°C/min in a programmable biofreezer, and thawing was carried out in a water bath at 25°C for 30 s.
Effects on cell stability were studied by carrying out serial time course measurements of viability following dilution 1:100 in different hypoosmotic solutions. The solutions were prepared at the beginning of the experimental period, stored at 220°C, and thawed at room temperature immediately before use. Different solutions were used in the two experiments: fresh water (3 6 1 mOsm/kg); Tris–NaCl buffer, pH 7.4 (10, 50, 150, and 300 mOsm/kg); activation solutions from Steyn et al. (20), Wheeler and Thorgaard (22), and Billard (2) (DIA532). Cell permeability to Hoechst 33258 was checked at different times after dilution and related to the nonpermeable (live) cell count before the hypoosmotic shock. Experimental Design
Fertility Trials Eggs were obtained from four females, pooled and fertilized with the sperm, using the wet method. The activation medium was water. The sperm/egg ratio was always in excess of 10 3 106 live cells/egg in order to guarantee sufficient live spermatozoa in the frozen/thawed samples. Eggs were placed in a vertical incubator for salmonids at 11 6 1°C, in the dark, and fertility was expressed as the percentage of eyed embryos. Cell Viability Sperm samples were fixed in 4% glutaraldehyde in phosphate-buffered saline (PBS) (at pH 7.4) for 1 min and the fixation was stopped by dilution 1:500 in PBS. Cell permeability to Hoechst 33258 was analyzed after 5 min of incubation at room temperature in the dark with equal volume of a solution of 20 mg/ml of Hoechst in sodium citrate buffer. Two smears from each sample (100 –120 cells each) were analyzed using an epifluorescence microscope. Unlabeled cells were designated as live cells, but when they displayed an intense fluorescence all around the head or close to the middle piece, they were noted as nonviable cells.
Trial I Fresh sperm. Immediately after it was extracted from four males, milt was used to carry out the standard semen analysis and the time course in viability after hypoosmotic shock. Water and all the sodium chloride solutions were used and viability was assessed at 30 s and 2, 5, 10, and 30 min after dilution. Diluted sperm. Semen from each of three individual males was diluted 1:3 in the extender No. 6 from Erdhal and Graham (10) containing different cryoprotectants (CPTs) as follows: 7% Me2SO, 7% DMA, 5% methanol. After 30 min of equilibration, the viability of the cells and their stability in hypoosmotic solutions were assessed under the same conditions that were used for the undiluted sperm. Frozen/thawed sperm. After freezing and thawing, viability by the Hoechst dye, fertility, and viability in hypoosmotic solutions were assessed for semen from three individual males. Two hypoosmotic solutions usually used to activate the motility of the frozen/thawed sperm were also checked: one from Steyn et al. (20) and the other from Wheeler and Thorgaard (22). Viability was also checked 10 sec after the hypoosmotic shock.
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Trial II After the standard parameters were checked, the milt from five individual males were separately diluted in four different media: two extenders (No. 6 from Erdhal and Graham (10) and Lahnsteiner (15)) containing 7% Me2SO, each with and without 10% egg yolk. Osmolality of the dilution media ranged between 1630 to 1770 mOms/kg without significant differences between extenders. The dilution ratio was 1:3 (sperm/extender) and freezing was carried out after equilibration for 30 min. After freezing and thawing the viability, fertility, and time course of viability in hypoosmotic solutions were measured. The hypoosmotic solutions tested were the 50 mOsm/kg, 150 mOsm/kg, and 300 mOsm/kg sodium chloride solutions and the activation solution DIA532 from Billard (2). Viability was assessed 10 and 30 s and 5, 10, and 30 min after hypoosmotic shock. All experiments were conducted in triplicate. Biometrics Results were examined using a one-way analysis of variance with the Student–Neuman– Keuls multiple range test. The significance level was 5%. RESULTS
Trial I—Evaluation of Cell Fragility during the Cryopreservation Procedure Unfrozen milt samples showed a high percentage of viable cells, ranging between 85 and 100%. Nevertheless, after dilution in hypoosmotic solutions between 30 s and 10 min the viability of these cells suffered a significant decrease (Fig. 1A). The cells exposed to water and the 10 mOsm/kg sodium chloride solution displayed a dramatic drop in viability at 5 min. Loss of viability took place later in 50 and 150 mOsm/kg solutions (10 min after dilution) and was not observed in the isoosmotic solution at 300 mOsm/kg. Dilution of sperm in the CPTs caused a slight but significant increase in fragility. The permeability of live cells to the probe increased more
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rapidly after the hypoosmotic shock and the viability in isoosmotic media was significantly reduced compared to untreated controls (Fig. 1B). Even in this solution (300 mOsm/kg), a decrease of viability with time was noticed. Sperm diluted in the extender containing Me2SO displayed significantly better results when compared with the other cryoprotectants that were tested. Freezing and thawing caused further significant losses in milt quality. The percentage of live cells suffered a significant decrease after the freeze/thaw process (Fig. 2), most pronounced when methanol was the cryoprotectant: with methanol only 3.9% of the postthawed cells were viable. Higher percentages of viable cells were observed when Me2SO or DMA were used (17.3 and 17.7%). Fertility was achieved only in samples frozen with Me2SO (10.4%): no fertility was observed in samples containing methanol or DMA, despite the samples containing live cells sufficient to fertilize the eggs. After thawing a dramatic increase in the fragility of the cells was also observed; live spermatozoa were dead in a few seconds after their contact with the hypoosmotic solutions (Fig. 3). Of the tested solutions, those formulated to activate cell motility gave significantly poorer results. Lower survival rates were registered after dilution with the Steyn solution, but with the medium from Wheeler and Thorgaard, the cells survived for a few minutes following freezing with Me2SO (Fig. 3A). These samples displayed significantly better results than those frozen with DMA (Fig. 3B) or methanol (Fig. 3C). Trial II—Effect of Egg Yolk on Cell Integrity Significant losses of semen quality were also noticed after thawing but the decrease was significantly smaller in samples frozen with media containing egg yolk: these gave 36 –38% of nonpermeable cells, whereas media without egg yolk reached only 21–23% live cells. No differences in viability were observed between extenders. Fertility dropped in all treatments in relation to the controls (98%) and the addition of egg yolk promoted a small but insignificant increase in eyed embryos when the extender
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FIG. 1. Permeability to Hoechst dye in fresh (A) and diluted sperm (B) (n 5 3). (A) Time course of permeability of fresh sperm in solutions of differing osmolality. (B) Time course of permeability of sperm after equilibration in different CPTs and dilution in the 300 mOsm/kg solution. *, no data available.
from Erdhal and Graham was used (6 and 4%, respectively). No fertilization occurred with the samples frozen with Lahnsteiner’s extender. The effect of egg yolk was more noticeable when resistance to hypoosmotic shock was analyzed (Fig. 4). Cells frozen in media without egg yolk did not show a significant proportion of live cells after 30 s in the solutions that were tested (Figs. 4A and 4C), but a significant resistance was observed when egg yolk was added to the extender (Figs. 4B and 4D). In these samples, viability decreased with the time, but a reasonable percentage of nonpermeable cells was observed even 5 min after dilution in the 10 mOsm/kg solution. The use of this stabilizer with the extender from Erdahl and Graham (Fig.
4B) provided significantly better results than the extender from Lahnsteiner (Fig. 4D). In relation to the hypoosmotically tested solutions, the cells displayed similar behavior in the 150 mOsm/kg solution and in DIA523, which was designed to activate trout sperm motility. DISCUSSION
The postthaw viability of cryopreserved spermatozoa has often given low fertility rates during insemination trials, despite the maintenance of adequate motility and structural integrity (viability) after thawing (12). The possibility exists that damage to the sperm membrane might occur despite preservation of other semen param-
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FIG. 2. Viability and fertility of fresh and frozen and thawed sperm (n 5 3). Statistically significant differences are noted by asterisks.
eters, but there is no published functional analysis that assesses cryopreserved fish sperm quality under physiological conditions. Motility in the spermatozoa of freshwater fishes with external fertilization is activated only after dilution in appropriate solutions that are usually hypoosmotic, and they then remain motile for very short periods—a few seconds in salmonids (4). The assessment of semen quality by biochemical studies, structural analysis, or permeability to fluorescence probes has always been carried out before activation, but this ignores the important stress to which the sperm is to be exposed in order to reach the egg. Functional tests should measure functionality under physiological conditions. Such measurements of membrane functional integrity performed in undiluted fresh semen, in semen diluted in CPTs, and in frozen/thawed semen could thus contribute to the assessment of functional damage during the cryopreservation process. Our results indicate that unfrozen, undiluted sperm show a progressive loss of viability with time of contact with hypoosmotic solutions. The cells display a behavior significantly different in water or 10 mOsm/kg solution, in which viability drops dramatically after 5 min. In 50 and 150 mOsm/kg solutions loss of viability takes place later. No loss is observed under isoosmotic conditions (300 mOsm/kg). Similar results were obtained with carp sperm (17), which showed a
high survival in solutions more concentrated than 250 mOsm/kg and an increase of dead cells in more hypoosmotic media. Nevertheless, the membrane is sufficiently resistant to allow cells to survive for the few minutes required to reach the egg after activation. Dilution in the CPTs causes a significant increase in fragility when the cells are subjected to hypoosmotic shock. Sperm diluted in extender containing Me2SO display significantly better results after 30 min equilibration than sperm exposed to the other cryoprotectants that were tested. In addition to direct effects of the CPTs on cells, the extender– cryoprotectant has a higher osmolality than the semen samples, so that subsequent dilution in hypoosmotic solutions will cause greater osmotic stress when the cells are first immersed in a hypertonic and then in a hypoosmotic medium. Curry and Watson (7) demonstrated that ram and human sperm show a high resistance to hyperosmotic stress, but their further dilution to isoosmotic conditions promotes loss of viability. The rapid influx of water into the cells under these conditions was proposed as the mechanism of cell damage. In the present study our results show that even when Me2SO was used as the CPT, the increase in sperm damage when exposed to hypoosmotic solutions was significant. The osmolality of the CPT solutions containing Me2SO used in the two experiments ranged from 1625 mOsm/kg to
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FIG. 3. Time course of permeability to Hoechst dye of frozen and thawed sperm after dilution in hypoosmotic solutions (n 5 3). (A) Samples frozen with Me2SO. (B) Samples frozen with DMA. (C) Samples frozen with methanol. Hypoosmotic solutions: A, Steyn et al. (20); B, Wheeler and Thorgaard (22); C, 50 mOsm/Kg; D, 150 mOsm/Kg; E, 300 mOsm/Kg.
1778 mOsm/kg, but cell viability did not appear to be affected by this exposure; loss of viability was observed only after hypoosmotic shock and could be explained by possible detrimental effects of the CPTs on the membranes. After freezing and thawing about 20% of the cells frozen with Me2SO or DMA were still nonpermeable to the Hoechst dye. A similar percentage of undamaged cells after thawing was reported by Ogier de Baulny et al. (19). In their study, Me2SO was also found to be the most favourable CPT as judged by membrane integrity after thawing. However, in the present study not only was the number of dead cells increased following cryopreservation, but there was also a dramatic increase in the fragility of the live cells. A few seconds after their contact with the hypoosmotic solutions they became permeable to the
probe. Samples frozen with Me2SO showed a higher resistance of the cell membrane than those frozen with DMA or methanol, which explains the superiority of this agent as a cryoprotectant for salmonid sperm. The fertility rates achieved in the present study are consistent with the observed increase in susceptibility to hypoosmotic shock, but not with the proportion of live cells measured before hypotonic exposure, demonstrating that sublethal cryodamage that is not detected by standard procedures had been caused. Donoghue et al. (9), working with turkey sperm, have also reported that there is a population of labile spermatozoa more sensitive to hypoosmotic stress, which is undetected if fluorescent probes are used alone. Membrane destabilization has been pointed out as the main factor causing damage to mammalian sperm during freezing and thawing (8).
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FIG. 4. Time course of permeability to Hoechst dye of frozen and thawed sperm after dilution in hypoosmotic solutions (n 5 5). Samples frozen with (A) No. 6 –Me2SO; (B) No. 6 –Me2SO– egg yolk; (C) Lahnsteiner– Me2SO; (D) Lahnsteiner–Me2SO– egg yolk. Hypoosmotic solutions: A, 10 mOsm/Kg; B, 150 mOsm/Kg; C, 300 mOsm/Kg; D, DIA532.
Alvarez and Storey (1) hypothesized two models to explain sublethal cryodamage in human sperm: peroxidation-related events due to lipid peroxidation of the plasma membrane; mechanical stress in the membrane, involving membrane embrittlement during phase transitions occurring during the freezing–thawing cycle. The use of lipid peroxidation inhibitors and membrane stabilizers, such as egg yolk or polyols, allowed them to confirm the stress-related proposal as the main contributor to sublethal cryodamage. Phase transitions were not detected during freezing and thawing of trout sperm (13) but our results show a significant increase of susceptibility to osmotic stress during the process, as well as a beneficial effect of egg yolk, suggesting that membrane embrittlement during the freezing/thawing process can
be significantly reduced by using membrane stabilizers. The sperm membrane of carp shows a significant lose of amino acids during cryopreservation and this could induce a destabilization of hydrophobic interactions between the proteins and lipids of the membrane during freezing (18). These results suggest that the decrease of sperm membrane stability in trout could also be due to changes in molecular interactions between membrane components. In the present study the use of egg yolk promoted significantly increased membrane resistance and sperm motility, but a similar increase in fertility was not observed. The cryoprotective action offered by the complex mixture that composes egg yolk is well known in mammals (8). The low density lipoprotein fraction is an extracellular cryoprotectant that
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renders the membrane more stable at low temperatures (21). The mode of action still remains unclear but the use of egg yolk in some species decreases the fertility rate, which has been explained by a possible interference between egg yolk lipids and other seminal plasma components during fertilization (6). In the present study, a similar interference by egg yolk could affect the sperm entry through the micropile or other unknown mechanisms involving the interaction of sperm and egg membrane. The low fertility rates achieved with postthawed semen in all trials could be exacerbated by the use of water as the sperm activator. As we have demonstrated, dilution of thawed sperm in such a medium causes cell death within a few seconds. All the tested solutions usually used to activate the cell motility, except DIA 532, were also hazardous for the stability of thawed cells. Our data suggest that new extenders containing different membrane stabilizers and motility activators with osmolalities exceeding 150 mOsm/kg are needed to minimize sublethal cryodamage and to improve fertility rates of trout sperm after freezing and thawing. CONCLUSION
The hypoosmotic solutions usually used to activate motility in artificial fertilization cause cell death within a few minutes in fresh sperm. Susceptibility to these solutions is slightly higher after dilution with cryoprotectants, but dramatically increases after freezing and thawing, showing sublethal cryodamage that is not demonstrated by ultrastructural or biochemical studies or by counting the live cells immediately after thawing. The results suggest that the addition of components that are able to increase the membrane stability and the use of higher osmolalities to activate motility should be analyzed in order to improve fertilizing ability of cryopreserved sperm. REFERENCES 1. Alvarez, J. G., and Storey, B. T. Evidence that membrane stress contributes more than lipid peroxidation to sublethal cryodamage in cryopreserved human
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SUBLETHAL DAMAGE IN CRYOPRESERVED FISH SPERM 15. Lahnsteiner, F., Weismann, T., and Patzner, R. A. A uniform method for cryopreservation of semen of the salmonid fishes O. mykiss, S. trutta f. fario, S. trutta f. lacustris, Coregonus sp. Aquacult. Res. 26, 801– 807 (1995). 16. Malejac, M., Loir, M., and Maisse, G. Qualite´ de la membrane des spermatozoides de truite arc-en-ciel; relation avec l’aptitude du sperme a la congelation. Aquat. Living Resour. 3, 43–54 (1990). 17. Ma´ria´n, T., Krazsnai, Z., Balkay, L., Bala´zs, M., Emry, M., Bene, L., and Tro´n, L. Hypo-osmotic shock induces an osmolality-dependent permeabilization and structural changes in the membrane of carp sperm. J. Histochem. Cytochem. 41(2), 291–297 (1993). 18. Nauc, V., and Boronciuc, Gh. Cryogenic changes in carp spermatozoa content. Proc. Cryo ’97, 34th
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