Action: Freeze sperm or eggs for future use
- Nine replicated studies (including three controlled studies) in Austria, Australia, Russia, the UK and USA found that following freezing frog and toad sperm viability depended on species and/or cryoprotectant used. One found that although sperm viability was low following freezing, it could be frozen for up to 58 weeks. Five of the studies and one additional replicated study in Australia found that following freezing, viability of sperm and in one case eggs, also depended on storage temperature, storage method, freezing or thawing rate.
- Seven replicated studies (including three controlled studies) in Austria, Australia, the UK and USA found that frog and toad sperm viability was greatest following freezing with the cryoprotectant dimethyl sulfoxide, glycerol, sucrose or dimethyl formamide.
Conservation breeding programmes are being used more frequently for threatened amphibian species. However, captive breeding often results in loss of genetic variation. This can mean that animals that were bred for release back in to the wild have reduced fitness. Freezing, or ‘cryopreservation’, of sperm and eggs, allows them to be stored until they are needed. Gene banks can therefore be created for amphibians ensuring that species’ genetic variation is preserved. It also means that the number of a particular species needed in captivity can be reduced and genes can be swapped between captive facilities. Fewer animals in captivity means that fewer amphibians need to be taken from the wild. Freezing can damage cells and so a cryoprotectant, such as dimethyl sulphoxide or glycerol is usually required to protect the cells.
Supporting evidence from individual studies
A replicated study in 1997–1998 of captive amphibians in the USA (Beesley, Costanzo & Lee 1998) found that recovery of viable sperm following freezing was significantly lower for leopard frogs Rana pipiens and American toads Bufo americanus compared to freeze-tolerant wood frogs Rana sylvatica. Sperm recovery was 59%, 48% and 81% respectively. Survival and viability of wood frog sperm was significantly greater using the cryoprotectant dimethyl sulfoxide and supplement of fetal bovine serum (survival: 96%; viability: 45%) than the other three protectants with glutathione (survival: 34–54%; viability with methanol: 10%) or without protectants (survival: 44–54%; viability with methanol: 16%). Testes from wild or commercially obtained males were macerated in a buffer solution. Sperm solutions from wood frogs were mixed with 0.5 M cryoprotectant (dimethyl sulfoxide, methanol, glycerol or ethylene glycol), a supplement (fetal bovine serum or glutathione) or a combination of these. Using the most successful cryopreservation treatment, sperm from each species was incubated on ice for 15 minutes, then frozen to -80°C for 1 hour (rate: 130°C/minute). Thawing was in warm water.
A replicated study of cane toads Bufo marinus in Australia (Browne et al. 1998) found that sperm retained motility and fertilizing capacity following cryopreservation, provided that cryoprotectants were used. Sperm frozen in sucrose alone retained no motility. The highest rates of recovery of sperm motility and fertilizing capacity were observed following storage with 15% dimethyl sulfoxide (motility: 69%; fertilization: 61%) and 20% glycerol (motility: 58%; fertilization: 81%). However, storage with different concentrations of dimethyl sulfoxide or glycerol all showed some motility (dimethyl sulfoxide: 35–69%; glycerol: 15–58%) and fertilizing capacity (dimethyl sulfoxide: 33-61%; glycerol: 15–81%). Sperm from macerated testes of four toads were cryopreserved in suspensions of 10% sucrose alone or with 10, 15 or 20% dimethyl sulfoxide or glycerol. Suspensions were cooled slowly to −196 °C. Sperm was thawed in air and tested within five minutes for motility and fertilization capacity (eggs from two females).
A replicated, controlled study in 1997 of captive wood frogs Rana sylvatica in the USA (Mugnano et al. 1998) found that some sperm recovered following freezing for up to 58 weeks provided that the cryoprotectant dimethyl sulfoxide or glycerol was used. Sperm viability was significantly reduced after freezing compared to chilling for 1–30 hours with glycerol (13–17 vs 50–55%) or dimethyl sulfoxide (10–13 vs 60%). However, viability was zero without a cryoprotectant. Viability was not significantly affected by cryoprotectant concentration. There was no significant difference in viability following freezing for 1–30 hours compared to 58 weeks. Whole testes frozen in dimethyl sulfoxide had significantly higher sperm viability than those in glycerol (14 vs 5%). When chilled, sperm in had lower survival than controls and so glucose was excluded. Testes from five wild-caught frogs were macerated in a buffer. Sperm solutions from each were mixed with glucose (2 M), glycerol or dimethyl sulfoxide (1.5 or 3 M), chilled for 20 minutes and then half were frozen to -80°C in ethanol/dry ice (rate 130°C/minute) for 1–30 hours or 58 weeks. Thawing was in a 30°C water bath. Four intact testes were frozen at −80°C in glucose or dimethyl sulfoxide for five days.
A replicated study of captive cane toads Bufo marinus in Australia (Browne, Clulow & Mahony 2001) found that storage method and temperature affected sperm and egg viability. Sperm stored in testes showed greater than 50% motility for seven days at 0°C and five days at 4°C. By day 15 only sperm stored at 0°C showed any motility (3%). In suspension, the longest retention of motility and fertilizing capacity was following storage in concentrated (1:1 dilution) anaerobic suspensions (up to 25–30 days). However, fertilization rates were significantly higher following storage in 1:5 dilution (day 5: 85% vs 55% for other concentrations). Egg viability was significantly higher following storage at 15°C compared to other temperatures (8 hours: 90% vs 0–60%). Storage at 5°C resulted in a decline to 0% viability after two hours. Sperm from wild toads were stored in intact testes at 0 or 4°C for 15 days (n = 6/treatment) or in suspension (macerated testes; n = 24) with Simplified amphibian Ringer solution at 0°C for 30 days. Dilutions were 1:1, 1:5 or 1:10 (testes:solution) and storage tubes were either opened or sealed. Immediately after ovulation, eggs from three females were stored in simplified amphibian Ringer solution at 5, 10, 15, 20 and 25°C (1,500 eggs/female). Fertilization rate was monitored up until 12 hours.
A replicated study of captive frogs in Australia (Browne, Clulow & Manony 2002) found that following storage at −80°C, sperm from tree frog species (Hylidae) showed greater motility than myobatrachid species (0–100 vs 1–20%). For tree frogs, sperm storage at −80°C in 15% dimethyl sulfoxide resulted in the highest motility (15%: 45–100%; 20%: 80%; glycerol 15%: 0–100%; glycerol 20%: 10–87%). Striped marsh frog Limnodynastes peronii sperm maintained higher motility when stored at 0°C in suspension compared to testes (three days: 41 vs 6%). Motility of whistling treefrog Litoria verreauxi sperm did not differ with storage method (three days: 83 vs 86%; six days: 41 vs 40%). Recovery of tree frog sperm did not differ with testes weight. Sperm from six frogs of two species were stored in intact testes and sperm from four frogs of three species were stored in suspension (macerated testes) for three or six days at 0°C. Sperm from nine tree frog and four myobatrachid species were cryopreserved in suspensions of 10% sucrose with dimethyl sulfoxide or glycerol (15 or 20%). Sperm were frozen slowly to −80 °C, thawed in air and observed for three minutes.
A replicated study of captive Puerto Rican frogs Eleutherodactylus coqui in the USA (Michael & Jones 2004) found that cryopreservation of sperm was successful with a cryoprotectant and fetal bovine serum (FSB). FBS alone resulted in only 8% viability. However, sperm viability was significantly higher with addition of sucrose or glycerol to FBS (sucrose: 28%; glycerol: 30%; dimethyl sulfoxide: 20%). Viability did not differ significantly with dimethyl sulfoxide. Prior to freezing sperm had a viability of 56% and so normalized viabilities were: 14% for FBS alone and 35%, 50% and 54% with added dimethyl sulfoxide, sucrose and glycerol respectively. Testes of wild caught frogs were macerated in solution. Sperm was then mixed with a cryoprotectant solution (six replicates/treatment): heat inactivated FBS alone, FBS with 2M sucrose, FBS with 2M glycerol or FBS with 2M dimethyl sulfoxide. Mixtures were frozen at −80°C for 24 hours and then thawed rapidly in a 20°C water bath. Fluorescent dye was used to examine sperm.
A replicated, controlled study in 2004 of captive African clawed frog Xenopus laevis and western clawed frog Xenopus tropicalis in the UK (Sargent & Mohun 2005) found that although sperm lost viability following freezing to −80°C, sufficient survived to fertilize eggs. Relative sperm motility after freezing, compared to a control was 30–40% (141–178 days) for African clawed frog and 39–70% (22–182 days) for western clawed frog. Optimum motility was obtained with a cooling rate of 10°C/minute in 0.2 m sucrose. Sodium bicarbonate was less effective and pentoxyfylline not effective at protecting sperm during a freeze-thaw cycle. Frozen sperm half-life was approximately one year for both species. Fertilization efficiency was greater in sodium chloride solution concentrations of 0.4 compared to 0.1 for western clawed frogs. Fertilization was similar with varying concentrations (4–40 mM) for African clawed frogs. Testes were macerated in sodium chloride solutions. Cryoprotectants (with egg yolk) were: 0.2, 0.4 or 0.6M sucrose, sodium bicarbonate or pentoxyfylline. Sperm was frozen to −80°C at rates of 0.5–50°C/minute. Samples were defrosted rapidly in a water bath at 30°C. Fresh eggs (40–100/test) were fertilized and success recorded after 5 hours.
A replicated study in 2008 of captive African clawed frog Xenopus laevis in Austria (Mansour, Lahnsteiner & Patzner 2009) found that the most effective cryopreservation protocol was sperm in motility-inhibiting saline (MIS) with 5% dimethyl sulfoxide and sucrose, frozen 10 cm above liquid nitrogen and thawed at room temperature for 40 seconds. Sperm motility and viability was significantly higher following incubation (>10 mins) at 4°C in 10% dimethyl sulfoxide (motility: 40–50%; viability: 65–75%) than in 5% glycerol (10–30%; 15–55%) or 10% methanol (0–15%; 0–35%). Sperm in 10% dimethyl sulfoxide frozen 10 cm above liquid nitrogen (motility: 20%; viability: 50%) and thawed at room temperature for 40 seconds (20%; 48%) had significantly higher motility and viability than sperm frozen 5 cm (1%; 8%) or 8 cm (8%; 16%) above liquid nitrogen and thawed at 5, 25, or 30°C for 10, 15 or 60 seconds respectively (1–8%; 6–20%). Sperm frozen in MIS with 5% dimethyl sulfoxide resulted in higher hatching rate (29%) than sperm frozen in sucrose or glucose (300 mmol/L) containing 5% or 10% dimethyl sulfoxide (6–19%) or in MIS containing 10% dimethyl sulfoxide (9%). Viability did not differ (24–38%). Addition of 73 mmol/L sucrose to MIS with 5% dimethyl sulfoxide increased sperm motility (18 to 46%) and hatching rate (29 to 48%). Testes from three males were macerated and tested/treatment. Fertilization was tested using 25–30 eggs at 18°C.
A replicated study in 2009 of captive European common frogs Rana temporaria in Austria (Mansour, Lahnsteiner & Patzner 2010) found that the most effective cryopreservation protocol was sperm in motility-inhibiting saline (MIS) with 5% glycerol, 2.5% sucrose and 5% hen egg yolk, frozen 10 cm above liquid nitrogen and thawed at 22 °C for 40 seconds. Sperm motility was maintained following incubation for 40 minutes at 4°C in MIS with 10% dimethyl sulfoxide (71%), 5% glycerol (69%) or 10% methanol (59%), but not 10% propandiol (0%). When frozen, in combination with sucrose, dimethyl sulfoxide resulted in significantly greater sperm motility and viability (10%; 42% respectively) than glycerol (8%; 25%). With MIS, motility and viability was similar with either dimethyl sulfoxide (13%; 27%) or glycerol (10%; 29%). Sperm frozen in MIS with sucrose and methanol had no motility. Sperm frozen 5 cm above liquid nitrogen had no motility, whereas at 10 cm motility was 30–35%. Addition of 5% (vs 10%) egg yolk and 2.5% sucrose to MIS with glycerol significantly increased hatching rate compared to all other treatments (23 vs 2–12%). Motility and viability did not differ. Testes from wild males were macerated (3/treatment). Sperm was frozen in liquid nitrogen. Fertilization was tested using 25–30 eggs.
A replicated, controlled study in 2009 of European common frogs Rana temporaria in the Moscow Region, Russia (Shishova et al. 2010) found that recovery of sperm after cryopreservation was high with certain cryoprotectants. Sperm motility was significantly greater with the cryoprotectant dimethyl formamide (motility: 65%; fertilization: 90%) compared to dimethyl sulphoxide (36–44%; 82–90%). High concentrations of dimethyl sulphoxide (6 vs 2–4%) significantly reduced hatching (54 vs 80%) and larval survival (49 vs 70–76%), but not fertilization (80 vs 86–90%). Motility-inhibiting saline and glycerol cryoprotectant resulted in low motility (28%) and zero fertility. Tris buffer in cryoprotectants did not significantly increase motility (43–48 vs 45%) or fertilization (70–81 vs 84%). Maximum fertilization was achieved with spermic urine from hormonally induced males (luteinizing hormone-releasing hormone) at concentrations of 15 x 106/ml (93%). Spermic urine or macerated testes from wild frogs were mixed with simplified amphibian Ringer solution or saline and cryodiluents: 2–12% dimethyl sulphoxide or 12% dimethyl formamide or motility-inhibiting saline and 5% glycerol, with 2.5, 6.5 or 10% sucrose with or without Tris buffer or 5–10% egg yolk. Spermic urine (1.0 x 108 cell/ml) and cryodiluents were frozen at 5–7°C/minute and then stored in liquid nitrogen. Thawing was in a 40°C water bath. Spermic urine, sperm from macerated testes (different concentrations) or thawed sperm in cryodiluents were added to eggs from hormonally induced wild females. Fertilization was assessed after 4–6 hours.
- Beesley S.G., Costanzo J.P. & Lee R.E. (1998) Cryopreservation of spermatozoa from freeze-tolerant and intolerant anurans. Cryobiology, 37, 155–162
- Browne R.K., Clulow J., Mahony M. & Clark A. (1998) Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology, 37, 339-345
- Mugnano J.A., Costanzo J.P., Beesley S.G. & Lee R.E. (1998) Evaluation of glycerol and dimethyl sulfoxide for the cryopreservation of spermatozoa from the wood frog (Rana sylvatica). Cryo-Letters, 19, 249-254
- Browne R.K., Clulow J. & Mahony M. (2001) Short-term storage of cane toad (Bufo marinus) gametes. Reproduction, 121, 167-173
- Browne R.K., Clulow J. & Manony M. (2002) The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. Cryo Letters, 23, 129-136
- Michael S.F. & Jones C. (2004) Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui. Cryobiology, 48, 90-94
- Sargent M.G. & Mohun T.J. (2005) Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis. Genesis, 41, 41–46
- Mansour N., Lahnsteiner F. & Patzner R.A. (2009) Optimization of the cryopreservation of African clawed frog (Xenopus laevis>) sperm. Theriogenology, 72, 1221-1228
- Mansour N., Lahnsteiner F. & Patzner R.A. (2010) Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria. Theriogenology, 74, 724-732
- Shishova N.R., Uteshev V.K., Kaurova S.A., Browne R.K. & Gakhova E.N. (2010) Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species. Theriogenology, 75, 220-232