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Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm
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Theriogenology 72 (2009) 1221–1228 www.theriojournal.com
Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm N. Mansour *, F. Lahnsteiner, R.A. Patzner Department of Organismic Biology, Faculty of Natural Sciences, University of Salzburg, Salzburg, Austria Received 4 June 2009; received in revised form 29 June 2009; accepted 3 July 2009
Abstract Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg2SO4, 1 mmol/L CaCl2, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 8C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 8C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 106 to 8 106/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm. # 2009 Elsevier Inc. All rights reserved. Keywords: Amphibians; Sperm cryopreservation; Sperm motility; Viability; Xenopus laevis
1. Introduction Global amphibian declines have encouraged research efforts to prevent amphibian extinctions. When compared with DNA banks, sperm banks have the advantage of easy use in breeding programs. Recovered sperm can maintain genetic variation in extant populations or reestablish species through special
* Corresponding author. Tel.: +43 662 8044 5630; fax: +43 662 8044 5698. E-mail address: [email protected] (N. Mansour). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.07.013
techniques including androgenesis. However, past techniques have not fully recovered the motility of fresh sperm and have been variable [1–4], and techniques need further optimization. Generally, successful semen cryopreservation is a complex mechanism and depends on several factors that affect the freezing/thawing process either separately or in combination [5]. Simple nonbuffered amphibian Ringer or sucrose solutions have been used as sperm diluents [1–4,6,7]. The most effective sperm cryoprotectants were sucrose or glycerol for Rana spp. [6], glycerol or dimethyl sulfoxide (DMSO) for Bufo spp. [1–3], mixtures of fetal bovine serum (FBS) and either DMSO
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or glycerol for Rana, Bufo, Hyla, and Myobatrachia spp. [7], and mixture of FBS and either sucrose or glycerol for Eleutherodactylus spp. [4]. The African clawed frog, Xenopus laevis, is the most used laboratory amphibian for cellular, molecular, and developmental studies. Additionally, this species has importance in studies on reproductive biology as it produces gametes throughout the year. Cryopreservation techniques of sperm have benefits in this species for laboratory purposes as for studies of artificial fertilization processes and selective cross-breeding. Moreover, this species can serve as a basis to develop reliable cryopreservation protocols that can in the future be transferred to endangered, native amphibian species. In Xenopus spp., Buchholz et al. [8] and Sargent and Mohun [9] were the first to try the sperm cryopreservation. The tested sperm diluents were either the sucrose [8,9] or 50% of FBS [8] with or without addition of penetrating cryoprotectants. The results of these experiments were nonconclusive: frozen-thawed sperm were nonfertile when using the protocol of Buchholz et al. [8]; the freezing, thawing, and fertilization steps have not been mentioned and described in detail [9]. Therefore, more studies are urgently needed to develop a reliable cryopreservation protocol for sperm of X. laevis by standardization of dilution rate, extender composition, rate of freezing and thawing processes, and fertilization steps. In the current study, a sperm extender, dilution rate, and fertilization conditions have been established. 2. Materials and methods Mature African claw frogs, X. laevis, were purchased from Horst Ka¨hler (Hamburg, Germany), transferred to the University of Salzburg, and kept in Fiberglas channels with a length of 220 cm and a width and a height of 50 cm. Twenty animals were kept in each channel. Water temperature was 18 2 8C. About 50% of the water was changed every 2 wk. Frogs were fed commercial pellets and bovine meat three times weekly. For stimulation of gamete production, frogs were injected in the dorsal lymph sacs with Ovopel pellets (one pellet consisted of 10 to 15 mg artificial GnRHethylamide D-ala6, Pro9 Net, and 2.5 to 3 mg of watersoluble dopamine antagonist metoclopramide; Interfish, Pudapest, Hungary). The pellets were ground, dissolved in 0.7% NaCl, and injected using a dose of one-half pellet/female and one-third pellet/male. For egg collection, 24 to 36 h after injection when some eggs were seen in the tank, the animals were anesthetized in 200 ppm MS 222 (Ethyl-m-aminobenzoate, methane-
sulfonate) for 10 to 20 min, and the eggs were collected by gentle abdominal massage. To collect semen, the treated males were killed 16 to 18 h after injection by an overdose of MS 222, and the testes were surgically removed. Testes of each male were macerated on ice in 1.5 mL of a motility inhibiting saline (MIS) composed of 150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg2SO4, 1 mmol/L CaCl2, and 20 mmol/L Tris, pH 8.0. Sperm cell concentration was counted in a Burker Tu¨rk counting chamber (Milian-AG, Basel, Switzerland) and it was 20 106 to 25 106/ mL. The sperm macerates were immediately cooled to 4 8C and used within 30 min. Preliminary studies showed that storage of Xenopus sperm at 4 8C prolonged its viability. 2.1. Sperm cryopreservation The experimental protocol is presented in Fig. 1. In Experiment 1, the toxicity of three penetrating cryoprotectants, glycerol (5%), dimethyl sulfoxide (DMSO; 10%), and methanol (10%) was tested after equilibration periods of 1, 10, and 20 min and incubation at 4 8C. After incubation was terminated, the sperm motility and viability were assessed as described later. Then in Experiment 2, freezing and thawing conditions were standardized The diluted sperm macerates (1:2 in MIS containing 10% DMSO) was frozen in acrylic hematocrit tubes of 50 mL volume (Hematlon; Hayashi-Rikagaku Co., Tokyo, Japan) for 7 min in the vapor of liquid nitrogen 5, 8, or 10 cm above the level of liquid nitrogen on a tray in an insulated self-made freezing box [10]. The temperature decrease at each freezing level was measured by placing a thermocouple probe at 48 C that was attached to a digital two-canal data-logger (PeakTech 5080; Heinz-Gu¨nter Lau GmbH, Ahrensburg, Germany) in the freezing box at the required freezing level. Rate of temperature decrease at the three freezing levels above the surface of liquid nitrogen are shown in Fig. 2. After freezing, the tubes were plunged into liquid nitrogen for storage. Thawing was performed in a water bath of 5, 25, and 30 8C for different time periods (10 to 60 sec). Slow thawing at room temperature for 40 sec was also tested. Immediately after thawing, sperm was used for motility and viability analyses. In Experiment 3, MIS, 300 mmol/L sucrose, or 300 mmol/L glucose were tested as basic sperm extenders with 5% or 10% DMSO as a penetrating cryoprotectant. In Experiment 4, we tested if addition of sucrose, as an external cryoprotectant, could improve the success of semen cryopreservation in MIS containing 5% DMSO. In each experiment, sperm macerates from three different males was frozen and examined separately.
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Fig. 1. Development of a cryopreservation protocol for X. laevis sperm.
2.2. Activation and measurement of sperm motility
Fig. 2. Temperature (8C) decrease during sperm freezing of X. laevis at different freezing levels above the surface of liquid nitrogen. Circle, freezing at 10 cm; square, freezing at 8 cm; triangle, freezing at 5 cm.
Motility measurements were performed at room temperature with computer-assisted cell motility analysis whereby the motility was recorded by using an inverse phase contrast microscope coupled with a Hitachi CCD color camera (Hitachi Kokusai Electric Inc., Tokyo, Japan) at 200 magnification and then analyzed with CellTrack cell motility analysis program (Motion Analysis Cooperation, Santa Rosa, CA, USA). For motility activation, 50 mL of distilled water was added into the investigation chamber (Makler type: depth 10 mm, volume of 100 mL), and 25 mL of diluted sperm was added and mixed. The chamber was closed with the coverslip whereupon excess sperm suspension was
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drained. Then the sample was quickly transferred to the microscope, and the motility was recorded directly on the computer by using the WinTV2000 program (Hauppauge Computer Works GmbH, Mo¨nchengladbach, Germany). When motility measurements are performed with testicular semen, there is always a risk of artifacts as it contains not only sperm but also other cell types. Therefore, in the motility analysis software, the parameter particle recognition was adjusted exactly to the size of Xenopus sperm, and smaller particles and larger cells as spermatids or blood cells were not included in the analysis. The following program setup has been established: Edge detection method, Sobel. Centroid parameters: neighborhood size, 10 pixels; maximum object size, 55 pixels; minimum object size, 20 pixels. Tracking parameters: maximum object speed, 15 pixel/frame. The following sperm parameters were measured: immotile (velocity < 5 mm sec-1), locally motile (velocity of 5 to 10 mm sec-1), motile (velocity >10 mm sec-1), average sperm velocity (mm sec-1), linear motile (rate of change of direction <250 degree sec-1), circular motile (rate of change of direction 250 to 900 degree sec-1), and nonlinear motile (rate of change of direction >900 degree sec-1). 2.3. Measurement of sperm viability Sperm viability was measured according to the modified procedures of Garner et al. [11]. Briefly, 50 mL diluted semen was mixed with 1 mL 6-carboxyfluorescein diacetate (CFDA) solution, 4 mg CFDA in 1 mL DMSO, in an Eppendorf tube on ice. After 4 min, 3 mL propidium iodide (8 mg in 1 mL distilled water) was added and mixed well. After 1 min, 20 mL stained semen was pipette on a slide, covered with a coverslip, and examined under a fluorescence microscope at a magnification of 400. Several pictures were photographed by using CellA Imaging Software for Life Science Microscopy (Soft Imaging System GmbH, Mu¨nster, Germany). Number of red (membranedamaged sperm) and green (live intact-membrane sperm) was recorded and counted from the pictures (Fig. 3). One hundred to 150 sperm were counted per each treatment. 2.4. Fertilization experiment Fertilization was performed in 50 mL glass beakers: 25 to 30 eggs were placed in the beakers and 16 mL of fresh sperm macerate (= control) or and 50 mL of frozenthawed sperm was added. Then, 2 mL of 20 mmol/L NaCl solution of 20 8C was added and gently shaken. After 1 to 2 min, the beakers were filled with 18 2 8C dechlorinated water. The egg:sperm ratio was 1:(15 to 16)
Fig. 3. Live (green) and dead (red) sperm of X. laevis after viability staining.
103. It was standardized in preliminary experiments, and the sperm:egg ratio used results in a fertilization rate of 70% to 80% when fresh sperm macerate is used. After fertilization was finished, the beakers were incubated at 18 2 8C, and the water was continuously aerated using aquarium air stones marked. After 48 h, the hatched tadpoles were counted. The hatching rate was calculated as the number of hatched larvae divided by the total number of eggs. Three replicates of fertilization trials were applied for each sperm treatment. 2.5. Statistical analysis Fertility and viability data were arcsine transformed and tested for normality. Analysis of variance (ANOVA) with subsequent Tukey’s b-test was used for comparison of mean values of the various treatments, and results are represented as mean SD. Differences were considered significant at P values less than 0.05. 3. Results Sperm collected in MIS was immotile and could be activated when the osmolality was decreased 50
N. Mansour et al. / Theriogenology 72 (2009) 1221–1228
mOsmol/kg. Additionally, during the course of our measurements and freezing experiments, about 3 to 4 h after testes maceration, the sperm did not show a decrease in motility when kept on ice. Untreated, freshly collected sperm had a motility rate of about 50% and swimming velocity of 21 mm sec-1 within the course of our experiment. The sperm motility patterns were of nonlinear, linear, and circular motility types with ratios of 59.6 14.6, 19.4 6.6, and 20.8 5.1, respectively. The rate of motile, nonmotile, and locally motile sperm, sperm velocity, and motility pattern did not change during the course of the experiments. 3.1. Toxicity of the tested penetrating cryoprotectants (Experiment 1)
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sperm had significantly lower percentages of motile and viable sperm and higher percentage of immotile sperm than that of the fresh sperm control. Sperm frozen 10 cm above the surface of liquid nitrogen had significantly higher percentages of motile and viable sperm and lower percentage of immotile sperm than that frozen 5 cm or 8 cm above the surface of liquid nitrogen (Fig. 5). When the cryopreserved sperm (freezing level 10 cm above the surface of liquid nitrogen) was thawed at room temperature for 40 sec, higher percentages of sperm motility and viability and lower percentage of immotile sperm were obtained than that when thawing at 5, 25, or 30 8C for 10, 15, or 60 sec (Fig. 6). The percentages of locally motile sperm, swimming velocity, and the motility pattern did not show changes
Diluted testicular sperm had higher percentages of motility and viability and lower percentage of immotile sperm after incubation in MIS containing 10% DMSO than that in MIS containing 10% methanol or 5% glycerol (Fig. 4). This effect was significant after incubation periods 10 min. The percentages of locally motile sperm, swimming velocity, and the motility pattern (linear, nonlinear, or circular) did not show changes during the course of this experiment and between all treatments. 3.2. Effect of different freezing and thawing conditions on sperm motility and viability (Experiment 2) In this experiment, MIS with 10% DMSO was used as an extender. After cryopreservation, the thawed
Fig. 4. Postincubation rates of sperm motility and viability of X. laevis in MIS containing different penetrating cryoprotectants and kept at 4 8C. Bars represent the motility rate, and lines represent the viability rate. Bars and lines with same letter are not significantly different. Horizontal shading, 10% DMSO; diagonal shading, 10% methanol; checkered shading, 5% glycerol; square, 10% DMSO; triangle, 10% methanol; circle, 5% glycerol.
Fig. 5. Postthaw rates of motility and viability rate of X. laevis sperm diluted in MIS containing 10% DMSO, frozen at different freezing levels above the surface of liquid nitrogen, and thawed at room temperature for 40 sec. Bars, motility rate; line, viability rate. Bars and lines with same letter are not significantly different.
Fig. 6. Postthaw rates of motility and viability of X. laevis sperm diluted in MIS containing 10% DMSO, frozen at 10 cm above the surface of liquid nitrogen, and thawed at different thawing temperatures. Bars, motility rate; line, viability rate. Bars and lines with same letter are not significantly different.
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Table 1 Effect of semen diluents on the motility of frozen-thawed sperm in X. laevis*. Extender
Immotile (%)
Local motile (%)
Motile (%)
Velocity (mm sec-1)
Fresh sperm control 300 mmol/L glucose + 5% DMSO 300 mmol/L glucose + 10% DMSO 300 mmol/L sucrose + 5% DMSO 300 mmol/L sucrose + 10% DMSO MIS + 10% DMSO MIS + 5% DMSO
22.5 7.1 b 65.4 12.5a 50.2 4.2 a 54.3 6.9 a 57.3 9.7 a 69.1 13.0a 61.3 7.2 a
21.8 4.2 a 21.7 6.2 a 28.8 4.8 a 24.9 7.6 a 18.7 4.5 a 30.2 9.2 a 26.8 6.1 a
55.6 9.1a 12.0 17.7b 20.8 6.6b 20.0 3.9b 23.0 5.8b 18.9 6.5b 25.8 2.1b
21.6 2.7a 21.3 5.2a 21.7 4.1a 18.8 3.7a 21.1 1.3a 19.9 5.0a 22.1 5.8a
*Data are means SD, n = 3. a,b Values within a column superscripted by the same letter are not significantly different.
Table 2 Effect of semen diluent on the viability and fertility of frozen-thawed sperm in X. laevis*. Extender Fresh sperm control 300 mmol/L glucose + 5% DMSO 300 mmol/L glucose + 10% DMSO 300 mmol/L sucrose + 5% DMSO 300 mmol/L sucrose + 10% DMSO MIS + 10% DMSO MIS + 5% DMSO
Live (%)
Hatching rate (%) a
90.8 5.4 25.5 3.4b
68.7 13.9a 12.4 3.4 c
23.5 5.1b
18.7 2.2 c
30.6 7.5b
5.8 5.1 c
31.2 5.4b
12.8 4.5 c
34.6 7.4b 37.5 6.6b
9.4 4.5 c 28.7 5.7 b
*Data are means SD, n = 3. a–c Values within a column superscripted by the same letter are not significantly different.
during the course of this experiment and between all treatments. 3.3. Effect of other sperm diluents on postthaw sperm motility, viability, and fertility (Experiment 3) The rates of sperm motility, viability, and hatching were higher and percentage of immotile sperm was lower in fresh sperm control than that in frozen-thawed sperm, similar for all treatments (Tables 1 and 2).
Addition of 10% DMSO to nonelectrolyte solutions of 300 mmol/L glucose or 300 mmol/L sucrose instead of the electrolyte solution MIS as extender base had no effect on the postthaw sperm motility, viability, and fertility (hatching rate). Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than the hatching rates of all other frozen semen (Table 2). Additionally, the percentage of locally motile sperm, the swimming velocity, and the swimming pattern did not show differences between the treatments and were similar to those of the fresh sperm control (Table 1). 3.4. Effect of addition of sucrose on postthaw sperm motility, viability, and fertility (Experiment 4) In this experiment, the highest postthaw motility rate, viability, and fertility (hatching rate) were obtained in the extender consisting of MIS with 5% DMSO and 73 mmol/L sucrose. Percentage of locally motile sperm, swimming velocity, and the sperm motility pattern did not show significant differences between all treatments (Table 3). When 73 mmol/L sucrose was added to an extender consisting of MIS with 5% DMSO, the postthaw motility rate and hatching rate were significantly higher and the postthaw percentage of immotile sperm was significantly lower than that in the extender containing MIS with 5% DMSO only (Tables 3 and 4). However, after addition of 30 mmol/L
Table 3 Effect of addition of sucrose on the motility of frozen-thawed sperm in X. laevis*. Extender Fresh sperm control MIS + 5% DMSO MIS + 5% DMSO + 30 mmol/L sucrose MIS + 5% DMSO + 73 mmol/L sucrose MIS + 5% DMSO + 146 mmol/L sucrose
Immotile (%) b
22.5 7.1 58.1 9.7 a 41.9 6.1a,b 30.5 6.8 b 43.6 7.2a,b
Local motile (%) a
21.8 4.2 23.8 9.0 a 24.5 5.1 a 24.7 6.2 a 24.5 6.1 a
*Data are means SD, n = 3. a,b Values within a column superscripted by the same letter are not significantly different.
Motile (%) a
55.6 9.1 18.0 0.8b 37.0 12.0a,b 45.8 4.8a 35.8 6.1a,b
Velocity (mm sec-1) 21.6 2.7a 22.6 6.2a 16.9 5.2a 20.3 2.7a 20.1 5.8a
N. Mansour et al. / Theriogenology 72 (2009) 1221–1228 Table 4 Effect of addition of sucrose on the motility, viability, and fertility of frozen-thawed sperm in X. laevis*. Extender
Live (%)
Hatching rate (%)
Fresh sperm control MIS + 5% DMSO MIS + 5% DMSO + 30 mmol/L sucrose MIS + 5% DMSO + 73 mmol/L sucrose MIS + 5% DMSO + 146 mmol/L sucrose
81.8 5.4a 33.7 17.4b,c 43.3 19.1b,c
68.7 13.9a 28.7 5.7c 27.9 6.9c
60.3 6.9b
48.4 4.3b
34.5 6.7b,c
26.5 4.7c
*Data are means SD, n = 3. a–c Values within a column superscripted by the same letter are not significantly different.
or 146 mmol/L sucrose to the extender consisting of MIS with 5% DMSO, the postthaw motility rate and viability showed non-significant increase and the percentage of immotile sperm had a non-significant decrease compared with that in semen frozen in the extender containing MIS with 5% DMSO only (Tables 3 and 4). 4. Discussion This is the first study broadly testing all parameters of cryopreservation methodology and describing in detail the responses of motility, viability, and fertility of sperm cryopreservation in the African clawed frog, X. laevis. The study demonstrates that successful cryopreservation of X. laevis sperm is possible whereby hatching rate, motility rate, and viability rate of about 70%, 80%, and 75%, respectively, of fresh hormonally induced sperm is achieved. The best cryopreservation protocol for X. laevis sperm is dilution of semen in MIS solution (to have a final concentration of 6.5 106 to 8 106/ mL) containing 5% DMSO and 73 mmol/L sucrose, freezing the diluted sperm in the vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec. The sperm of X. laevis collected in MIS solution were immotile, and when the collected sperm was diluted to an osmolality of 50 mOsmol/kg, the sperm motility was activated. Anuran sperm is generally immotile at the osmolality level of >100 mOsmol/kg, and sperm motility is activated by decreasing this osmolality [12,13]. More than 50% of X. laevis fresh sperm had nonlinear motility type and the remaining showed circular and linear motility types. Moreover, the swimming velocity was 15 to 25 mm sec-1, which indicates that the sperm motility is generally slow. In accordance with Sargent and Mohun [9] with a
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subjective method, most of Xenopus sperm motility was slow and of corkscrew-like and circular movements. However, the process of freezing-thawing of Xenopus sperm did not affect the sperm velocity and the type of motility, the sperm motility was decreased. This decrease in motility rate is probably due to variabilities in the sperm resistance during the freezing and thawing process. Incubation of the diluted sperm with the penetrating cryoprotectants DMSO, glycerol, or methanol showed that methanol was the most toxic cryoprotectant for Xenopus sperm. Methanol has not been tested previously in Xenopus species, but similar results have been obtained with sperm of other anurans [2,6]. Dimethyl sulfoxide was the least toxic cryoprotectant and had the highest cryoprotective effect. This is consistent with data of Browne et al. [2] on cane toad (Bufo marinus) sperm. The most effective DMSO concentration for Xenopus sperm was 5%, whereas in other amphibian cryopreservation studies, concentration of 10% to 20% were used [1,2,6]. Buchholz et al. [8] tested the cryopreservation of Xenopus sperm in a solution of 10% sucrose with 15% DMSO, however no fertilization was obtained with the cryopreserved sperm. Probably 15% DMSO acted toxic on sperm, as in our experiments 10% DMSO already decreased the motility and hatching rates. In our study for cryopreservation of X. laevis sperm, a freezing rate of 20 to 25 8C/min was optimal (Fig. 2). This freezing rate was higher than that used before for X. laevis and X. tropicalis sperm (10 8C/min) by Sargent and Mohun [9]. However, the freezing techniques are not comparable between the two studies as these authors froze the semen in Eppendorf tubes in a freezing box with dry ice and measured the temperature during freezing with a probe immersed in the tubes containing a standard cryoprotectant mixture. However, the thawing condition at room temperature (22 2 8C) for 40 sec resulted in higher sperm survival (% motile and % viable) than that of frozen sperm thawed at 25 8C and 30 8C for 15 and 10 sec, respectively. The exact cause for this phenomenon is unknown. Probably, the thawing process of cryopreserved X. laevis sperm is critical and needs special conditions of warm temperature and a relatively long period of time. Previously, thawing at 30 8C for about 10 sec was used to thaw Xenopus semen diluted in sucrose diluent without addition of penetrating cryoprotectant and frozen in Eppendorf tubes [9]. Generally, a successful freezing and thawing protocol is a complex procedure and depends not only on freezing and thawing rates but also on the freezing diluent and cryoprotectants used [5].
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In our results, MIS as a sperm diluent was superior to 300 mmol/L of either sucrose or glucose diluents when mixed with 5% DMSO. The previously published cryopreservation protocols use either sucrose [8,9] or 50% of fetal bovine serum [8] as extender base with or without penetrating cryoprotectants. In contrast with the current results, in the cane toad (Bufo marinus), the organic diluent sucrose resulted in higher cryopreservation success than that of inorganic salt solution [2]. Sucrose and glucose must be considered as nonpenetrating cryoprotectants that dehydrate sperm during freezing and thawing processes, lower the extracellular ionic strength, and may provide protection to sperm membranes [14]. In our cryopreservation protocol, 15 103 to 16 103 sperm were used to fertilize an egg. With this ratio, a fertility of 68.7% was obtained in fresh semen control and of 48.4% in semen frozen in MIS containing 5% DMSO and 73 mmol/L sucrose. Previous studies used higher sperm-to-egg ratios. Sargent and Mohun [9] have used a ratio of about 1:1 105 (egg:sperm) to obtain just 50% fertilization. However, Hollinger and Corton [12] used a dose of 3.3 106 sperm/mL to have about 50% fertilization rate, but the exact number of eggs used in their fertilization experiments has not been mentioned. In accordance with previous studies [9,12], sperm motility of the fresh sperm was about 50%. The postthaw motility rate obtained in the current study was 18% to 42%, whereas only 5% to 10% was obtained by Sargent and Mohun [9]. This difference might be due to the fact that these authors did not use a penetrating cryoprotectant, and, consequently, more sperm damage occurred during the freezing and thawing process. Additionally, a higher sperm:egg ratio was necessary in the former study [9] than that in our experiments proving that the penetrating cryoprotectant DMSO has a positive effect on the cryopreservation of X. laevis sperm. In conclusion, dilution of testicular sperm 6.5 106 to 8 106/mL in MIS containing 5% DMSO and 73 mmol/L sucrose, then freezing in the vapor of liquid nitrogen at a freezing rate of 20 to 25 8C/min and
thawing at room temperature for 40 sec is a reliable freezing and thawing protocol for X. laevis sperm. Acknowledgments This research was supported by the Austrian FWF, Der Wissenschaftsfonds, project no. P20109-B17. References [1] Browne PK, Clulow J, Mahony M, Clark A. Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology 1998;37:339–45. [2] Browne PK, Mahony M, Clulow J. A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) sperm. Cryobiology 2002;44:251–7. [3] Browne PK, Clulow J, Mahony M. The effect of saccharides on the post-thaw recovery of cane toad (Bufo marinus) sperm. Cryo Lett 2002;23:121–8. [4] Michael SF, Jones C. Cryopreservation of sperm of the terrestrial Puerto Rican frog, Eleutherodactylus coqui. Cryobiology 2004;48:90–4. [5] Holt WV. Basic aspects of frozen storage of semen. Anim Reprod Sci 2000;62:3–22. [6] Beesley SG, Costanzo JP, Lee RE. Cryopreservation of sperm from freeze-tolerant and -intolerant anurans. Cryobiology 1998;37:155–62. [7] Browne PK, Clulow J, Mahony M. The short term storage and cryopreservation of sperm from hylid and myobatrachid frogs. Cryo Lett 2002;23:129–36. [8] Buchholz DR, Fu L, Shi YB. Cryopreservation of Xenopus transgenic lines. Mol Reprod Dev 2004;67:65–9. [9] Sargent MG, Mohun TJ. Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis. Gensis 2005;41:41–6. [10] Lahnsteiner F, Mansour N. Protocols for the cryopreservation of Salmonidae semen, Lota lota (Gadidae) and Esox lucius (Esocidae). In: Methods in Reproductive Aquaculture, Marine and Freshwater Species. CRC Press, Taylor & Francis Group; 2008. p. 373–84. [11] Garner DL, Pinkel D, Johnson LA, Pace MM. Assessment of sperml fuction using dual fluorescent staining and flow cytometric analyses. Biol Reprod 1986;34:127–38. [12] Hollinger TG, Corton GL. Artificial fertilization of gametes from south African clawed frog, Xenopus laevis. Gamete Res 1980;3: 45–57. [13] Kouba AJ, Vance CK, Frommeyer MA, Roth TL. Structural and functional aspects of Bufo americanus sperm: effects of inactivation and reactivation. J Exp Zool 2003;295A:172–82. [14] Gao DC. Mechanisms of cryoinjury in living cells. ILAR J 2000;41:187–96.
Theriogenology 72 (2009) 1221–1228 www.theriojournal.com
Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm N. Mansour *, F. Lahnsteiner, R.A. Patzner Department of Organismic Biology, Faculty of Natural Sciences, University of Salzburg, Salzburg, Austria Received 4 June 2009; received in revised form 29 June 2009; accepted 3 July 2009
Abstract Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg2SO4, 1 mmol/L CaCl2, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 8C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 8C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 106 to 8 106/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm. # 2009 Elsevier Inc. All rights reserved. Keywords: Amphibians; Sperm cryopreservation; Sperm motility; Viability; Xenopus laevis
1. Introduction Global amphibian declines have encouraged research efforts to prevent amphibian extinctions. When compared with DNA banks, sperm banks have the advantage of easy use in breeding programs. Recovered sperm can maintain genetic variation in extant populations or reestablish species through special
* Corresponding author. Tel.: +43 662 8044 5630; fax: +43 662 8044 5698. E-mail address: [email protected] (N. Mansour). 0093-691X/$ – see front matter # 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2009.07.013
techniques including androgenesis. However, past techniques have not fully recovered the motility of fresh sperm and have been variable [1–4], and techniques need further optimization. Generally, successful semen cryopreservation is a complex mechanism and depends on several factors that affect the freezing/thawing process either separately or in combination [5]. Simple nonbuffered amphibian Ringer or sucrose solutions have been used as sperm diluents [1–4,6,7]. The most effective sperm cryoprotectants were sucrose or glycerol for Rana spp. [6], glycerol or dimethyl sulfoxide (DMSO) for Bufo spp. [1–3], mixtures of fetal bovine serum (FBS) and either DMSO
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or glycerol for Rana, Bufo, Hyla, and Myobatrachia spp. [7], and mixture of FBS and either sucrose or glycerol for Eleutherodactylus spp. [4]. The African clawed frog, Xenopus laevis, is the most used laboratory amphibian for cellular, molecular, and developmental studies. Additionally, this species has importance in studies on reproductive biology as it produces gametes throughout the year. Cryopreservation techniques of sperm have benefits in this species for laboratory purposes as for studies of artificial fertilization processes and selective cross-breeding. Moreover, this species can serve as a basis to develop reliable cryopreservation protocols that can in the future be transferred to endangered, native amphibian species. In Xenopus spp., Buchholz et al. [8] and Sargent and Mohun [9] were the first to try the sperm cryopreservation. The tested sperm diluents were either the sucrose [8,9] or 50% of FBS [8] with or without addition of penetrating cryoprotectants. The results of these experiments were nonconclusive: frozen-thawed sperm were nonfertile when using the protocol of Buchholz et al. [8]; the freezing, thawing, and fertilization steps have not been mentioned and described in detail [9]. Therefore, more studies are urgently needed to develop a reliable cryopreservation protocol for sperm of X. laevis by standardization of dilution rate, extender composition, rate of freezing and thawing processes, and fertilization steps. In the current study, a sperm extender, dilution rate, and fertilization conditions have been established. 2. Materials and methods Mature African claw frogs, X. laevis, were purchased from Horst Ka¨hler (Hamburg, Germany), transferred to the University of Salzburg, and kept in Fiberglas channels with a length of 220 cm and a width and a height of 50 cm. Twenty animals were kept in each channel. Water temperature was 18 2 8C. About 50% of the water was changed every 2 wk. Frogs were fed commercial pellets and bovine meat three times weekly. For stimulation of gamete production, frogs were injected in the dorsal lymph sacs with Ovopel pellets (one pellet consisted of 10 to 15 mg artificial GnRHethylamide D-ala6, Pro9 Net, and 2.5 to 3 mg of watersoluble dopamine antagonist metoclopramide; Interfish, Pudapest, Hungary). The pellets were ground, dissolved in 0.7% NaCl, and injected using a dose of one-half pellet/female and one-third pellet/male. For egg collection, 24 to 36 h after injection when some eggs were seen in the tank, the animals were anesthetized in 200 ppm MS 222 (Ethyl-m-aminobenzoate, methane-
sulfonate) for 10 to 20 min, and the eggs were collected by gentle abdominal massage. To collect semen, the treated males were killed 16 to 18 h after injection by an overdose of MS 222, and the testes were surgically removed. Testes of each male were macerated on ice in 1.5 mL of a motility inhibiting saline (MIS) composed of 150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg2SO4, 1 mmol/L CaCl2, and 20 mmol/L Tris, pH 8.0. Sperm cell concentration was counted in a Burker Tu¨rk counting chamber (Milian-AG, Basel, Switzerland) and it was 20 106 to 25 106/ mL. The sperm macerates were immediately cooled to 4 8C and used within 30 min. Preliminary studies showed that storage of Xenopus sperm at 4 8C prolonged its viability. 2.1. Sperm cryopreservation The experimental protocol is presented in Fig. 1. In Experiment 1, the toxicity of three penetrating cryoprotectants, glycerol (5%), dimethyl sulfoxide (DMSO; 10%), and methanol (10%) was tested after equilibration periods of 1, 10, and 20 min and incubation at 4 8C. After incubation was terminated, the sperm motility and viability were assessed as described later. Then in Experiment 2, freezing and thawing conditions were standardized The diluted sperm macerates (1:2 in MIS containing 10% DMSO) was frozen in acrylic hematocrit tubes of 50 mL volume (Hematlon; Hayashi-Rikagaku Co., Tokyo, Japan) for 7 min in the vapor of liquid nitrogen 5, 8, or 10 cm above the level of liquid nitrogen on a tray in an insulated self-made freezing box [10]. The temperature decrease at each freezing level was measured by placing a thermocouple probe at 48 C that was attached to a digital two-canal data-logger (PeakTech 5080; Heinz-Gu¨nter Lau GmbH, Ahrensburg, Germany) in the freezing box at the required freezing level. Rate of temperature decrease at the three freezing levels above the surface of liquid nitrogen are shown in Fig. 2. After freezing, the tubes were plunged into liquid nitrogen for storage. Thawing was performed in a water bath of 5, 25, and 30 8C for different time periods (10 to 60 sec). Slow thawing at room temperature for 40 sec was also tested. Immediately after thawing, sperm was used for motility and viability analyses. In Experiment 3, MIS, 300 mmol/L sucrose, or 300 mmol/L glucose were tested as basic sperm extenders with 5% or 10% DMSO as a penetrating cryoprotectant. In Experiment 4, we tested if addition of sucrose, as an external cryoprotectant, could improve the success of semen cryopreservation in MIS containing 5% DMSO. In each experiment, sperm macerates from three different males was frozen and examined separately.
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Fig. 1. Development of a cryopreservation protocol for X. laevis sperm.
2.2. Activation and measurement of sperm motility
Fig. 2. Temperature (8C) decrease during sperm freezing of X. laevis at different freezing levels above the surface of liquid nitrogen. Circle, freezing at 10 cm; square, freezing at 8 cm; triangle, freezing at 5 cm.
Motility measurements were performed at room temperature with computer-assisted cell motility analysis whereby the motility was recorded by using an inverse phase contrast microscope coupled with a Hitachi CCD color camera (Hitachi Kokusai Electric Inc., Tokyo, Japan) at 200 magnification and then analyzed with CellTrack cell motility analysis program (Motion Analysis Cooperation, Santa Rosa, CA, USA). For motility activation, 50 mL of distilled water was added into the investigation chamber (Makler type: depth 10 mm, volume of 100 mL), and 25 mL of diluted sperm was added and mixed. The chamber was closed with the coverslip whereupon excess sperm suspension was
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drained. Then the sample was quickly transferred to the microscope, and the motility was recorded directly on the computer by using the WinTV2000 program (Hauppauge Computer Works GmbH, Mo¨nchengladbach, Germany). When motility measurements are performed with testicular semen, there is always a risk of artifacts as it contains not only sperm but also other cell types. Therefore, in the motility analysis software, the parameter particle recognition was adjusted exactly to the size of Xenopus sperm, and smaller particles and larger cells as spermatids or blood cells were not included in the analysis. The following program setup has been established: Edge detection method, Sobel. Centroid parameters: neighborhood size, 10 pixels; maximum object size, 55 pixels; minimum object size, 20 pixels. Tracking parameters: maximum object speed, 15 pixel/frame. The following sperm parameters were measured: immotile (velocity < 5 mm sec-1), locally motile (velocity of 5 to 10 mm sec-1), motile (velocity >10 mm sec-1), average sperm velocity (mm sec-1), linear motile (rate of change of direction <250 degree sec-1), circular motile (rate of change of direction 250 to 900 degree sec-1), and nonlinear motile (rate of change of direction >900 degree sec-1). 2.3. Measurement of sperm viability Sperm viability was measured according to the modified procedures of Garner et al. [11]. Briefly, 50 mL diluted semen was mixed with 1 mL 6-carboxyfluorescein diacetate (CFDA) solution, 4 mg CFDA in 1 mL DMSO, in an Eppendorf tube on ice. After 4 min, 3 mL propidium iodide (8 mg in 1 mL distilled water) was added and mixed well. After 1 min, 20 mL stained semen was pipette on a slide, covered with a coverslip, and examined under a fluorescence microscope at a magnification of 400. Several pictures were photographed by using CellA Imaging Software for Life Science Microscopy (Soft Imaging System GmbH, Mu¨nster, Germany). Number of red (membranedamaged sperm) and green (live intact-membrane sperm) was recorded and counted from the pictures (Fig. 3). One hundred to 150 sperm were counted per each treatment. 2.4. Fertilization experiment Fertilization was performed in 50 mL glass beakers: 25 to 30 eggs were placed in the beakers and 16 mL of fresh sperm macerate (= control) or and 50 mL of frozenthawed sperm was added. Then, 2 mL of 20 mmol/L NaCl solution of 20 8C was added and gently shaken. After 1 to 2 min, the beakers were filled with 18 2 8C dechlorinated water. The egg:sperm ratio was 1:(15 to 16)
Fig. 3. Live (green) and dead (red) sperm of X. laevis after viability staining.
103. It was standardized in preliminary experiments, and the sperm:egg ratio used results in a fertilization rate of 70% to 80% when fresh sperm macerate is used. After fertilization was finished, the beakers were incubated at 18 2 8C, and the water was continuously aerated using aquarium air stones marked. After 48 h, the hatched tadpoles were counted. The hatching rate was calculated as the number of hatched larvae divided by the total number of eggs. Three replicates of fertilization trials were applied for each sperm treatment. 2.5. Statistical analysis Fertility and viability data were arcsine transformed and tested for normality. Analysis of variance (ANOVA) with subsequent Tukey’s b-test was used for comparison of mean values of the various treatments, and results are represented as mean SD. Differences were considered significant at P values less than 0.05. 3. Results Sperm collected in MIS was immotile and could be activated when the osmolality was decreased 50
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mOsmol/kg. Additionally, during the course of our measurements and freezing experiments, about 3 to 4 h after testes maceration, the sperm did not show a decrease in motility when kept on ice. Untreated, freshly collected sperm had a motility rate of about 50% and swimming velocity of 21 mm sec-1 within the course of our experiment. The sperm motility patterns were of nonlinear, linear, and circular motility types with ratios of 59.6 14.6, 19.4 6.6, and 20.8 5.1, respectively. The rate of motile, nonmotile, and locally motile sperm, sperm velocity, and motility pattern did not change during the course of the experiments. 3.1. Toxicity of the tested penetrating cryoprotectants (Experiment 1)
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sperm had significantly lower percentages of motile and viable sperm and higher percentage of immotile sperm than that of the fresh sperm control. Sperm frozen 10 cm above the surface of liquid nitrogen had significantly higher percentages of motile and viable sperm and lower percentage of immotile sperm than that frozen 5 cm or 8 cm above the surface of liquid nitrogen (Fig. 5). When the cryopreserved sperm (freezing level 10 cm above the surface of liquid nitrogen) was thawed at room temperature for 40 sec, higher percentages of sperm motility and viability and lower percentage of immotile sperm were obtained than that when thawing at 5, 25, or 30 8C for 10, 15, or 60 sec (Fig. 6). The percentages of locally motile sperm, swimming velocity, and the motility pattern did not show changes
Diluted testicular sperm had higher percentages of motility and viability and lower percentage of immotile sperm after incubation in MIS containing 10% DMSO than that in MIS containing 10% methanol or 5% glycerol (Fig. 4). This effect was significant after incubation periods 10 min. The percentages of locally motile sperm, swimming velocity, and the motility pattern (linear, nonlinear, or circular) did not show changes during the course of this experiment and between all treatments. 3.2. Effect of different freezing and thawing conditions on sperm motility and viability (Experiment 2) In this experiment, MIS with 10% DMSO was used as an extender. After cryopreservation, the thawed
Fig. 4. Postincubation rates of sperm motility and viability of X. laevis in MIS containing different penetrating cryoprotectants and kept at 4 8C. Bars represent the motility rate, and lines represent the viability rate. Bars and lines with same letter are not significantly different. Horizontal shading, 10% DMSO; diagonal shading, 10% methanol; checkered shading, 5% glycerol; square, 10% DMSO; triangle, 10% methanol; circle, 5% glycerol.
Fig. 5. Postthaw rates of motility and viability rate of X. laevis sperm diluted in MIS containing 10% DMSO, frozen at different freezing levels above the surface of liquid nitrogen, and thawed at room temperature for 40 sec. Bars, motility rate; line, viability rate. Bars and lines with same letter are not significantly different.
Fig. 6. Postthaw rates of motility and viability of X. laevis sperm diluted in MIS containing 10% DMSO, frozen at 10 cm above the surface of liquid nitrogen, and thawed at different thawing temperatures. Bars, motility rate; line, viability rate. Bars and lines with same letter are not significantly different.
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Table 1 Effect of semen diluents on the motility of frozen-thawed sperm in X. laevis*. Extender
Immotile (%)
Local motile (%)
Motile (%)
Velocity (mm sec-1)
Fresh sperm control 300 mmol/L glucose + 5% DMSO 300 mmol/L glucose + 10% DMSO 300 mmol/L sucrose + 5% DMSO 300 mmol/L sucrose + 10% DMSO MIS + 10% DMSO MIS + 5% DMSO
22.5 7.1 b 65.4 12.5a 50.2 4.2 a 54.3 6.9 a 57.3 9.7 a 69.1 13.0a 61.3 7.2 a
21.8 4.2 a 21.7 6.2 a 28.8 4.8 a 24.9 7.6 a 18.7 4.5 a 30.2 9.2 a 26.8 6.1 a
55.6 9.1a 12.0 17.7b 20.8 6.6b 20.0 3.9b 23.0 5.8b 18.9 6.5b 25.8 2.1b
21.6 2.7a 21.3 5.2a 21.7 4.1a 18.8 3.7a 21.1 1.3a 19.9 5.0a 22.1 5.8a
*Data are means SD, n = 3. a,b Values within a column superscripted by the same letter are not significantly different.
Table 2 Effect of semen diluent on the viability and fertility of frozen-thawed sperm in X. laevis*. Extender Fresh sperm control 300 mmol/L glucose + 5% DMSO 300 mmol/L glucose + 10% DMSO 300 mmol/L sucrose + 5% DMSO 300 mmol/L sucrose + 10% DMSO MIS + 10% DMSO MIS + 5% DMSO
Live (%)
Hatching rate (%) a
90.8 5.4 25.5 3.4b
68.7 13.9a 12.4 3.4 c
23.5 5.1b
18.7 2.2 c
30.6 7.5b
5.8 5.1 c
31.2 5.4b
12.8 4.5 c
34.6 7.4b 37.5 6.6b
9.4 4.5 c 28.7 5.7 b
*Data are means SD, n = 3. a–c Values within a column superscripted by the same letter are not significantly different.
during the course of this experiment and between all treatments. 3.3. Effect of other sperm diluents on postthaw sperm motility, viability, and fertility (Experiment 3) The rates of sperm motility, viability, and hatching were higher and percentage of immotile sperm was lower in fresh sperm control than that in frozen-thawed sperm, similar for all treatments (Tables 1 and 2).
Addition of 10% DMSO to nonelectrolyte solutions of 300 mmol/L glucose or 300 mmol/L sucrose instead of the electrolyte solution MIS as extender base had no effect on the postthaw sperm motility, viability, and fertility (hatching rate). Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than the hatching rates of all other frozen semen (Table 2). Additionally, the percentage of locally motile sperm, the swimming velocity, and the swimming pattern did not show differences between the treatments and were similar to those of the fresh sperm control (Table 1). 3.4. Effect of addition of sucrose on postthaw sperm motility, viability, and fertility (Experiment 4) In this experiment, the highest postthaw motility rate, viability, and fertility (hatching rate) were obtained in the extender consisting of MIS with 5% DMSO and 73 mmol/L sucrose. Percentage of locally motile sperm, swimming velocity, and the sperm motility pattern did not show significant differences between all treatments (Table 3). When 73 mmol/L sucrose was added to an extender consisting of MIS with 5% DMSO, the postthaw motility rate and hatching rate were significantly higher and the postthaw percentage of immotile sperm was significantly lower than that in the extender containing MIS with 5% DMSO only (Tables 3 and 4). However, after addition of 30 mmol/L
Table 3 Effect of addition of sucrose on the motility of frozen-thawed sperm in X. laevis*. Extender Fresh sperm control MIS + 5% DMSO MIS + 5% DMSO + 30 mmol/L sucrose MIS + 5% DMSO + 73 mmol/L sucrose MIS + 5% DMSO + 146 mmol/L sucrose
Immotile (%) b
22.5 7.1 58.1 9.7 a 41.9 6.1a,b 30.5 6.8 b 43.6 7.2a,b
Local motile (%) a
21.8 4.2 23.8 9.0 a 24.5 5.1 a 24.7 6.2 a 24.5 6.1 a
*Data are means SD, n = 3. a,b Values within a column superscripted by the same letter are not significantly different.
Motile (%) a
55.6 9.1 18.0 0.8b 37.0 12.0a,b 45.8 4.8a 35.8 6.1a,b
Velocity (mm sec-1) 21.6 2.7a 22.6 6.2a 16.9 5.2a 20.3 2.7a 20.1 5.8a
N. Mansour et al. / Theriogenology 72 (2009) 1221–1228 Table 4 Effect of addition of sucrose on the motility, viability, and fertility of frozen-thawed sperm in X. laevis*. Extender
Live (%)
Hatching rate (%)
Fresh sperm control MIS + 5% DMSO MIS + 5% DMSO + 30 mmol/L sucrose MIS + 5% DMSO + 73 mmol/L sucrose MIS + 5% DMSO + 146 mmol/L sucrose
81.8 5.4a 33.7 17.4b,c 43.3 19.1b,c
68.7 13.9a 28.7 5.7c 27.9 6.9c
60.3 6.9b
48.4 4.3b
34.5 6.7b,c
26.5 4.7c
*Data are means SD, n = 3. a–c Values within a column superscripted by the same letter are not significantly different.
or 146 mmol/L sucrose to the extender consisting of MIS with 5% DMSO, the postthaw motility rate and viability showed non-significant increase and the percentage of immotile sperm had a non-significant decrease compared with that in semen frozen in the extender containing MIS with 5% DMSO only (Tables 3 and 4). 4. Discussion This is the first study broadly testing all parameters of cryopreservation methodology and describing in detail the responses of motility, viability, and fertility of sperm cryopreservation in the African clawed frog, X. laevis. The study demonstrates that successful cryopreservation of X. laevis sperm is possible whereby hatching rate, motility rate, and viability rate of about 70%, 80%, and 75%, respectively, of fresh hormonally induced sperm is achieved. The best cryopreservation protocol for X. laevis sperm is dilution of semen in MIS solution (to have a final concentration of 6.5 106 to 8 106/ mL) containing 5% DMSO and 73 mmol/L sucrose, freezing the diluted sperm in the vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec. The sperm of X. laevis collected in MIS solution were immotile, and when the collected sperm was diluted to an osmolality of 50 mOsmol/kg, the sperm motility was activated. Anuran sperm is generally immotile at the osmolality level of >100 mOsmol/kg, and sperm motility is activated by decreasing this osmolality [12,13]. More than 50% of X. laevis fresh sperm had nonlinear motility type and the remaining showed circular and linear motility types. Moreover, the swimming velocity was 15 to 25 mm sec-1, which indicates that the sperm motility is generally slow. In accordance with Sargent and Mohun [9] with a
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subjective method, most of Xenopus sperm motility was slow and of corkscrew-like and circular movements. However, the process of freezing-thawing of Xenopus sperm did not affect the sperm velocity and the type of motility, the sperm motility was decreased. This decrease in motility rate is probably due to variabilities in the sperm resistance during the freezing and thawing process. Incubation of the diluted sperm with the penetrating cryoprotectants DMSO, glycerol, or methanol showed that methanol was the most toxic cryoprotectant for Xenopus sperm. Methanol has not been tested previously in Xenopus species, but similar results have been obtained with sperm of other anurans [2,6]. Dimethyl sulfoxide was the least toxic cryoprotectant and had the highest cryoprotective effect. This is consistent with data of Browne et al. [2] on cane toad (Bufo marinus) sperm. The most effective DMSO concentration for Xenopus sperm was 5%, whereas in other amphibian cryopreservation studies, concentration of 10% to 20% were used [1,2,6]. Buchholz et al. [8] tested the cryopreservation of Xenopus sperm in a solution of 10% sucrose with 15% DMSO, however no fertilization was obtained with the cryopreserved sperm. Probably 15% DMSO acted toxic on sperm, as in our experiments 10% DMSO already decreased the motility and hatching rates. In our study for cryopreservation of X. laevis sperm, a freezing rate of 20 to 25 8C/min was optimal (Fig. 2). This freezing rate was higher than that used before for X. laevis and X. tropicalis sperm (10 8C/min) by Sargent and Mohun [9]. However, the freezing techniques are not comparable between the two studies as these authors froze the semen in Eppendorf tubes in a freezing box with dry ice and measured the temperature during freezing with a probe immersed in the tubes containing a standard cryoprotectant mixture. However, the thawing condition at room temperature (22 2 8C) for 40 sec resulted in higher sperm survival (% motile and % viable) than that of frozen sperm thawed at 25 8C and 30 8C for 15 and 10 sec, respectively. The exact cause for this phenomenon is unknown. Probably, the thawing process of cryopreserved X. laevis sperm is critical and needs special conditions of warm temperature and a relatively long period of time. Previously, thawing at 30 8C for about 10 sec was used to thaw Xenopus semen diluted in sucrose diluent without addition of penetrating cryoprotectant and frozen in Eppendorf tubes [9]. Generally, a successful freezing and thawing protocol is a complex procedure and depends not only on freezing and thawing rates but also on the freezing diluent and cryoprotectants used [5].
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In our results, MIS as a sperm diluent was superior to 300 mmol/L of either sucrose or glucose diluents when mixed with 5% DMSO. The previously published cryopreservation protocols use either sucrose [8,9] or 50% of fetal bovine serum [8] as extender base with or without penetrating cryoprotectants. In contrast with the current results, in the cane toad (Bufo marinus), the organic diluent sucrose resulted in higher cryopreservation success than that of inorganic salt solution [2]. Sucrose and glucose must be considered as nonpenetrating cryoprotectants that dehydrate sperm during freezing and thawing processes, lower the extracellular ionic strength, and may provide protection to sperm membranes [14]. In our cryopreservation protocol, 15 103 to 16 103 sperm were used to fertilize an egg. With this ratio, a fertility of 68.7% was obtained in fresh semen control and of 48.4% in semen frozen in MIS containing 5% DMSO and 73 mmol/L sucrose. Previous studies used higher sperm-to-egg ratios. Sargent and Mohun [9] have used a ratio of about 1:1 105 (egg:sperm) to obtain just 50% fertilization. However, Hollinger and Corton [12] used a dose of 3.3 106 sperm/mL to have about 50% fertilization rate, but the exact number of eggs used in their fertilization experiments has not been mentioned. In accordance with previous studies [9,12], sperm motility of the fresh sperm was about 50%. The postthaw motility rate obtained in the current study was 18% to 42%, whereas only 5% to 10% was obtained by Sargent and Mohun [9]. This difference might be due to the fact that these authors did not use a penetrating cryoprotectant, and, consequently, more sperm damage occurred during the freezing and thawing process. Additionally, a higher sperm:egg ratio was necessary in the former study [9] than that in our experiments proving that the penetrating cryoprotectant DMSO has a positive effect on the cryopreservation of X. laevis sperm. In conclusion, dilution of testicular sperm 6.5 106 to 8 106/mL in MIS containing 5% DMSO and 73 mmol/L sucrose, then freezing in the vapor of liquid nitrogen at a freezing rate of 20 to 25 8C/min and
thawing at room temperature for 40 sec is a reliable freezing and thawing protocol for X. laevis sperm. Acknowledgments This research was supported by the Austrian FWF, Der Wissenschaftsfonds, project no. P20109-B17. References [1] Browne PK, Clulow J, Mahony M, Clark A. Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology 1998;37:339–45. [2] Browne PK, Mahony M, Clulow J. A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) sperm. Cryobiology 2002;44:251–7. [3] Browne PK, Clulow J, Mahony M. The effect of saccharides on the post-thaw recovery of cane toad (Bufo marinus) sperm. Cryo Lett 2002;23:121–8. [4] Michael SF, Jones C. Cryopreservation of sperm of the terrestrial Puerto Rican frog, Eleutherodactylus coqui. Cryobiology 2004;48:90–4. [5] Holt WV. Basic aspects of frozen storage of semen. Anim Reprod Sci 2000;62:3–22. [6] Beesley SG, Costanzo JP, Lee RE. Cryopreservation of sperm from freeze-tolerant and -intolerant anurans. Cryobiology 1998;37:155–62. [7] Browne PK, Clulow J, Mahony M. The short term storage and cryopreservation of sperm from hylid and myobatrachid frogs. Cryo Lett 2002;23:129–36. [8] Buchholz DR, Fu L, Shi YB. Cryopreservation of Xenopus transgenic lines. Mol Reprod Dev 2004;67:65–9. [9] Sargent MG, Mohun TJ. Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis. Gensis 2005;41:41–6. [10] Lahnsteiner F, Mansour N. Protocols for the cryopreservation of Salmonidae semen, Lota lota (Gadidae) and Esox lucius (Esocidae). In: Methods in Reproductive Aquaculture, Marine and Freshwater Species. CRC Press, Taylor & Francis Group; 2008. p. 373–84. [11] Garner DL, Pinkel D, Johnson LA, Pace MM. Assessment of sperml fuction using dual fluorescent staining and flow cytometric analyses. Biol Reprod 1986;34:127–38. [12] Hollinger TG, Corton GL. Artificial fertilization of gametes from south African clawed frog, Xenopus laevis. Gamete Res 1980;3: 45–57. [13] Kouba AJ, Vance CK, Frommeyer MA, Roth TL. Structural and functional aspects of Bufo americanus sperm: effects of inactivation and reactivation. J Exp Zool 2003;295A:172–82. [14] Gao DC. Mechanisms of cryoinjury in living cells. ILAR J 2000;41:187–96.