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A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) sperm
Cryobiology 44 (2002) 251–257 www.academicpress.com
A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) spermq R.K. Browne,* M. Mahony, and J. Clulow Department of Biological Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia Received 11 February 2002; accepted 21 May 2002
Abstract Previous studies on cane toad (Bufo marinus; Bufonidae; Anura) sperm cryopreservation were extended to compare the effects of cryopreservation in established sucrose (non-ionic) diluents with cryopreservation in ionic diluents containing amphibian Ringer solutions (with and without egg-yolk). In addition, methanol was tested as a cryoprotectant for B. marinus sperm for the first time. Twenty-seven cryoprotective solutions were trialled, with each containing one of the three diluents [10% (w/v) sucrose, simplified amphibian Ringer (SAR) or SAR/egg-yolk], with one of the three cryoprotectants (Me2 SO, glycerol, or methanol) at one of the three concentrations (10%, 15%, or 20% v/v). Sperm were collected by maceration of testes into cryoprotective solutions with post-thaw recovery assessed as the percentage of motile sperm and the degree (vigour) of motility. Percentage motility was the most sensitive measure of post-thaw recovery. The recovery of motility was lowest in Ringer (SAR) diluents and highest in sucrose diluents, with improved motility in SAR diluents when egg-yolk was added. Methanol was the poorest cryoprotectant and Me2 SO the most effective. Methanol at high concentrations was shown to support recovery in sucrose diluent but not in SAR, although its effectiveness in SAR was improved by egg-yolk. Overall, the efficacy of diluents in supporting a high percentage of sperm recovery was in declining order: sucrose > SAR/eggyolk > SAR diluents, and with cryoprotectants: Me2 SO > glycerol > methanol. In conclusion, SAR offers less potential as a diluent than sucrose, presumably due to the presence of inorganic ions. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Amphibian; Sperm; Frog; Cryopreservation; Saccharide; Saline
A relatively few studies have investigated the effects of freeze–thaw on amphibian sperm [5,9–11,15,23,25]. Nevertheless, successful cryopreservation has been reported with sperm frozen in suspensions derived from macerated testes q
This work was funded by institutional sources. Corresponding author. Fax: +61-2-4921-6923. E-mail address: [email protected] (R.K. Browne). *
[9–11,15]. Those suspensions contained organic compounds derived from interstitial fluid, cytoplasm, and blood, in addition to added cryoprotectants. In other vertebrates, organic compounds such as amino acids, lipids, and lipoproteins, added independently or as components in cytoplasmic and reproductive tract fluids [6,24,28,29], have contributed to successful sperm cryopreservation. In the future, the major applied use of amphibian sperm cryopreservation is likely to be
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in the management of endangered species. In such animals, sperm would be collected non-invasively from cloacal fluids, following the hormonal induction of sperm release from the testes via the kidney ducts into the urine [20]. The contents of urine will be relatively higher in inorganic electrolytes and lower in organic compounds than the suspensions derived from macerated testes. Our laboratory is now developing protocols for collecting and cryopreserving amphibian sperm collected in cloacal fluids. Our previous studies derived successful protocols for the cryopreservation of amphibian sperm from testicular macerates employing disaccharides (principally sucrose) as the osmotic solutes in cryodiluents [9–11]. The current study was undertaken as a systematic comparison of the response of the sperm of one amphibian species (Bufo marinus) to cryopreservation in non-polar, saccharide (sucrose) diluents and saline diluents containing inorganic electrolytes. The purpose of the study was to assess the relative effectiveness of organic versus saline diluents in cryopreserving amphibian sperm. Such knowledge will be useful in producing protocols for cryopreserving cloacal fluid sperm, in particular, for establishing whether the electrolytes in cloacal fluid might be beneficial or detrimental to sperm cryopreservation, and whether the cryodiluents used with cloacal sperm should be based primarily on organic solutes or inorganic electrolytes. As in other vertebrates, there may be variation between frog species in the response of sperm to cryopreservation protocols [26,29], including to different cryoprotectants. Consequently, we included methanol as a cryoprotectant in this study to expand the number of cryoprotectants which might be considered for use with amphibian species, in addition to the cryoprotectants we employed in previous studies on B. marinus (Me2 SO and glycerol). Methanol was examined as a cryoprotectant because, in common with glycerol and Me2 SO, it shows a high variation in toxicity between species, has a different range of cryoprotective actions, and in a limited number of fish species is the preferred cryoprotectant [12,13,26,27]. In this study, the effects were investigated of all combinations of each of the three diluents (10% (w/v) sucrose; Simplified Amphibian Ringer (SAR); SAR with egg-yolk) with each of the three cryoprotectants (Me2 SO, methanol, and glycerol) at each of the three concentrations (10%, 15%, and 20% v/v) on the freeze–thaw recovery of testicular cane toad (Bufo marinus) sperm using the
cooling protocol of Browne et al. [9]. As stated above, one of the reasons for this study was to understand the possible effects of urine electrolytes on sperm cryopreservation. However, the current experiments were conducted with testicular sperm, as testicular sperm are more readily collected in large numbers, and to facilitate comparison of sperm responses between inorganic electrolytes and those of saccharides using testicular sperm [9–11]. Future reports will present results of cryopreservation studies on cloacal sperm.
Materials and methods General Cane toads were obtained from Mareeba, North Queensland, Australia. Toads were collected during the breeding season, maintained in plastic vivaria at 28 °C with access to water, and fed high protein dog feed pellets [15% (w/w) protein, Luv Tender Chunks, Friskies Pet Care, Sydney, Australia] twice weekly. Anaesthetic was prepared as a 0.4% (w/v) aqueous solution of Tricaine Methane-Sulfonate (MS222) (Ruth Consolidated Industries P/L., Annandale, NSW, Australia) adjusted to pH 7.2 by addition of 0.05 M NaOH. Toads were injected through the dorsal lymph sac with 5 ml MS222 solution per 100 g body weight. The heart was removed after anaesthesia to ensure death. Excised testes were placed in 10 ml SAR in petri dishes at 0 °C (on ice) and held less than 30 min before sperm suspensions were prepared by maceration of testes in cryoprotective diluents containing various cryoprotectants. Preparation of suspensions and cryopreservation Twenty-seven cryoprotective solutions (treatments) were prepared from three diluents [10% (w/v) sucrose (294 mOsmol kg1 ], simplified amphibian Ringer (SAR; 113.0 mM NaCl, 1 mM CaCl2 , 2.0 mM KCl, 3.6 mM NaHCO3 ; 220 m Osmol kg1 ), and SAR with 10% (v/v) egg-yolk) and three cryoprotectants: 10%, 15%, and 20% (v/ v) Me2 SO, methanol or glycerol. Sperm suspensions were prepared by maceration of individual testes at a 1:1 ratio of testis weight (g) to volume (ml) of cryoprotective solution. Immediately after preparation, five 0.025 ml aliquots were sampled from each suspension prepared from each animal replicate and introduced into 0.125 ml cryo-straws
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(n ¼ 15 straws per treatment, i.e., five straws from each of the three animal replicates per treatment). Suspensions in straws were then freeze–thawed using the freezing ramps developed by Browne et al. [9]: starting temperature 2 °C; )1 °C min1 to )8 °C, hold 2 min; )3 °C min1 to )16°C; )5 °C min1 to )80 °C; LN2 quench and thawed in air. Sub-samples of suspensions were activated before and after freeze–thaw by dilution of one part suspension to two parts distilled H2 O. In activated samples, the percentage of motility and degree of motility of sperm were estimated under a phase contrast microscope (400). The degree of motility was graded from 0 to 4 using the scale of Emmens [18]. The percentage of motile sperm recorded after freeze–thaw was expressed as a relative percentage, as a substantial and variable proportion (range 0–40%) of unfrozen sperm were immotile in sub-samples activated immediately after preparation of testicular macerates. Relative percentage motility of each sample after cryopreservation was calculated as RM ¼ PM/IM 100 (RM, relative percentage motility; PM, post-thaw motility; IM, initial motility).
Statistical analyses Means and standard errors were calculated for the relative percentage of motile sperm for all treatments. Percentage motility data were arcsine transformed before testing for normality and homogeneity of variance. The data were subjected to analysis of variance and Tukey–Kramer multiplerange tests were employed for the comparison of means. All analyses were performed using the JMP 3.2 software package (SAS Institute Inc.).
Results Taken together, the data in Figs. 1 and 2 show that SAR-based diluents were less effective than sucrose-based diluents in supporting the recovery of sperm after freeze–thaw. However, the addition of egg-yolk to SAR substantially improved recovery compared with SAR alone. Percentage motility was a more sensitive indicator of treatment response than the degree of motility (sperm vigour) (Fig. 1). Consequently, only the analyses of percentage motility are presented in detail. Nevertheless, the mean data for sperm vigour (which is a measure of the strength of forward
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Fig. 1. A scatter plot of post-thaw degree of motility (vigour) against post-thaw relative percentage motile sperm plotted using treatment means as a response. Sucrose (), SAR/egg-yolk (d), and SAR (m).
progressive motility) are presented in Fig. 1 as it demonstrates that sperm cryopreserved in SAR diluents, which had a low recovery in percentage motility, also had a poor recovery of forward progression in the relatively few sperm that were motile. Comparisons of percentage motility data between treatments pooled by diluent showed that the mean recovery of motility was significantly higher (P < 0:01) in sucrose (40:4 2:3%) than in SAR and egg-yolk (32:0 1:9%Þ which, in turn, was significantly higher (P < 0:001) than SAR (13:7 1:2%). Comparisons between treatments pooled by cryoprotectant type showed that recovery was significantly higher (P < 0:01) in Me2 SO (37:2 2:0%) than in glycerol (29:8 1:9%) which was higher (P < 0:01) than methanol (19:7 2:1%). Comparisons between treatments pooled by cryoprotectant concentration show that recovery declined significantly (P < 0:001) from 20% and 15% (35:2 2:3%, 31:3 2:0%), respectively, to 10% (v/v) (19:5 1:6%) concentrations. Fig. 2 (lower case superscripts) shows recovery of relative percentage motility in treatments grouped within each of the three diluents: (a) 10% (w/v) sucrose, (b) SAR with 10% (v/v) egg-yolk, and (c) SAR. Fig. 2a shows that in sucrose diluent, recovery was highest in the 15% Me2 SO and 20% glycerol treatments and lowest in the 15% methanol and 10% glycerol treatments. The 15% Me2 SO treatment showed a significantly (P < 0:01) higher recovery than all other 15% and 10% cryoprotectant concentrations. Fig. 2b shows that in SAR with egg-yolk, recovery was significantly (P < 0:05) higher in 20% Me2 SO than in any other
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Fig. 2. The relative percentage motility of B. marinus sperm cryopreserved in 27 CPAs prepared from three diluents: (a) 10% (w/v) sucrose, (b) SAR with 10% (v/v) egg-yolk, (c) SAR, with 10%, 15%, or 20% (v/v) Me2 SO , methanol , or glycerol as cryoprotectants (mean SE, n ¼ 15). Within each diluent, means sharing the same lower case superscript are not significantly different (P < 0:05). Within each cryoprotectant at the same concentration, means sharing the same upper case superscript are not significantly different between diluents (P < 0:05).
treatment. Recovery of motility in other treatments was similar, about 30% lower than 20% Me2 SO, except for 10% Me2 SO and methanol for which it was significantly (P < 0:05) lower than other treatments. Fig. 2c shows that in SAR alone, where recovery was generally poor, 15% Me2 SO gave the highest mean recovery and had a significantly (P < 0:05) higher motility than all the methanol and glycerol treatments, but not other Me2 SO treatments. Methanol at all concentrations gave a very low motility (<5%). Fig. 2 (upper case superscripts) also includes comparisons between diluents for each concen-
tration of each cryoprotectant. The recovery of motility in all cryoprotectants at all concentrations was significantly (P < 0:05) higher in sucrose than in SAR, except in the 10% glycerol treatment, in which recovery in SAR alone was not significantly different to sucrose (P < 0:05). Recovery was significantly (P < 0:05) higher in all SAR/egg-yolk treatments than in SAR except for 10% and 15% Me2 SO. There were fewer significant differences in recovery between SAR with egg-yolk and sucrose diluents than between sucrose and SAR alone, as a result of recovery generally being higher in SAR with egg-yolk than
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in SAR alone. However, recovery was still higher in a number of sucrose treatments than in the equivalent SAR/egg-yolk treatment, e.g., 10% and 15% Me2 SO and 20% glycerol (P < 0:01). The exceptional treatment was 10% glycerol in which there was significantly (P < 0:05) higher recovery in the SAR/egg-yolk treatment than in the equivalent sucrose or SAR treatments. This latter finding indicated that at low concentrations of cryoprotectant, there was a beneficial interaction between glycerol and egg-yolk in the presence of SAR that made glycerol the most effective cryoprotectant (an effect not seen at higher cryoprotectant concentrations). In summary, the experiments in this study compared the response of B. marinus sperm to cryopreservation in organic and inorganic diluents (sucrose, SAR, and SAR/egg-yolk) combined with three different cryoprotectants. Overall, the most effective diluent was sucrose and the most effective cryoprotectants were 15–20% (v/v) Me2 SO and glycerol. SAR and methanol were a poor diluent and cryoprotectant, respectively, particularly when combined. Nevertheless, the addition of eggyolk substantially improved the effectiveness of SAR.
Discussion The cryodiluents used in this study contained both non-permeating (sucrose, SAR, egg-yolk) and permeating components (glycerol, Me2 SO, methanol). As indicated from the results, the effects of and interactions between these components may be quite complex. Non-permeating components dehydrate sperm during freeze–thaw, lower extracellular ionic strength, and may provide protection to membranes. Permeating cryoprotectants dehydrate cytoplasm and suppress intracellular ice formation during freeze–thaw [21]. As well, glycerol, Me2 SO, and egg-yolk alter membrane fluidity and act as anti-oxidants reducing peroxidation reactions affecting cell membrane integrity [22]. The higher recovery of sperm in sucrose, and the beneficial effect of egg-yolk in improving the recovery of sperm in SAR, indicates that inorganic electrolytes exert deleterious effects on B. marinus sperm during cryopreservation and that non-polar organic compounds both support cryopreservation and reverse the deleterious effects of inorganic electrolytes. This study did not resolve the reasons for these opposing effects, but
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it may be hypothesized from the results that organic compounds are involved in the preservation of membrane integrity. Studies in fish have generally shown that ionic diluents without added cryoprotectants support only low recovery after freezing [7], but that Me2 SO or glycerol added to the ionic diluents improve recovery [1]. Similarly, our results with B. marinus sperm showed almost no recovery in SAR alone (data not presented) and moderate recovery in SAR in the presence of these cryoprotectants (but not methanol). The mechanisms of action of glycerol and Me2 SO, and their positive interactions with organic compounds in cryodiluents, may involve effects on cell membrane fluidity and antioxidant effects [4,22], as well as direct effects on the formation of intracellular ice. Sucrose without other cryoprotectants has been reported to support cryopreservation of mammalian sperm under some conditions [29]. However, when sucrose is used as the sole cryoprotectant for B. marinus sperm, there is almost no freeze–thaw recovery (Browne et al., unpublished) and a permeating cryoprotectant is required to achieve recovery. Therefore, it may be suggested that sucrose acts as a non-penetrating osmolyte during cryopreservation of B. marinus sperm. The superior cryopreservation with sucrose in comparison to SAR also suggests that sucrose may dilute extracellular ions during the viscous phase of freezing [30] and stabilise plasma membranes [8,10,16]. The addition of egg-yolk to SAR improved recovery with all cryoprotectants, particularly at high cryoprotectant concentrations. Egg-yolk has been reported to improve the post-thaw recovery of sperm by diluting ions and stabilising membranes, possibly through replacement of lost membrane components. In mammalian sperm, the beneficial effect of egg-yolk in diluents is attributed to membrane stabilisation by phosphatidylcholine and polyunsaturated fatty acids and replacement of membrane proteins during thawing [28]. Various protein/lipid formulations in cryodiluents have improved recovery of sperm of fish [19] and mammals [28]. Protein in diluents improves recovery of unfrozen fish sperm stored at 0 °C in Me2 SO/saline diluents [14]. Nevertheless, the improved recovery achieved by these formulations is species- and extender-specific and implies that a complex set of interactions and successful formulations vary with species [2,3,17].
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The current study supported our previous findings on B. marinus [9,10] which demonstrated that sucrose and other saccharides such as trehalose were effective in cryodiluents. A preliminary study of several amphibian species in taxa unrelated to B. marinus supported this finding [11]. The current study has also shown that other organic compounds contained in egg-yolk also provide beneficial effects during cryopreservation. There is considerable scope for further work on amphibian sperm to establish mechanisms by which organic compounds improve cryopreservation and to identify the specific compounds in complex mixtures such as egg-yolk which are beneficial. This would allow the optimisation of the freezing and storage protocols which incorporate such compounds. In practical terms, there are implications from this study for the design of protocols for freezing sperm collected in amphibian cloacal fluids. The results suggest that when freezing sperm from amphibian cloacal fluids, inorganic electrolytes should be removed, or their levels should be low in comparison to the levels of organic compounds in the cryodiluents.
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A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) spermq R.K. Browne,* M. Mahony, and J. Clulow Department of Biological Sciences, University of Newcastle, University Drive, Callaghan, NSW 2308, Australia Received 11 February 2002; accepted 21 May 2002
Abstract Previous studies on cane toad (Bufo marinus; Bufonidae; Anura) sperm cryopreservation were extended to compare the effects of cryopreservation in established sucrose (non-ionic) diluents with cryopreservation in ionic diluents containing amphibian Ringer solutions (with and without egg-yolk). In addition, methanol was tested as a cryoprotectant for B. marinus sperm for the first time. Twenty-seven cryoprotective solutions were trialled, with each containing one of the three diluents [10% (w/v) sucrose, simplified amphibian Ringer (SAR) or SAR/egg-yolk], with one of the three cryoprotectants (Me2 SO, glycerol, or methanol) at one of the three concentrations (10%, 15%, or 20% v/v). Sperm were collected by maceration of testes into cryoprotective solutions with post-thaw recovery assessed as the percentage of motile sperm and the degree (vigour) of motility. Percentage motility was the most sensitive measure of post-thaw recovery. The recovery of motility was lowest in Ringer (SAR) diluents and highest in sucrose diluents, with improved motility in SAR diluents when egg-yolk was added. Methanol was the poorest cryoprotectant and Me2 SO the most effective. Methanol at high concentrations was shown to support recovery in sucrose diluent but not in SAR, although its effectiveness in SAR was improved by egg-yolk. Overall, the efficacy of diluents in supporting a high percentage of sperm recovery was in declining order: sucrose > SAR/eggyolk > SAR diluents, and with cryoprotectants: Me2 SO > glycerol > methanol. In conclusion, SAR offers less potential as a diluent than sucrose, presumably due to the presence of inorganic ions. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: Amphibian; Sperm; Frog; Cryopreservation; Saccharide; Saline
A relatively few studies have investigated the effects of freeze–thaw on amphibian sperm [5,9–11,15,23,25]. Nevertheless, successful cryopreservation has been reported with sperm frozen in suspensions derived from macerated testes q
This work was funded by institutional sources. Corresponding author. Fax: +61-2-4921-6923. E-mail address: [email protected] (R.K. Browne). *
[9–11,15]. Those suspensions contained organic compounds derived from interstitial fluid, cytoplasm, and blood, in addition to added cryoprotectants. In other vertebrates, organic compounds such as amino acids, lipids, and lipoproteins, added independently or as components in cytoplasmic and reproductive tract fluids [6,24,28,29], have contributed to successful sperm cryopreservation. In the future, the major applied use of amphibian sperm cryopreservation is likely to be
0011-2240/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 0 0 1 1 - 2 2 4 0 ( 0 2 ) 0 0 0 3 1 - 7
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R.K. Browne et al. / Cryobiology 44 (2002) 251–257
in the management of endangered species. In such animals, sperm would be collected non-invasively from cloacal fluids, following the hormonal induction of sperm release from the testes via the kidney ducts into the urine [20]. The contents of urine will be relatively higher in inorganic electrolytes and lower in organic compounds than the suspensions derived from macerated testes. Our laboratory is now developing protocols for collecting and cryopreserving amphibian sperm collected in cloacal fluids. Our previous studies derived successful protocols for the cryopreservation of amphibian sperm from testicular macerates employing disaccharides (principally sucrose) as the osmotic solutes in cryodiluents [9–11]. The current study was undertaken as a systematic comparison of the response of the sperm of one amphibian species (Bufo marinus) to cryopreservation in non-polar, saccharide (sucrose) diluents and saline diluents containing inorganic electrolytes. The purpose of the study was to assess the relative effectiveness of organic versus saline diluents in cryopreserving amphibian sperm. Such knowledge will be useful in producing protocols for cryopreserving cloacal fluid sperm, in particular, for establishing whether the electrolytes in cloacal fluid might be beneficial or detrimental to sperm cryopreservation, and whether the cryodiluents used with cloacal sperm should be based primarily on organic solutes or inorganic electrolytes. As in other vertebrates, there may be variation between frog species in the response of sperm to cryopreservation protocols [26,29], including to different cryoprotectants. Consequently, we included methanol as a cryoprotectant in this study to expand the number of cryoprotectants which might be considered for use with amphibian species, in addition to the cryoprotectants we employed in previous studies on B. marinus (Me2 SO and glycerol). Methanol was examined as a cryoprotectant because, in common with glycerol and Me2 SO, it shows a high variation in toxicity between species, has a different range of cryoprotective actions, and in a limited number of fish species is the preferred cryoprotectant [12,13,26,27]. In this study, the effects were investigated of all combinations of each of the three diluents (10% (w/v) sucrose; Simplified Amphibian Ringer (SAR); SAR with egg-yolk) with each of the three cryoprotectants (Me2 SO, methanol, and glycerol) at each of the three concentrations (10%, 15%, and 20% v/v) on the freeze–thaw recovery of testicular cane toad (Bufo marinus) sperm using the
cooling protocol of Browne et al. [9]. As stated above, one of the reasons for this study was to understand the possible effects of urine electrolytes on sperm cryopreservation. However, the current experiments were conducted with testicular sperm, as testicular sperm are more readily collected in large numbers, and to facilitate comparison of sperm responses between inorganic electrolytes and those of saccharides using testicular sperm [9–11]. Future reports will present results of cryopreservation studies on cloacal sperm.
Materials and methods General Cane toads were obtained from Mareeba, North Queensland, Australia. Toads were collected during the breeding season, maintained in plastic vivaria at 28 °C with access to water, and fed high protein dog feed pellets [15% (w/w) protein, Luv Tender Chunks, Friskies Pet Care, Sydney, Australia] twice weekly. Anaesthetic was prepared as a 0.4% (w/v) aqueous solution of Tricaine Methane-Sulfonate (MS222) (Ruth Consolidated Industries P/L., Annandale, NSW, Australia) adjusted to pH 7.2 by addition of 0.05 M NaOH. Toads were injected through the dorsal lymph sac with 5 ml MS222 solution per 100 g body weight. The heart was removed after anaesthesia to ensure death. Excised testes were placed in 10 ml SAR in petri dishes at 0 °C (on ice) and held less than 30 min before sperm suspensions were prepared by maceration of testes in cryoprotective diluents containing various cryoprotectants. Preparation of suspensions and cryopreservation Twenty-seven cryoprotective solutions (treatments) were prepared from three diluents [10% (w/v) sucrose (294 mOsmol kg1 ], simplified amphibian Ringer (SAR; 113.0 mM NaCl, 1 mM CaCl2 , 2.0 mM KCl, 3.6 mM NaHCO3 ; 220 m Osmol kg1 ), and SAR with 10% (v/v) egg-yolk) and three cryoprotectants: 10%, 15%, and 20% (v/ v) Me2 SO, methanol or glycerol. Sperm suspensions were prepared by maceration of individual testes at a 1:1 ratio of testis weight (g) to volume (ml) of cryoprotective solution. Immediately after preparation, five 0.025 ml aliquots were sampled from each suspension prepared from each animal replicate and introduced into 0.125 ml cryo-straws
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(n ¼ 15 straws per treatment, i.e., five straws from each of the three animal replicates per treatment). Suspensions in straws were then freeze–thawed using the freezing ramps developed by Browne et al. [9]: starting temperature 2 °C; )1 °C min1 to )8 °C, hold 2 min; )3 °C min1 to )16°C; )5 °C min1 to )80 °C; LN2 quench and thawed in air. Sub-samples of suspensions were activated before and after freeze–thaw by dilution of one part suspension to two parts distilled H2 O. In activated samples, the percentage of motility and degree of motility of sperm were estimated under a phase contrast microscope (400). The degree of motility was graded from 0 to 4 using the scale of Emmens [18]. The percentage of motile sperm recorded after freeze–thaw was expressed as a relative percentage, as a substantial and variable proportion (range 0–40%) of unfrozen sperm were immotile in sub-samples activated immediately after preparation of testicular macerates. Relative percentage motility of each sample after cryopreservation was calculated as RM ¼ PM/IM 100 (RM, relative percentage motility; PM, post-thaw motility; IM, initial motility).
Statistical analyses Means and standard errors were calculated for the relative percentage of motile sperm for all treatments. Percentage motility data were arcsine transformed before testing for normality and homogeneity of variance. The data were subjected to analysis of variance and Tukey–Kramer multiplerange tests were employed for the comparison of means. All analyses were performed using the JMP 3.2 software package (SAS Institute Inc.).
Results Taken together, the data in Figs. 1 and 2 show that SAR-based diluents were less effective than sucrose-based diluents in supporting the recovery of sperm after freeze–thaw. However, the addition of egg-yolk to SAR substantially improved recovery compared with SAR alone. Percentage motility was a more sensitive indicator of treatment response than the degree of motility (sperm vigour) (Fig. 1). Consequently, only the analyses of percentage motility are presented in detail. Nevertheless, the mean data for sperm vigour (which is a measure of the strength of forward
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Fig. 1. A scatter plot of post-thaw degree of motility (vigour) against post-thaw relative percentage motile sperm plotted using treatment means as a response. Sucrose (), SAR/egg-yolk (d), and SAR (m).
progressive motility) are presented in Fig. 1 as it demonstrates that sperm cryopreserved in SAR diluents, which had a low recovery in percentage motility, also had a poor recovery of forward progression in the relatively few sperm that were motile. Comparisons of percentage motility data between treatments pooled by diluent showed that the mean recovery of motility was significantly higher (P < 0:01) in sucrose (40:4 2:3%) than in SAR and egg-yolk (32:0 1:9%Þ which, in turn, was significantly higher (P < 0:001) than SAR (13:7 1:2%). Comparisons between treatments pooled by cryoprotectant type showed that recovery was significantly higher (P < 0:01) in Me2 SO (37:2 2:0%) than in glycerol (29:8 1:9%) which was higher (P < 0:01) than methanol (19:7 2:1%). Comparisons between treatments pooled by cryoprotectant concentration show that recovery declined significantly (P < 0:001) from 20% and 15% (35:2 2:3%, 31:3 2:0%), respectively, to 10% (v/v) (19:5 1:6%) concentrations. Fig. 2 (lower case superscripts) shows recovery of relative percentage motility in treatments grouped within each of the three diluents: (a) 10% (w/v) sucrose, (b) SAR with 10% (v/v) egg-yolk, and (c) SAR. Fig. 2a shows that in sucrose diluent, recovery was highest in the 15% Me2 SO and 20% glycerol treatments and lowest in the 15% methanol and 10% glycerol treatments. The 15% Me2 SO treatment showed a significantly (P < 0:01) higher recovery than all other 15% and 10% cryoprotectant concentrations. Fig. 2b shows that in SAR with egg-yolk, recovery was significantly (P < 0:05) higher in 20% Me2 SO than in any other
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Fig. 2. The relative percentage motility of B. marinus sperm cryopreserved in 27 CPAs prepared from three diluents: (a) 10% (w/v) sucrose, (b) SAR with 10% (v/v) egg-yolk, (c) SAR, with 10%, 15%, or 20% (v/v) Me2 SO , methanol , or glycerol as cryoprotectants (mean SE, n ¼ 15). Within each diluent, means sharing the same lower case superscript are not significantly different (P < 0:05). Within each cryoprotectant at the same concentration, means sharing the same upper case superscript are not significantly different between diluents (P < 0:05).
treatment. Recovery of motility in other treatments was similar, about 30% lower than 20% Me2 SO, except for 10% Me2 SO and methanol for which it was significantly (P < 0:05) lower than other treatments. Fig. 2c shows that in SAR alone, where recovery was generally poor, 15% Me2 SO gave the highest mean recovery and had a significantly (P < 0:05) higher motility than all the methanol and glycerol treatments, but not other Me2 SO treatments. Methanol at all concentrations gave a very low motility (<5%). Fig. 2 (upper case superscripts) also includes comparisons between diluents for each concen-
tration of each cryoprotectant. The recovery of motility in all cryoprotectants at all concentrations was significantly (P < 0:05) higher in sucrose than in SAR, except in the 10% glycerol treatment, in which recovery in SAR alone was not significantly different to sucrose (P < 0:05). Recovery was significantly (P < 0:05) higher in all SAR/egg-yolk treatments than in SAR except for 10% and 15% Me2 SO. There were fewer significant differences in recovery between SAR with egg-yolk and sucrose diluents than between sucrose and SAR alone, as a result of recovery generally being higher in SAR with egg-yolk than
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in SAR alone. However, recovery was still higher in a number of sucrose treatments than in the equivalent SAR/egg-yolk treatment, e.g., 10% and 15% Me2 SO and 20% glycerol (P < 0:01). The exceptional treatment was 10% glycerol in which there was significantly (P < 0:05) higher recovery in the SAR/egg-yolk treatment than in the equivalent sucrose or SAR treatments. This latter finding indicated that at low concentrations of cryoprotectant, there was a beneficial interaction between glycerol and egg-yolk in the presence of SAR that made glycerol the most effective cryoprotectant (an effect not seen at higher cryoprotectant concentrations). In summary, the experiments in this study compared the response of B. marinus sperm to cryopreservation in organic and inorganic diluents (sucrose, SAR, and SAR/egg-yolk) combined with three different cryoprotectants. Overall, the most effective diluent was sucrose and the most effective cryoprotectants were 15–20% (v/v) Me2 SO and glycerol. SAR and methanol were a poor diluent and cryoprotectant, respectively, particularly when combined. Nevertheless, the addition of eggyolk substantially improved the effectiveness of SAR.
Discussion The cryodiluents used in this study contained both non-permeating (sucrose, SAR, egg-yolk) and permeating components (glycerol, Me2 SO, methanol). As indicated from the results, the effects of and interactions between these components may be quite complex. Non-permeating components dehydrate sperm during freeze–thaw, lower extracellular ionic strength, and may provide protection to membranes. Permeating cryoprotectants dehydrate cytoplasm and suppress intracellular ice formation during freeze–thaw [21]. As well, glycerol, Me2 SO, and egg-yolk alter membrane fluidity and act as anti-oxidants reducing peroxidation reactions affecting cell membrane integrity [22]. The higher recovery of sperm in sucrose, and the beneficial effect of egg-yolk in improving the recovery of sperm in SAR, indicates that inorganic electrolytes exert deleterious effects on B. marinus sperm during cryopreservation and that non-polar organic compounds both support cryopreservation and reverse the deleterious effects of inorganic electrolytes. This study did not resolve the reasons for these opposing effects, but
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it may be hypothesized from the results that organic compounds are involved in the preservation of membrane integrity. Studies in fish have generally shown that ionic diluents without added cryoprotectants support only low recovery after freezing [7], but that Me2 SO or glycerol added to the ionic diluents improve recovery [1]. Similarly, our results with B. marinus sperm showed almost no recovery in SAR alone (data not presented) and moderate recovery in SAR in the presence of these cryoprotectants (but not methanol). The mechanisms of action of glycerol and Me2 SO, and their positive interactions with organic compounds in cryodiluents, may involve effects on cell membrane fluidity and antioxidant effects [4,22], as well as direct effects on the formation of intracellular ice. Sucrose without other cryoprotectants has been reported to support cryopreservation of mammalian sperm under some conditions [29]. However, when sucrose is used as the sole cryoprotectant for B. marinus sperm, there is almost no freeze–thaw recovery (Browne et al., unpublished) and a permeating cryoprotectant is required to achieve recovery. Therefore, it may be suggested that sucrose acts as a non-penetrating osmolyte during cryopreservation of B. marinus sperm. The superior cryopreservation with sucrose in comparison to SAR also suggests that sucrose may dilute extracellular ions during the viscous phase of freezing [30] and stabilise plasma membranes [8,10,16]. The addition of egg-yolk to SAR improved recovery with all cryoprotectants, particularly at high cryoprotectant concentrations. Egg-yolk has been reported to improve the post-thaw recovery of sperm by diluting ions and stabilising membranes, possibly through replacement of lost membrane components. In mammalian sperm, the beneficial effect of egg-yolk in diluents is attributed to membrane stabilisation by phosphatidylcholine and polyunsaturated fatty acids and replacement of membrane proteins during thawing [28]. Various protein/lipid formulations in cryodiluents have improved recovery of sperm of fish [19] and mammals [28]. Protein in diluents improves recovery of unfrozen fish sperm stored at 0 °C in Me2 SO/saline diluents [14]. Nevertheless, the improved recovery achieved by these formulations is species- and extender-specific and implies that a complex set of interactions and successful formulations vary with species [2,3,17].
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The current study supported our previous findings on B. marinus [9,10] which demonstrated that sucrose and other saccharides such as trehalose were effective in cryodiluents. A preliminary study of several amphibian species in taxa unrelated to B. marinus supported this finding [11]. The current study has also shown that other organic compounds contained in egg-yolk also provide beneficial effects during cryopreservation. There is considerable scope for further work on amphibian sperm to establish mechanisms by which organic compounds improve cryopreservation and to identify the specific compounds in complex mixtures such as egg-yolk which are beneficial. This would allow the optimisation of the freezing and storage protocols which incorporate such compounds. In practical terms, there are implications from this study for the design of protocols for freezing sperm collected in amphibian cloacal fluids. The results suggest that when freezing sperm from amphibian cloacal fluids, inorganic electrolytes should be removed, or their levels should be low in comparison to the levels of organic compounds in the cryodiluents.
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