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  Supporting the Sustainable Management of Amphibian and Reptile Biodiversity

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Amphibian Sperm Cryopreservation Reference List

Here we list articles that concern amphibian sperm cryopreservation by date, then by author) with notes of milestones in research. Some of these articles will also be listed in "Short-term Storage of Amphibian Sperm and Oocytes". 

Sperm can be sampled from testis in all amphibians, or by hormonal induction, which does not require the sacrifice of males.

The sacrifice of males is often a lesser ethical and conservation problem with threatened species, and particularly critically endangered species, as when they are reproduced in Conservation Breeding Programs (CBPs) many hundreds are available and often do not have habitat for release. Under current conventions they often cannot be given to private individuals and so are killed.

Amphibian and Conservation journal is working toward improved management of threatened species in CBPs including the rights of privates to be a part of CBPs and the release of surplus amphibians to privates.

With externally fertilising salamanders such as giant salamanders sperm is sampled from semen as in fish. The males of internally fertilising salamanders release sperm in spermatophores which are then taken up by females.

The ultimate achievement of the use of cryopreserved hormonally induced sperm for fertilisation of anuran oocytes was by Edith Gakova's team at the Institute of Cell Biophysics, Russian Federation.

The frontier in amphibian cryobiology is now with salamanders led for internally fertilising salamanders by Chester Figiel, National Fish Strain Registry, USA, and for cryptobranchids by Dale McGinnity, Nashville Zoo, USA. 

Recovery after cryopreservation is assessed by membrane integrity, motility, DNA integrity, and ultimately by fertilisation. Below we have listed the type of sperm sampling and the assessment of recovery.

By date

Dates are publication dates and consequently after the time of research.

1946 until 1973 - Early work - methods for both cryopreservation and recovery vague. 

Rostrand J. 1946. Biologie experimentale – glycerine et resistance du sperme ex basses temperatures. Comptes Rendus de L’Académie des Sciences Série III 222:1524-1525. Testicular sperm Recovery vague

Barton HL, Guttman SI 1972. Low temperature preservation of toad spermatozoa (Genus Bufo). Texas Journal of Science. 23:363-370. Testicular sperm Recovery vague

>>>

1996 to 1997 - anuran (frog or toad) sperm first cryopreserved with subsequent fertilisation. These articles were difficult to find or access in the west.

Kaurova S. A., Chekurova N. R., Melnikova E. V., Uteshev V. K., and Gakhova E. N. (1996), “Cryopreservation of frog Rana temporaria sperm without loss of fertilizing capacity,” in: Genetic Resource Conservation. Proc. of XIV Working Meeting, May 13 – 15, 1996, Pushchino [in Russian], pp. 106 - 108. Testicular sperm Fertilisation

Kaurova S. A., Uteshev V. K., Chekurova N. R., and Gakhova E. N. (1997), “Cryopreservation of testis of frog Rana temporaria,” Infusionsther. Transfusionsmed. 24(5):78- 79. Testicular sperm Fertilisation

>>>

1998 - physiological studies of basic cryobiology using freeze tolerant frogs. 

Beesley SG, Jon P. Costanzo JP, Lee, Jr. RE. 1998. Cryopreservation of spermatozoa from freeze-tolerant and-intolerant anuran's. Cryobiology 37(2):155-162. Testicular sperm Membrane integrity

Costanzo JP, Mugnano JA, Wehrheim HM. 1998. Osmotic and freezing tolerance in spermatozoa of freeze-tolerant and -intolerant frogs. American Journal of Physiology 44:713-719. Testicular sperm  Membrane integrity

>>>

1998 - Mugnano, Costanzo, and Beesley directly study amphibian sperm cryopreservation using membrane staining as a metric for viability.

Mugnano JA, Costanzo JP, Beesley SG, Lee Jr RE. 1998. Evaluation of glycerol and dimethyl sulfoxide for the cryopreservation of spermatozoa from the wood frog (Rana sylvatica). Cryo-Letters. 19:249-254. Testicular sperm Membrane integrity

>>>

1998 to 2002 - The University of Newcastle, Australia, provides PhD scholarship for cryopreservation of amphibian sperm  - Application of technologies developed in aquaculture to amphibian sperm cryopreservation and a series of articles using testicular sperm from the introduced pest (in Australia) species the Cane toad (Bufo marinus) are published.

Browne RK. Clulow J. Mahony M. Clark A. 1998. Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology. 37(4):339-345. Testicular sperm  Fertilisation

Browne RK. Mahony M. Clulow J. 2002. A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) sperm. Cryobiology. 44(3):251-257. Testicular sperm Motility

Browne RK. Davis J. Pomering M. Clulow J. 2002. Storage of cane toad (Bufo marinus) sperm for 6 days at 0ºC with subsequent cryopreservation. Reproduction, Fertility and Development. 14(5):267-273. Testicular sperm Motility

Browne, R.K., Clulow, J., Manony, M., 2002. The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. Cryo Letters 23(2):129-136. Testicular sperm Motility

Browne RK. Clulow J. Mahony M. 2002. The effect of saccharides on the post-thaw recovery of cane toad (Bufo marinus) spermatozoa. Cryo Letters. 23(2):121-128. Testicular sperm Motility

>>>

2004 to 2010 - a series of studies test and apply cryopreservation methods to an extended range of species; commercial Xenopus species and Eleutherodactylus coqui. Sperm still generally sampled by testes maceration with fertilisation only achieved from sperm from testes maceration.

Hopkins BK. Herr C. 2004. Cryopreservation of frog (Rana pipiens) sperm collected by non-lethal methods. Canadian Journal of Zoology. 82:555–563. Hormonal Induction Membrane integrity

Michael SF, Jones C. 2004. Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui. Cryobiology. 48(1):90-94. Membrane integrity

Buchholz D, Li F, Shi Y. 2004. Cryopreservation of Xenopus transgenic lines.  Molecular Reproduction and Development 67:65-69. Testicular sperm Intracytoplasmic Sperm Injection (ICSI)

Sargent MG. Mohun TJ. 2005. Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis. Genesis. 41:41−46. Testicular Sperm Testicular sperm Fertilisation

Mansour N, Lahnsteiner F, Robert A, Patzer RA. 2009. Successful cryopreservation protocol for the sperm of African Clawed Frog, Xenopus laevis. Theriogenology. 72:1221-1228. Testicular Sperm Fertilisation

Mansour N, Lahnsteiner F, Patzner R A. 2009. Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm. Theriogenology. 72:1221-1228. Testicular Sperm Fertilisation

Mansour N, Lahnsteiner F, Patzner RA. 2010. Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria. Theriogenology. 74:724-732. Testicular sperm Fertilisation

>>>

2010. The Institute of Cell Biophysics, Russian Federation, achieves the ultimate goal of amphibian sperm cryopreservation by high rates of fertility from hormonally induced sperm.

Shishova NV, Uteshev VK., Kaurova SA., Browne RK., Gakhova EN. 2010. Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species. Theriogenology. 75(2):220-232. Hormonally induced sperm Fertilisation

>>>

2010 to 2011. Cryopreservation of salamander sperm begins - Nashville Zoo samples sperm from captive and wild North American giant salamanders (Cryptobranchus alleganiensis) and immediately achieves high rates of motility and sperm integrity. However, another research team finds poor rates of recovery of sperm morphology are shown with the sperm of the Critically Endangered, Chinese giant salamander (Andrias davidianus)

Michigan State University. Freezing, preserving sperm vital to saving ‘snot otter’ salamanders. 2010.  http://msutoday.msu.edu/news/2010/freezing-preserving-sperm-vital-to-saving-/#sthash.QHR70uqE.dpuf   Sperm from semen Motility

National Geographic. 2010. “Snot Otter” Sperm to save Giant Salamander. http://news.nationalgeographic.com/news/2010/08/100820-hellbenders-snot-otters-sperm-amphibians-science-environment/   Sperm from semen Motility

Peng L. Yue YM, Liu Y. 2011. Effect of cryopreservation and short-term storage of Chinese giant salamander sperm. Acta Hydrobiologica Sinica 35:325-332. Sperm from semen Motility

>>>

2013. Applying techniques from Shishova (2010) low (Langhorne et al.) to moderate (Uteshev et al.) rates of fertilisation are shown for hormonally induced sperm for a toad and a frog species.

Langhorne CJ, Calatayud NE, Kouba AJ, Feugang JM, Vance CK, Willard ST. 2013. Cryoconservation: Successful sperm cryopreservation and developmental outcomes using endangered North American amphibians. Cryobiology. 67(3):405. Hormonally induced sperm Fertilisation

Uteshev VK, Shishova  N, Kaurova  S, Manokhin A, Gakhova E. 2013. Collection and cryopreservation of hormonally induced sperm of pool frog (Pelophylax lessonae). Russian Journal of Herpetology. 20(2): http://www.folium.ru/rjh/index.php/rjh/article/view/528   Hormonally induced sperm Fertilisation

Figiel CR Jr. 2013. Cryopreservation of sperm from the axolotl Ambystoma mexicanum: Implications for conservation. Herpetological Conservation and Biology. 8(3):748-755.

By author

Barton HL, Guttman SI 1972. Low temperature preservation of toad spermatozoa (Genus Bufo). Texas Journal of Science. 23:363-370.

Beesley SG, Jon P. Costanzo JP, Lee, Jr. RE. 1998. Cryopreservation of spermatozoa from freeze-tolerant and-intolerant anurans. Cryobiology 37(2):155-162.

Browne RK. Clulow J. Mahony M. Clark A. 1998. Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology. 37(4):339-345.

Costanzo JP, Mugnano JA, Wehrheim HM. 1998. Osmotic and freezing tolerance in spermatozoa of freeze-tolerant and -intolerant frogs. American Journal of Physiology 44:713-719.

Browne RK. Mahony M. Clulow J. 2002. A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) sperm. Cryobiology. 44(3):251-257.

Browne RK. Davis J. Pomering M. Clulow J. 2002. Storage of cane toad (Bufo marinus) sperm for 6 days at 0ºC with subsequent cryopreservation. Reproduction, Fertility and Development. 14(5):267-273.

Browne, R.K., Clulow, J., Manony, M., 2002. The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. Cryo Letters 23(2):129-136.

Browne RK. Clulow J. Mahony M. 2002. The effect of saccharides on the post-thaw recovery of cane toad (Bufo marinus) spermatozoa. Cryo Letters. 23(2):121-128.

Buchholz D, Li F, Shi Y. 2004. Cryopreservation of Xenopus transgenic lines.  Molecular Reproduction and Development 67:65-69.

Figiel CR Jr. 2013. Cryopreservation of sperm from the axolotl Ambystoma mexicanum: Implications for conservation. Herpetological Conservation and Biology. 8(3):748-755.

Hopkins BK. Herr C. 2004. Cryopreservation of frog (Rana pipiens) sperm collected by non-lethal methods. Canadian Journal of Zoology. 82:555–563.

Kaurova S. A., Chekurova N. R., Melnikova E. V., Uteshev V. K., and Gakhova E. N. (1996), “Cryopreservation of frog Rana temporaria sperm without loss of fertilizing capacity,” in: Genetic Resource Conservation. Proc. of XIV Working Meeting, May 13 – 15, 1996, Pushchino [in Russian], pp. 106 - 108.

Kaurova S. A., Uteshev V. K., Chekurova N. R., and Gakhova E. N. (1997), “Cryopreservation of testis of frog Rana temporaria,” Infusionsther. Transfusionsmed. 24(5):78- 79.

Langhorne CJ, Calatayud NE, Kouba AJ, Feugang JM, Vance CK, Willard ST. 2013. Cryoconservation: Successful sperm cryopreservation and develop-mental outcomes using endangered North American amphibians. Cryobiology. 67(3):405.

Michael SF, Jones C. 2004. Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui. Cryobiology. 48(1):90-94.

Mansour N, Lahnsteiner F, Robert A, Patzer RA. 2009. Successful cryopreservation protocol for the sperm of African Clawed Frog, Xenopus laevis. Theriogenology. 72:1221-1228.

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 RA. 2010. Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria. Theriogenology. 74:724-732.

Michigan State University. Freezing, preserving sperm vital to saving ‘snot otter’ salamanders. 2010.  http://msutoday.msu.edu/news/2010/freezing-preserving-sperm-vital-to-saving-/#sthash.QHR70uqE.dpuf

Mugnano JA, Costanzo JP, Beesley SG, Lee Jr RE. 1998. Evaluation of glycerol and dimethyl sulfoxide for the cryopreservation of spermatozoa from the wood frog (Rana sylvatica). Cryo-Letters. 19:249-254.

National Geographic. 2010. “Snot Otter” Sperm to save Giant Salamander. http://news.nationalgeographic.com/news/2010/08/100820-hellbenders-snot-otters-sperm-amphibians-science-environment/

Peng L. Yue YM, Liu Y. 2011. Effect of cryopreservation and short-term storage of Chinese giant salamander sperm. Acta Hydrobiologica Sinica 35:325-332.

Rostrand J. 1946. Biologie experimentale – glycerine et resistance du sperme ex basses temperatures. Comptes Rendus de L’Académie des Sciences Série III 222:1524-1525.

Sargent MG. Mohun TJ. 2005. Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis. Genesis. 41:41−46.

Shishova NV, Uteshev VK., Kaurova SA., Browne RK., Gakhova EN. 2010. Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species. Theriogenology. 75(2):220-232.

Uteshev VK, Shishova  N, Kaurova  S, Manokhin A, Gakhova E. 2013. Collection and cryopreservation of hormonally induced sperm of pool frog (Pelophylax lessonae). Russian Journal of Herpetology. 20(2): http://www.folium.ru/rjh/index.php/rjh/article/view/528  

 

 

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 Dr Robert Browne

Dr. Robert Browne Chairperson

Dr. Browne established the Internet based ARC in 2011 and expanded it globally in 2013. Robert is committed to achieving the ARC's goal to provide for the sustainable management of amphibians and reptiles. He has a wide international experience in herpetological conservation and has published over 40 scientific articles on amphibian and reptile conservation. see Biography