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Step-wise dilution for removal of glycerol from fresh and cryopreserved equine spermatozoa
Animal Reproduction Science 84 (2004) 147–156
Step-wise dilution for removal of glycerol from fresh and cryopreserved equine spermatozoa Myrthe T. Wessel1 , Barry A. Ball∗ Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, 1114 Tupper Hall, Davis, CA 95616, USA Received 5 September 2003; received in revised form 8 December 2003; accepted 10 December 2003
Abstract Osmotic stress is an important component of the damage to spermatozoa during cryopreservation. Osmotic injury, due to hyperosmolar freezing extenders, changes in relative solute concentration in the extra cellular medium during freezing and differences in the relative permeabilities of penetrating cryoprotectants, such as glycerol, and water occur when cryopreserved spermatozoa are diluted into isosmotic media or when spermatozoa are placed in the female reproductive tract. The purpose of the study reported here was to evaluate the effect of step-wise dilution for the removal of the permeating cryoprotectant, glycerol, from both fresh and cryopreserved equine spermatozoa on their motility and viability. There were significant (P < 0.05) effects of osmolality and dilution method on both total and progressive motility as well as viability in fresh spermatozoa. With the rapid, one-step dilution being significantly more detrimental to viability and motility, compared to the fixed molarity and fixed volume-dilutions. With frozen–thawed spermatozoa there were significant effects of stallion on total and progressive motility of spermatozoa after cryopreservation and thawing; however, treatment (type of dilution after thawing) did not affect post-thaw motility. These data indicate that rapid, single-step removal of glycerol from fresh equine spermatozoa results in a post-hyperosmotic stress that is characterized by a reduction in both motility and membrane integrity. This post-hyperosmotic stress can be reduced by a step-wise dilution for removal of glycerol, which improved the maintenance of both motility and membrane integrity. However a similar benefit for step-wise dilution for removal of glycerol was not observed in cryopreserved equine spermatozoa. © 2004 Published by Elsevier B.V. Keywords: Osmotic stress; Gradient dilution; Cryopreservation; Spermatozoa; Equine
∗ Corresponding author. Tel.: +1-530-752-1358; fax: +1-530-752-4278. E-mail addresses: [email protected] (M.T. Wessel), [email protected] (B.A. Ball). 1 Equatorplein 12, 3582 PJ, Utrecht, The Netherlands. Tel.: +31-36-5451511.
0378-4320/$ – see front matter © 2004 Published by Elsevier B.V. doi:10.1016/j.anireprosci.2003.12.004
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1. Introduction Osmotic stress is an important component of the damage to spermatozoa during cryopreservation (Gao et al., 1997; Watson, 2000). Osmotic stress results because of ice nucleation and the resulting concentration of solutes within the medium and because of the addition and removal of near molar concentrations of cryoprotectants (CPAs) (Gao et al., 1997). Osmotic injury, due to hyperosmolar freezing extenders, changes in relative solute concentration in the extra cellular medium during freezing and differences in the relative permeabilities of penetrating cryoprotectants, such as glycerol, and water occur when cryopreserved spermatozoa are diluted into isosmotic media or when spermatozoa are placed in the female reproductive tract. Osmotic tolerance of spermatozoa appears to differ between species, and the relative ability of spermatozoa to survive osmotic stress appears related, in part, to the ability of spermatozoa to survive cryopreservation. Until recently, there was limited information regarding the osmotic tolerance of equine spermatozoa. Based upon recent reports, it appears that equine spermatozoa have a relatively limited osmotic tolerance and appear similar to boar spermatozoa in their response to osmotic change (Ball and Vo, 2001; Gilmore et al., 1996; Pommer et al., 2002). The limited osmotic tolerance of equine spermatozoa may therefore be an important component of damage to these cells during cryopreservation. Although the rapid addition of molar amounts of glycerol to equine spermatozoa results in a decreased motility, there was no effect on the percentage of live, acrosome-intact spermatozoa or on their mitochondrial membrane potential (Ball and Vo, 2001; Pommer et al., 2002). In contrast, the rapid removal of glycerol by dilution in isotonic media resulted in a marked decline in motility, viability, and mitochondrial membrane potential of equine spermatozoa (Ball and Vo, 2001). Similar effects related to the rapid removal of permeating cryoprotectants have been characterized for human (Gao et al., 1995; Gilmore et al., 1997) and mouse (Phelps et al., 1999) spermatozoa. Equine spermatozoa appear to behave as linear osmometers within the range of 150–900 mOsm and if cell volume excursions exceed the lytic volume of the cell, then disruption of the plasma membrane results in a loss of cell viability (Pommer et al., 2002). Swelling of equine spermatozoa is more detrimental than shrinking which likely explains the more severe changes noted during the removal compared to addition of soluble cryoprotectants such as glycerol (Pommer et al., 2002).Step-wise dilution of spermatozoa to remove cryoprotectants has been used in attempts to limit changes in cell volume during the removal of permeating cryoprotectants and therefore limit osmotic damage (Rasch et al., 1996; Gilmore et al., 1997, 1998). The purpose of the study reported here was to evaluate the effect of step-wise dilution for the removal of the permeating cryoprotectant, glycerol, from both fresh and cryopreserved equine spermatozoa on their motility and viability. 2. Materials and methods 2.1. Semen collection and processing Semen was collected from five stallions with an artificial vagina and filtered to remove gel. Depending upon the experiment, raw semen samples were maintained at 37 ◦ C or semen was
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extended with a non-fat dried skim milk extender (NFDSM; EZ-Mixin, Animal Reproduction Systems, Chino, CA) and allowed to cool rapidly to room temperature. For experiment 1, spermatozoa (n = 2 ejaculates from five stallions) were layered onto a discontinuous (40%/80%) Percoll (Sigma Chemical Company, St. Louis, MO) gradient (Drobnis et al., 1991) and centrifuged (300 × g; 20 min) to isolate motile spermatozoa. The resultant motile population of spermatozoa was then resuspended in a modified Tyrode’s albumin, lactate, pyruvate medium (TALP) (Bavister, 1989), washed by centrifugation (300 × g; 10 min) and resuspended in TALP to a final concentration of 600 × 106 sperm/ml. For cryopreservation (experiment 2), spermatozoa (50 × 106 cells/ml) in non-fat dried skim milk extender were centrifuged (300 × g; 15 min), the supernatant was removed, and spermatozoa were resuspended in freezing medium. Freezing medium was INRA82 (4% glycerol) (Palmer, 1984). Extended spermatozoa (400 × 106 cells/ml) were packaged in 0.5 ml polyvinyl chloride straws and frozen in a controlled rate freezer (Kryo10/16; TS Scientific, Perkasie, PA) (Cochran et al., 1984). In all experiments, osmolality of media was confirmed by measurement with a freezing point depression osmometer (Model 5004: Precision System Inc., Natick, MA). 2.2. Evaluation of sperm motility and membrane integrity Sperm motility parameters were assessed by Computer-Assisted Sperm Analysis (CEROS System, Hamilton-Thorn, So. Hamilton, MA). A 7 l drop of sperm suspension was placed on a prewarmed slide (Cell-Vu counting chamber, Fertility Technology Resources, Marietta, GA) and cover-slipped. The slide was maintained at 37 ◦ C, and a minimum of 200 spermatozoa per sample was examined. Membrane integrity was assessed by exclusion of propidium iodide (Sigma Chemical Co., St. Louis, MO, USA), as previously described (Ball and Vo, 2001). 2.3. Experimental design In experiment 1, the effect of multistep dilution for removal of glycerol on the motility and viability of fresh equine spermatozoa was determined. Percoll-separated spermatozoa were diluted (1:5.3) in TALP with glycerol (1000 and 1500 mOsm final osmolality) or without glycerol (325 mOsm; isotonic) and incubated (10 min; 37 ◦ C). After incubation, spermatozoa in each group were diluted (1:5) in isotonic TALP in either a single-step or a four-step fixed volume (FV) or fixed molality (FM) dilution method (Gao et al., 1995). Spermatozoa from each group were also diluted (1:5) in the corresponding anisosmotic TALP with glycerol as a control. Step-wise dilutions were performed at 30 s intervals. After 15 min of incubation at 37 ◦ C, sperm motility and viability were determined. In experiment 2, the effect of multistep dilution for removal of glycerol on the motility and viability of cryopreserved equine spermatozoa was determined. Cryopreserved spermatozoa were thawed in a water bath (35 ◦ C; 30 s), and the contents of the straws were transferred to prewarmed 1.5 ml microcentrifuge tubes. Subsequently glycerol was removed by dilution of thawed semen with prewarmed non-fat dried skim milk extender for motility assessment or TALP for determination of viability. Dilution methods again consisted of a single-step or a four-step fixed volume or fixed molality dilution method along with dilution in a hypertonic
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osmotic buffer (sucrose) in NFDSM (four steps at 30 s intervals: 600, 400, 325, 325 mOsm). The samples were incubated for 15 min at 37 ◦ C prior to determination of motility by CASA. For viability assessment, TALP was used instead of NFDSM extender in the dilution steps for the single-step or a four-step fixed volume or fixed molality dilutions. In addition, an anisosmotic TALP with the corresponding concentration of glycerol was used for dilution. After dilution, propidium iodide was added to the samples and after 5 min of incubation at 37 ◦ C viability was determined. In experiment 3, a single-step dilution as well as eight-step fixed volume and fixed molarity dilutions were evaluated. After thawing, as described above, spermatozoa were
Fig. 1. Changes in progressive and total sperm motility after removal of glycerol from equine spermatozoa at 325 (control), 1000 and 1500 mOsm. After glycerol loading, equine spermatozoa were diluted in the corresponding anisosmotic TALP or glycerol was removed by a four-step fixed molarity, four-step fixed volume or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of motility via computer-assisted semen analysis. Data are normalized to control value (325 mOsm; which is assigned a value of 1.0) for analysis and are presented as least-squares mean ± SEM. Values with different letters (a, b) differ (P < 0.05).
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diluted in NFDSM extender, and the samples were incubated for 15 min at 37 ◦ C prior to determination of motility by CASA.
3. Statistical analysis Data were analyzed by analysis of variance (ANOVA), and differences between individual means were compared with the Fisher protected least significant difference (Statview, SAS Institute, Cary, NC). In some experiments data were normalized to control values prior to analysis. A value of P < 0.05 was taken as statistically significant.
4. Results In experiment 1, there were significant (P < 0.05) effects of osmolality and dilution method on both total and progressive motility as well as sperm viability (Figs. 1 and 2). For spermatozoa at 1000 mOsm, the removal of glycerol via a one-step dilution resulted in a greater decline in total and progressive motility than did removal of glycerol via a four-step fixed molarity dilution or maintenance of spermatozoa in the corresponding anisosmotic media (Fig. 1). For spermatozoa at 1000 mOsm, removal of glycerol via a one-step dilution resulted in a greater decline in viability than did multistep removal of glycerol or
Fig. 2. Changes in sperm viability assessed by exclusion of propidium iodide after removal of glycerol from equine spermatozoa at 325 (control), 1000 and 1500 mOsm. Data presented are normalized to control values (single dilution at 325 mOsm is assigned a value of 1.0) for each replicate. After glycerol loading, equine spermatozoa were diluted in the corresponding anisosmotic TALP or glycerol was removed by a four-step fixed molarity, four-step fixed volume or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of viability via exclusion of propidium iodide. Data are presented as least-squares mean ±SEM. Values with different letters (a, b, c, d) differ (P < 0.05).
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maintenance of spermatozoa in the corresponding anisosmotic media (Fig. 2). For spermatozoa at 1500 mOsm, one-step dilution for removal of glycerol resulted in a greater decline in viability than did maintenance of spermatozoa in anisosmotic media or multistep removal of glycerol (Fig. 2). In experiment 2, there was a significant effect of stallion on total and progressive motility of spermatozoa after cryopreservation and thawing; however, treatment (type of dilution after thawing) did not affect post-thaw motility (Fig. 3). Based upon exclusion of propidium iodide, removal of glycerol by either one-step or multistep dilution resulted in a
Fig. 3. Progressive and total sperm motility after removal of glycerol from frozen–thawed equine spermatozoa. After thawing, equine spermatozoa were diluted by a four-step fixed molarity, four-step fixed volume, four-step hypertonic or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of motility via computer-assisted semen analysis. Values for total and progressive motility are presented for semen from five stallions. Data are presented as least-squares mean ± SEM.
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Fig. 4. Changes in sperm viability assessed by exclusion of propidium iodide after removal of glycerol from frozen–thawed equine spermatozoa. After thawing, equine spermatozoa were diluted by a four-step fixed molarity, four-step fixed volume, four-step hypertonic or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of viability via exclusion of propidium iodide. Within stallions, values with different letters (a, b) differ (P < 0.01). Values for viability are presented for semen from four stallions. Data are presented as least-squares mean ± SEM.
greater reduction in sperm viability than did dilution of spermatozoa in media with glycerol (anisosmotic) (Fig. 4). In experiment 3, there were significant effects of stallion, but not of treatment, on both total and progressive motility after thawing (Fig. 5).
5. Discussion During cryopreservation, equine spermatozoa are exposed to changes in osmotic pressure associated with several factors. With the addition of permeable cryoprotectants, spermatozoa undergo a reduction in cell volume followed by an increase in cell volume as concentrations of CPA equilibrate. Conversely, when spermatozoa loaded with cryoprotectants are placed into isosmotic media or female genital tract secretions, there is an increase in cell volume due to the influx of water into the cell (Gao et al., 1997). When changes in cell volume exceed species-dependent tolerances, cell function and integrity are disrupted (Ball and Vo, 2001; Pommer et al., 2002). For equine spermatozoa, osmotic tolerance appears limited and post-hyperosmotic stress is characterized by a loss of motility, viability and mitochondrial membrane potential that fails to recover after spermatozoa are returned to isosmotic condition (Ball and Vo, 2001; Pommer et al., 2002). Equine spermatozoa behave as linear osmometers, decreasing in cell volume when exposed to hyperosmotic conditions and swelling after exposure to hyposmotic conditions (Pommer et al., 2002). Although motility of equine spermatozoa decreases rapidly under anisosmotic conditions, exposure to hyposmotic stress results in a more rapid alteration
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Fig. 5. Progressive and total sperm motility after removal of glycerol from frozen–thawed equine spermatozoa. After thawing, equine spermatozoa were diluted by an eight-step fixed molarity, eight-step fixed volume or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of motility via computer-assisted semen analysis. Values for total and progressive motility are presented for semen from four stallions. Data are presented as least-squares mean ± SEM.
in both membrane integrity and mitochondrial membrane potential (Ball and Vo, 2001; Pommer et al., 2002). These adverse affects appear to persist after spermatozoa are returned to isosmotic conditions. When equine spermatozoa are loaded with permeating cryoprotectants, such as glycerol, there is a decline in motility, with little alteration in membrane integrity or mitochondrial membrane potential (Ball and Vo, 2001). The rapid removal of permeating CPAs results in a relative hyposmotic shock upon dilution into isosmotic extenders or reproductive tract fluids. This is reflected in a rapid loss of sperm motility, membrane integrity and mitochondrial membrane potential (Ball and Vo, 2001; Pommer et al., 2002).
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In the present study, removal of glycerol from fresh equine spermatozoa suspended in a modified TALP via either a four-step FV or FM dilution, improved the maintenance of sperm motility compared to a rapid, single-step dilution. This observation is similar to that reported for spermatozoa from a number of other species including human (Gao et al., 1995; Gilmore et al., 1997), mouse (Phelps et al., 1999) and boar spermatozoa (Gilmore et al., 1998). Likewise, multistep removal of glycerol from equine spermatozoa improved viability as determined by exclusion of propidium iodide compared to rapid dilution. A fixed molarity dilution appeared superior for maintenance of motility and viability compared to the fixed volume-dilution for removal of glycerol; however, these differences were not statistically significant. Interestingly, neither the use of a multistep dilution nor the use of an osmotic buffer (sucrose) for removal of glycerol from frozen–thawed equine spermatozoa improved maintenance of motility or viability compared to a rapid, one-step dilution. Although a number of studies have detailed the theoretical benefits of a multistep dilution to remove permeating CPAs from cryopreserved spermatozoa, relatively few studies have examined this effect experimentally (Gao et al., 1995, 1997; Gilmore et al., 1997). Several factors may explain the differences observed in multistep removal of glycerol from fresh equine spermatozoa (experiment 1) or from cryopreserved spermatozoa (experiment 2). In experiment 1, spermatozoa were separated on a Percoll-density gradient and resuspended in a modified TALP with BSA whereas in experiment 2, spermatozoa were processed for cryopreservation without the use of a Percoll-density gradient and were maintained in a cryopreservation extender with skim milk and egg yolk. Percoll-density gradient centrifugation is known to alter the phospholipid composition of sperm membranes and has been shown to alter the hydraulic conductivity of mouse spermatozoa (Phelps et al., 1999). Likewise, exposure of spermatozoa to milk proteins and egg yolk increases the osmotic tolerance of murine (Agca et al., 2002) and canine (Songsasen et al., 2002) spermatozoa. Other studies have suggested that alterations in the lipid bilayer, caused by phase separation events during cryopreservation (Holt and North, 1984; De Leeuw et al., 1990) could well influence osmotic properties. This observation is supported for equine spermatozoa where cold shock induced by rapid cooling at suprazero temperatures affects membrane permeability (Devireddy et al., 2002). In summary, the rapid, single-step removal of glycerol from fresh equine spermatozoa results in a post-hyperosmotic stress that is characterized by a reduction in both motility and membrane integrity. This post-hyperosmotic stress can be reduced by a step-wise dilution for removal of glycerol, which improved the maintenance of both motility and membrane integrity. A similar benefit for step-wise dilution for removal of glycerol was not observed in cryopreserved equine spermatozoa.
Acknowledgements This research was supported by the John P. Hughes Endowment, by the Center for Equine Health with funds provided by the Oak Tree Racing Association, the State of California pari-mutuel fund, and contributions by private donors. The authors thank Anthony Vo for technical assistance.
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References Agca, Y., Gilmore, J., Byers, M., Woods, E.J., Liu, J., Critser, J.K., 2002. Osmotic characteristics of mouse spermatozoa in the presence of extenders and sugars. Biol. Reprod. 67, 1493–1501. Ball, B.A., Vo, A., 2001. Osmotic tolerance of equine spermatozoa and the effects of soluble cryoprotectants on equine sperm motility, viability, and mitochondrial membrane potential. J. Androl. 22, 1061–1069. Bavister, B.D., 1989. A consistently successful procedure for in vitro fertilization of golden hamster eggs. Gamete Res. 23, 139–158. Cochran, J.D., Amann, R.P., Froman, D.P., Pickett, B.W., 1984. Effects of centrifugation, glycerol level, cooling to 5 ◦ C, freezing rate and thawing rate on the post-thaw motility of equine sperm. Theriogenology 22, 25–37. De Leeuw, F.E., Chen, H.C., Colenbrander, B., Verkleij, A.J., 1990. Cold-induced ultrastructural changes in bull and boar sperm plasma membranes. Cryobiology 27, 171–183. Devireddy, R.V., Swanlund, D.J., Olin, T., Vincente, W., Troedsson, M.H.T., Bischof, J.C., Roberts, K.P., 2002. Cryopreservation of equine sperm: optimal cooling rates in the presence and absence of cryoprotective agents determined using differential scanning calorimetry. Biol. Reprod. 66, 222–231. Drobnis, E.Z., Zhong, C.Q., Overstreet, J.W., 1991. Separation of cryopreserved human semen using Sephadex columns, washing, or Percoll gradients. J. Androl. 12, 201–208. Gao, D.Y., Liu, J., Liu, C., McGann, L.E., Watson, P.F., Kleinhans, F.W., Mazur, P., Critser, E.S., Critser, J.K., 1995. Prevention of osmotic injury to human spermatozoa during addition and removal of glycerol. Hum. Reprod. 10, 1109–1122. Gao, D.Y., Mazur, P., Critser, J.K., 1997. Fundamental cryobiology of mammalian spermatozoa. In: Karow, A.M., Critser, J.K. (Eds.), Reproductive Tissue Banding: Scientific Principles. Academic Press, San Diego, pp. 263–328. Gilmore, J.A., Junying, D., Tao, J., Peter, A.T., Critser, J.K., 1996. Osmotic properties of boar spermatozoa and their relevance to cryopreservation. J. Reprod. Fertil. 107, 87–95. Gilmore, J.A., Liu, J., Gao, D.Y., Critser, J.K., 1997. Determination of optimal cryoprotectants and procedures for their addition and removal from human spermatozoa. Hum. Reprod. 12, 112–118. Gilmore, J.A., Liu, J., Peter, A.T., Critser, J.K., 1998. Determination of plasma membrane characteristics of boar spermatozoa and their relevance to cryopreservation. Biol. Reprod. 58, 28–36. Holt, W.V., North, R.D., 1984. Partially irreversible cold-induced lipid phase transitions in mammalian sperm plasma membrane domains: freeze-fracture study. J. Exp. Zool. 230, 473–483. Palmer, E., 1984. Factors affecting stallion semen survival and fertility. In: Proceedings of the 10th International Congress on Anim. Reprod. Art. Insem., vol. II, pp. 377–379. Phelps, M.J., Liu, J., Benson, J.D., Willoughby, C.E., Gilmore, J.A., Critser, J.K., 1999. Effects of Percoll separation, cryoprotective agents, and temperature on plasma membrane permeability characteristics of murine spermatozoa and their relevance to cryopreservation. Biol. Reprod. 61, 1031–1041. Pommer, A.C., Rutllant, J., Meyers, S.A., 2002. The role of osmotic resistance on equine spermatozoal function. Theriogenology 58, 1373–1384. Rasch, K., Schoon, H.A., Sieme, H., Klug, E., 1996. Histomorphological endometrial status and influence of oxytocin on the uterine drainage and pregnancy rate in mares. Equine. Vet. J. 28, 455–460. Songsasen, N., Yu, I., Murton, S., Paccamonti, D.L., Eilts, B.E., Godke, R.A., Leibo, S.P., 2002. Osmotic sensitivity of canine spermatozoa. Cryobiology 44, 79–90. Watson, P.F., 2000. The causes of reduced fertility with cryopreserved semen. Anim. Reprod. Sci. 60–61, 481–492.
Step-wise dilution for removal of glycerol from fresh and cryopreserved equine spermatozoa Myrthe T. Wessel1 , Barry A. Ball∗ Department of Population Health and Reproduction, School of Veterinary Medicine, University of California, 1114 Tupper Hall, Davis, CA 95616, USA Received 5 September 2003; received in revised form 8 December 2003; accepted 10 December 2003
Abstract Osmotic stress is an important component of the damage to spermatozoa during cryopreservation. Osmotic injury, due to hyperosmolar freezing extenders, changes in relative solute concentration in the extra cellular medium during freezing and differences in the relative permeabilities of penetrating cryoprotectants, such as glycerol, and water occur when cryopreserved spermatozoa are diluted into isosmotic media or when spermatozoa are placed in the female reproductive tract. The purpose of the study reported here was to evaluate the effect of step-wise dilution for the removal of the permeating cryoprotectant, glycerol, from both fresh and cryopreserved equine spermatozoa on their motility and viability. There were significant (P < 0.05) effects of osmolality and dilution method on both total and progressive motility as well as viability in fresh spermatozoa. With the rapid, one-step dilution being significantly more detrimental to viability and motility, compared to the fixed molarity and fixed volume-dilutions. With frozen–thawed spermatozoa there were significant effects of stallion on total and progressive motility of spermatozoa after cryopreservation and thawing; however, treatment (type of dilution after thawing) did not affect post-thaw motility. These data indicate that rapid, single-step removal of glycerol from fresh equine spermatozoa results in a post-hyperosmotic stress that is characterized by a reduction in both motility and membrane integrity. This post-hyperosmotic stress can be reduced by a step-wise dilution for removal of glycerol, which improved the maintenance of both motility and membrane integrity. However a similar benefit for step-wise dilution for removal of glycerol was not observed in cryopreserved equine spermatozoa. © 2004 Published by Elsevier B.V. Keywords: Osmotic stress; Gradient dilution; Cryopreservation; Spermatozoa; Equine
∗ Corresponding author. Tel.: +1-530-752-1358; fax: +1-530-752-4278. E-mail addresses: [email protected] (M.T. Wessel), [email protected] (B.A. Ball). 1 Equatorplein 12, 3582 PJ, Utrecht, The Netherlands. Tel.: +31-36-5451511.
0378-4320/$ – see front matter © 2004 Published by Elsevier B.V. doi:10.1016/j.anireprosci.2003.12.004
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1. Introduction Osmotic stress is an important component of the damage to spermatozoa during cryopreservation (Gao et al., 1997; Watson, 2000). Osmotic stress results because of ice nucleation and the resulting concentration of solutes within the medium and because of the addition and removal of near molar concentrations of cryoprotectants (CPAs) (Gao et al., 1997). Osmotic injury, due to hyperosmolar freezing extenders, changes in relative solute concentration in the extra cellular medium during freezing and differences in the relative permeabilities of penetrating cryoprotectants, such as glycerol, and water occur when cryopreserved spermatozoa are diluted into isosmotic media or when spermatozoa are placed in the female reproductive tract. Osmotic tolerance of spermatozoa appears to differ between species, and the relative ability of spermatozoa to survive osmotic stress appears related, in part, to the ability of spermatozoa to survive cryopreservation. Until recently, there was limited information regarding the osmotic tolerance of equine spermatozoa. Based upon recent reports, it appears that equine spermatozoa have a relatively limited osmotic tolerance and appear similar to boar spermatozoa in their response to osmotic change (Ball and Vo, 2001; Gilmore et al., 1996; Pommer et al., 2002). The limited osmotic tolerance of equine spermatozoa may therefore be an important component of damage to these cells during cryopreservation. Although the rapid addition of molar amounts of glycerol to equine spermatozoa results in a decreased motility, there was no effect on the percentage of live, acrosome-intact spermatozoa or on their mitochondrial membrane potential (Ball and Vo, 2001; Pommer et al., 2002). In contrast, the rapid removal of glycerol by dilution in isotonic media resulted in a marked decline in motility, viability, and mitochondrial membrane potential of equine spermatozoa (Ball and Vo, 2001). Similar effects related to the rapid removal of permeating cryoprotectants have been characterized for human (Gao et al., 1995; Gilmore et al., 1997) and mouse (Phelps et al., 1999) spermatozoa. Equine spermatozoa appear to behave as linear osmometers within the range of 150–900 mOsm and if cell volume excursions exceed the lytic volume of the cell, then disruption of the plasma membrane results in a loss of cell viability (Pommer et al., 2002). Swelling of equine spermatozoa is more detrimental than shrinking which likely explains the more severe changes noted during the removal compared to addition of soluble cryoprotectants such as glycerol (Pommer et al., 2002).Step-wise dilution of spermatozoa to remove cryoprotectants has been used in attempts to limit changes in cell volume during the removal of permeating cryoprotectants and therefore limit osmotic damage (Rasch et al., 1996; Gilmore et al., 1997, 1998). The purpose of the study reported here was to evaluate the effect of step-wise dilution for the removal of the permeating cryoprotectant, glycerol, from both fresh and cryopreserved equine spermatozoa on their motility and viability. 2. Materials and methods 2.1. Semen collection and processing Semen was collected from five stallions with an artificial vagina and filtered to remove gel. Depending upon the experiment, raw semen samples were maintained at 37 ◦ C or semen was
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extended with a non-fat dried skim milk extender (NFDSM; EZ-Mixin, Animal Reproduction Systems, Chino, CA) and allowed to cool rapidly to room temperature. For experiment 1, spermatozoa (n = 2 ejaculates from five stallions) were layered onto a discontinuous (40%/80%) Percoll (Sigma Chemical Company, St. Louis, MO) gradient (Drobnis et al., 1991) and centrifuged (300 × g; 20 min) to isolate motile spermatozoa. The resultant motile population of spermatozoa was then resuspended in a modified Tyrode’s albumin, lactate, pyruvate medium (TALP) (Bavister, 1989), washed by centrifugation (300 × g; 10 min) and resuspended in TALP to a final concentration of 600 × 106 sperm/ml. For cryopreservation (experiment 2), spermatozoa (50 × 106 cells/ml) in non-fat dried skim milk extender were centrifuged (300 × g; 15 min), the supernatant was removed, and spermatozoa were resuspended in freezing medium. Freezing medium was INRA82 (4% glycerol) (Palmer, 1984). Extended spermatozoa (400 × 106 cells/ml) were packaged in 0.5 ml polyvinyl chloride straws and frozen in a controlled rate freezer (Kryo10/16; TS Scientific, Perkasie, PA) (Cochran et al., 1984). In all experiments, osmolality of media was confirmed by measurement with a freezing point depression osmometer (Model 5004: Precision System Inc., Natick, MA). 2.2. Evaluation of sperm motility and membrane integrity Sperm motility parameters were assessed by Computer-Assisted Sperm Analysis (CEROS System, Hamilton-Thorn, So. Hamilton, MA). A 7 l drop of sperm suspension was placed on a prewarmed slide (Cell-Vu counting chamber, Fertility Technology Resources, Marietta, GA) and cover-slipped. The slide was maintained at 37 ◦ C, and a minimum of 200 spermatozoa per sample was examined. Membrane integrity was assessed by exclusion of propidium iodide (Sigma Chemical Co., St. Louis, MO, USA), as previously described (Ball and Vo, 2001). 2.3. Experimental design In experiment 1, the effect of multistep dilution for removal of glycerol on the motility and viability of fresh equine spermatozoa was determined. Percoll-separated spermatozoa were diluted (1:5.3) in TALP with glycerol (1000 and 1500 mOsm final osmolality) or without glycerol (325 mOsm; isotonic) and incubated (10 min; 37 ◦ C). After incubation, spermatozoa in each group were diluted (1:5) in isotonic TALP in either a single-step or a four-step fixed volume (FV) or fixed molality (FM) dilution method (Gao et al., 1995). Spermatozoa from each group were also diluted (1:5) in the corresponding anisosmotic TALP with glycerol as a control. Step-wise dilutions were performed at 30 s intervals. After 15 min of incubation at 37 ◦ C, sperm motility and viability were determined. In experiment 2, the effect of multistep dilution for removal of glycerol on the motility and viability of cryopreserved equine spermatozoa was determined. Cryopreserved spermatozoa were thawed in a water bath (35 ◦ C; 30 s), and the contents of the straws were transferred to prewarmed 1.5 ml microcentrifuge tubes. Subsequently glycerol was removed by dilution of thawed semen with prewarmed non-fat dried skim milk extender for motility assessment or TALP for determination of viability. Dilution methods again consisted of a single-step or a four-step fixed volume or fixed molality dilution method along with dilution in a hypertonic
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osmotic buffer (sucrose) in NFDSM (four steps at 30 s intervals: 600, 400, 325, 325 mOsm). The samples were incubated for 15 min at 37 ◦ C prior to determination of motility by CASA. For viability assessment, TALP was used instead of NFDSM extender in the dilution steps for the single-step or a four-step fixed volume or fixed molality dilutions. In addition, an anisosmotic TALP with the corresponding concentration of glycerol was used for dilution. After dilution, propidium iodide was added to the samples and after 5 min of incubation at 37 ◦ C viability was determined. In experiment 3, a single-step dilution as well as eight-step fixed volume and fixed molarity dilutions were evaluated. After thawing, as described above, spermatozoa were
Fig. 1. Changes in progressive and total sperm motility after removal of glycerol from equine spermatozoa at 325 (control), 1000 and 1500 mOsm. After glycerol loading, equine spermatozoa were diluted in the corresponding anisosmotic TALP or glycerol was removed by a four-step fixed molarity, four-step fixed volume or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of motility via computer-assisted semen analysis. Data are normalized to control value (325 mOsm; which is assigned a value of 1.0) for analysis and are presented as least-squares mean ± SEM. Values with different letters (a, b) differ (P < 0.05).
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diluted in NFDSM extender, and the samples were incubated for 15 min at 37 ◦ C prior to determination of motility by CASA.
3. Statistical analysis Data were analyzed by analysis of variance (ANOVA), and differences between individual means were compared with the Fisher protected least significant difference (Statview, SAS Institute, Cary, NC). In some experiments data were normalized to control values prior to analysis. A value of P < 0.05 was taken as statistically significant.
4. Results In experiment 1, there were significant (P < 0.05) effects of osmolality and dilution method on both total and progressive motility as well as sperm viability (Figs. 1 and 2). For spermatozoa at 1000 mOsm, the removal of glycerol via a one-step dilution resulted in a greater decline in total and progressive motility than did removal of glycerol via a four-step fixed molarity dilution or maintenance of spermatozoa in the corresponding anisosmotic media (Fig. 1). For spermatozoa at 1000 mOsm, removal of glycerol via a one-step dilution resulted in a greater decline in viability than did multistep removal of glycerol or
Fig. 2. Changes in sperm viability assessed by exclusion of propidium iodide after removal of glycerol from equine spermatozoa at 325 (control), 1000 and 1500 mOsm. Data presented are normalized to control values (single dilution at 325 mOsm is assigned a value of 1.0) for each replicate. After glycerol loading, equine spermatozoa were diluted in the corresponding anisosmotic TALP or glycerol was removed by a four-step fixed molarity, four-step fixed volume or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of viability via exclusion of propidium iodide. Data are presented as least-squares mean ±SEM. Values with different letters (a, b, c, d) differ (P < 0.05).
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maintenance of spermatozoa in the corresponding anisosmotic media (Fig. 2). For spermatozoa at 1500 mOsm, one-step dilution for removal of glycerol resulted in a greater decline in viability than did maintenance of spermatozoa in anisosmotic media or multistep removal of glycerol (Fig. 2). In experiment 2, there was a significant effect of stallion on total and progressive motility of spermatozoa after cryopreservation and thawing; however, treatment (type of dilution after thawing) did not affect post-thaw motility (Fig. 3). Based upon exclusion of propidium iodide, removal of glycerol by either one-step or multistep dilution resulted in a
Fig. 3. Progressive and total sperm motility after removal of glycerol from frozen–thawed equine spermatozoa. After thawing, equine spermatozoa were diluted by a four-step fixed molarity, four-step fixed volume, four-step hypertonic or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of motility via computer-assisted semen analysis. Values for total and progressive motility are presented for semen from five stallions. Data are presented as least-squares mean ± SEM.
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Fig. 4. Changes in sperm viability assessed by exclusion of propidium iodide after removal of glycerol from frozen–thawed equine spermatozoa. After thawing, equine spermatozoa were diluted by a four-step fixed molarity, four-step fixed volume, four-step hypertonic or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of viability via exclusion of propidium iodide. Within stallions, values with different letters (a, b) differ (P < 0.01). Values for viability are presented for semen from four stallions. Data are presented as least-squares mean ± SEM.
greater reduction in sperm viability than did dilution of spermatozoa in media with glycerol (anisosmotic) (Fig. 4). In experiment 3, there were significant effects of stallion, but not of treatment, on both total and progressive motility after thawing (Fig. 5).
5. Discussion During cryopreservation, equine spermatozoa are exposed to changes in osmotic pressure associated with several factors. With the addition of permeable cryoprotectants, spermatozoa undergo a reduction in cell volume followed by an increase in cell volume as concentrations of CPA equilibrate. Conversely, when spermatozoa loaded with cryoprotectants are placed into isosmotic media or female genital tract secretions, there is an increase in cell volume due to the influx of water into the cell (Gao et al., 1997). When changes in cell volume exceed species-dependent tolerances, cell function and integrity are disrupted (Ball and Vo, 2001; Pommer et al., 2002). For equine spermatozoa, osmotic tolerance appears limited and post-hyperosmotic stress is characterized by a loss of motility, viability and mitochondrial membrane potential that fails to recover after spermatozoa are returned to isosmotic condition (Ball and Vo, 2001; Pommer et al., 2002). Equine spermatozoa behave as linear osmometers, decreasing in cell volume when exposed to hyperosmotic conditions and swelling after exposure to hyposmotic conditions (Pommer et al., 2002). Although motility of equine spermatozoa decreases rapidly under anisosmotic conditions, exposure to hyposmotic stress results in a more rapid alteration
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Fig. 5. Progressive and total sperm motility after removal of glycerol from frozen–thawed equine spermatozoa. After thawing, equine spermatozoa were diluted by an eight-step fixed molarity, eight-step fixed volume or single-step dilution. Sperm samples were incubated for 15 min at 37 ◦ C prior to determination of motility via computer-assisted semen analysis. Values for total and progressive motility are presented for semen from four stallions. Data are presented as least-squares mean ± SEM.
in both membrane integrity and mitochondrial membrane potential (Ball and Vo, 2001; Pommer et al., 2002). These adverse affects appear to persist after spermatozoa are returned to isosmotic conditions. When equine spermatozoa are loaded with permeating cryoprotectants, such as glycerol, there is a decline in motility, with little alteration in membrane integrity or mitochondrial membrane potential (Ball and Vo, 2001). The rapid removal of permeating CPAs results in a relative hyposmotic shock upon dilution into isosmotic extenders or reproductive tract fluids. This is reflected in a rapid loss of sperm motility, membrane integrity and mitochondrial membrane potential (Ball and Vo, 2001; Pommer et al., 2002).
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In the present study, removal of glycerol from fresh equine spermatozoa suspended in a modified TALP via either a four-step FV or FM dilution, improved the maintenance of sperm motility compared to a rapid, single-step dilution. This observation is similar to that reported for spermatozoa from a number of other species including human (Gao et al., 1995; Gilmore et al., 1997), mouse (Phelps et al., 1999) and boar spermatozoa (Gilmore et al., 1998). Likewise, multistep removal of glycerol from equine spermatozoa improved viability as determined by exclusion of propidium iodide compared to rapid dilution. A fixed molarity dilution appeared superior for maintenance of motility and viability compared to the fixed volume-dilution for removal of glycerol; however, these differences were not statistically significant. Interestingly, neither the use of a multistep dilution nor the use of an osmotic buffer (sucrose) for removal of glycerol from frozen–thawed equine spermatozoa improved maintenance of motility or viability compared to a rapid, one-step dilution. Although a number of studies have detailed the theoretical benefits of a multistep dilution to remove permeating CPAs from cryopreserved spermatozoa, relatively few studies have examined this effect experimentally (Gao et al., 1995, 1997; Gilmore et al., 1997). Several factors may explain the differences observed in multistep removal of glycerol from fresh equine spermatozoa (experiment 1) or from cryopreserved spermatozoa (experiment 2). In experiment 1, spermatozoa were separated on a Percoll-density gradient and resuspended in a modified TALP with BSA whereas in experiment 2, spermatozoa were processed for cryopreservation without the use of a Percoll-density gradient and were maintained in a cryopreservation extender with skim milk and egg yolk. Percoll-density gradient centrifugation is known to alter the phospholipid composition of sperm membranes and has been shown to alter the hydraulic conductivity of mouse spermatozoa (Phelps et al., 1999). Likewise, exposure of spermatozoa to milk proteins and egg yolk increases the osmotic tolerance of murine (Agca et al., 2002) and canine (Songsasen et al., 2002) spermatozoa. Other studies have suggested that alterations in the lipid bilayer, caused by phase separation events during cryopreservation (Holt and North, 1984; De Leeuw et al., 1990) could well influence osmotic properties. This observation is supported for equine spermatozoa where cold shock induced by rapid cooling at suprazero temperatures affects membrane permeability (Devireddy et al., 2002). In summary, the rapid, single-step removal of glycerol from fresh equine spermatozoa results in a post-hyperosmotic stress that is characterized by a reduction in both motility and membrane integrity. This post-hyperosmotic stress can be reduced by a step-wise dilution for removal of glycerol, which improved the maintenance of both motility and membrane integrity. A similar benefit for step-wise dilution for removal of glycerol was not observed in cryopreserved equine spermatozoa.
Acknowledgements This research was supported by the John P. Hughes Endowment, by the Center for Equine Health with funds provided by the Oak Tree Racing Association, the State of California pari-mutuel fund, and contributions by private donors. The authors thank Anthony Vo for technical assistance.
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