Generation of reproductively mature offspring from the endangered green and golden bell frog Litoria aurea using cryopreserved spermatozoa
Rose Upton
A B G , Simon Clulow B C , Natalie E. Calatayud B D E , Kim Colyvas F , Rebecca G. Y. Seeto A , Lesley A. M. Wong A , Michael J. Mahony A B and John Clulow A B
+ Author Affiliations
- Author Affiliations
A The Conservation Biology Research Group, School of Environmental and Life Sciences, The University of Newcastle, Callaghan, NSW 2308, Australia.
B FAUNA Research Alliance, PO Box 5092, Kahibah, NSW 2290, Australia.
C Centre for Conservation Ecology and Genomics, Institute for Applied Ecology, University of Canberra, Bruce, ACT 2617, Australia.
D Taronga Institute of Science and Learning, Taronga Conservation Society Australia, Taronga Western Plains Zoo, Dubbo, NSW 2830, Australia.
E San Diego Zoo Global-Beckman Center for Conservation Research, 15600 San Pasqual Valley Road, Escondido, CA 92027, USA.
F School of Mathematical and Physical Sciences, University of Newcastle, Callaghan, NSW 2308, Australia.
G Corresponding author. Email: rose.upton@uon.edu.au
Reproduction, Fertility and Development 33(9) 562-572 https://doi.org/10.1071/RD20296
Submitted: 16 November 2020 Accepted: 25 February 2021 Published: 6 April 2021
Journal Compilation © CSIRO 2021 Open Access CC BY
Abstract
Amphibians are becoming increasingly reliant on captive breeding programs for continued survival. Assisted reproductive technologies including gamete cryopreservation and IVF can help reduce costs of breeding programs, provide insurance against extinction and assist genetic rescue in wild populations. However, the use of these technologies to produce reproductively mature offspring has only been demonstrated in a few non-model species. We aimed to optimise sperm cryopreservation in the threatened frog Litoria aurea and generate mature offspring from frozen–thawed spermatozoa by IVF. We tested three concentrations (1.4, 2.1 and 2.8 M) of the cryoprotectants dimethylsulfoxide (DMSO) and glycerol with 0.3 M sucrose. Using DMSO was more likely to result in recovery of sperm motility, vitality and acrosome integrity than glycerol, regardless of concentration, with forward progressive motility being most sensitive to damage. The lowest concentrations of 1.4 and 2.1 M provided the best protection regardless of cryoprotectant type. Spermatozoa cryopreserved in 2.1 M DMSO outperformed spermatozoa cryopreserved in equivalent concentrations of glycerol in terms of their ability to fertilise ova, resulting in higher rates of embryos hatching and several individuals reaching sexual maturity. We have demonstrated that sperm cryopreservation and subsequent offspring generation via IVF is a feasible conservation tool for L. aurea and other threatened amphibians.
Keywords: anuran, artificial fertilisation, captive survival-assurance colonies, cryoconservation, genome resource bank, green and golden bell frog, Pelodryadidae, spermatozoa.
References
Abu Bakar, A., Bower, D. S., Stockwell, M. P., Clulow, S., Clulow, J., and Mahony, M. J. (2016). Susceptibility to disease varies with ontogeny and immunocompetence in a threatened amphibian. Oecologia 181, 997–1009.| Susceptibility to disease varies with ontogeny and immunocompetence in a threatened amphibian.Crossref | GoogleScholarGoogle Scholar | 27021312PubMed |
Bates, D., Mächler, M., Bolker, B., and Walker, S. (2015). Fitting Linear Mixed-Effects Models Using lme4. J. Stat. Softw. 67, 1–48.
| Fitting Linear Mixed-Effects Models Using lme4.Crossref | GoogleScholarGoogle Scholar |
Beesley, S. G., Costanzo, J. P., and Lee, R. E. (1998). Cryopreservation of spermatozoa from freeze-tolerant and -intolerant anurans. Cryobiology 37, 155–162.
| Cryopreservation of spermatozoa from freeze-tolerant and -intolerant anurans.Crossref | GoogleScholarGoogle Scholar | 9769166PubMed |
Berger, L., Speare, R., Daszak, P., Green, D. E., Cunningham, A. A., Goggin, C. L., Slocombe, R., Ragan, M. A., Hyatt, A. D., McDonald, K. R., Hines, H. B., Lips, K. R., Marantelli, G., and Parkes, H. (1998). Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America. Proc. Natl Acad. Sci. USA 95, 9031–9036.
| Chytridiomycosis causes amphibian mortality associated with population declines in the rain forests of Australia and Central America.Crossref | GoogleScholarGoogle Scholar | 9671799PubMed |
Bishop, P. J., Angulo, A., Lewis, J. P., Moore, R. D., Rabb, G. B., and Moreno, J. G. (2012). The Amphibian Extinction Crisis: What will it take to put the action into the Amphibian Conservation Action Plan? S.A.P.I.EN.S 5, 97–111.
Bower, D. S., Lips, K. R., Schwarzkopf, L., Georges, A., and Clulow, S. (2017). Amphibians on the brink. Science 357, 454.
| Amphibians on the brink.Crossref | GoogleScholarGoogle Scholar | 28774916PubMed |
Browne, R. K., Clulow, J., Mahony, M. J., and Clark, A. K. (1998). Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology 37, 339–345.
| Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm.Crossref | GoogleScholarGoogle Scholar | 9917350PubMed |
Browne, R. K., Clulow, J., and Mahony, M. J. (2002a). The effect of saccharides on the post-thaw recovery of cane toad (Bufo marinus) spermatozoa. Cryo Letters 23, 121–128.
| 12050780PubMed |
Browne, R. K., Clulow, J., and Mahony, M. J. (2002b). The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. Cryo Letters 23, 129–136.
| 12050781PubMed |
Browne, R. K., Davis, J., Clulow, J., and Pomering, M. (2002c). Storage of cane toad Bufo marinus sperm for 6 days at 0°C with subsequent cryopreservation. Reprod. Fertil. Dev. 14, 267–273.
| Storage of cane toad Bufo marinus sperm for 6 days at 0°C with subsequent cryopreservation.Crossref | GoogleScholarGoogle Scholar | 12467350PubMed |
Browne, R. K., Mahony, M. J., and Clulow, J. (2002d). A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) sperm. Cryobiology 44, 251–257.
| A comparison of sucrose, saline, and saline with egg-yolk diluents on the cryopreservation of cane toad (Bufo marinus) sperm.Crossref | GoogleScholarGoogle Scholar | 12237090PubMed |
Browne, R. K., Silla, A. J., Upton, R., Della-Togna, G., Marcec-Greaves, R., Shishova, N. V., Uteshev, V. K., Proano, B., Perez, O. D., Mansour, N., Kaurova, S. A., Gakhova, E. N., Cosson, J., Dyzuba, B., Kramarova, L. I., McGinnity, D., Gonzalez, M., Clulow, J., and Clulow, S. (2019). Sperm collection and storage for the sustainable management of amphibian biodiversity. Theriogenology 133, 187–200.
| Sperm collection and storage for the sustainable management of amphibian biodiversity.Crossref | GoogleScholarGoogle Scholar | 31155034PubMed |
Campbell, L., Bower, D. S., Clulow, S., Stockwell, M., Clulow, J., and Mahony, M. (2019). Interaction between temperature and sublethal infection with the amphibian chytrid fungus impacts a susceptible frog species. Sci. Rep. 9, 83.
| Interaction between temperature and sublethal infection with the amphibian chytrid fungus impacts a susceptible frog species.Crossref | GoogleScholarGoogle Scholar | 30643160PubMed |
Clulow, J., and Clulow, S. (2016). Cryopreservation and other assisted reproductive technologies for the conservation of threatened amphibians and reptiles: Bringing the ARTs up to speed. Reprod. Fertil. Dev. 28, 1116–1132.
| Cryopreservation and other assisted reproductive technologies for the conservation of threatened amphibians and reptiles: Bringing the ARTs up to speed.Crossref | GoogleScholarGoogle Scholar |
Clulow, J., Trudeau, V. L., and Kouba, A. J. (2014). Amphibian declines in the twenty-first century: Why we need assisted reproductive technologies. Adv. Exp. Med. Biol. 753, 275–316.
| Amphibian declines in the twenty-first century: Why we need assisted reproductive technologies.Crossref | GoogleScholarGoogle Scholar | 25091914PubMed |
Clulow, S., Gould, J., James, H., Stockwell, M., Clulow, J., and Mahony, M. (2018). Elevated salinity blocks pathogen transmission and improves host survival from the global amphibian chytrid pandemic: Implications for translocations. J. Appl. Ecol. 55, 830–840.
| Elevated salinity blocks pathogen transmission and improves host survival from the global amphibian chytrid pandemic: Implications for translocations.Crossref | GoogleScholarGoogle Scholar |
Clulow, J., Upton, R., Trudeau, V. L., and Clulow, S. (2019). Amphibian assisted reproductive technologies: moving from technology to application. In ‘Reproductive Sciences in Animal Conservation’. (Eds P. Comizzoli, J. L. Brown, and W. V. Holt) pp. 413–463. (Springer International Publishing: Cham.)
Daly, J., Zuchowicz, N., Nuñez Lendo, C. I., Khosla, K., Lager, C., Henley, M., Bischof, J., Kleinhans, F. W., Lin, C., Peters, E., and Hagedorn, M. (2018). Successful cryopreservation of coral larvae using vitrification and laser warming. Cryobiology 85, 161–162.
| Successful cryopreservation of coral larvae using vitrification and laser warming.Crossref | GoogleScholarGoogle Scholar |
Della Togna, G., Howell, L. G., Clulow, J., Langhorne, C. J., Marcec-Greaves, R., and Calatayud, N. E. (2020). Evaluating amphibian biobanking and reproduction for captive breeding programs according to the Amphibian Conservation Action Plan objectives. Theriogenology 150, 412–431.
| Evaluating amphibian biobanking and reproduction for captive breeding programs according to the Amphibian Conservation Action Plan objectives.Crossref | GoogleScholarGoogle Scholar | 32127175PubMed |
Gillespie, G. R., Roberts, J. D., Hunter, D., Hoskin, C. J., Alford, R. A., Heard, G. W., Hines, H., Lemckert, F., Newell, D., and Scheele, B. C. (2020). Status and priority conservation actions for Australian frog species. Biol. Conserv. 247, 108543.
| Status and priority conservation actions for Australian frog species.Crossref | GoogleScholarGoogle Scholar |
Gosner, K. L. (1960). A Simplified Table for Staging Anuran Embryos and Larvae with Notes on Identification. Herpetologica 16, 183–190.
Hinkson, K. M., Baecher, J. A., and Poo, S. (2019). Cryopreservation and hormonal induction of spermic urine in a novel species: The smooth-sided toad (Rhaebo guttatus). Cryobiology 89, 109–111.
| Cryopreservation and hormonal induction of spermic urine in a novel species: The smooth-sided toad (Rhaebo guttatus).Crossref | GoogleScholarGoogle Scholar | 31078579PubMed |
Howell, L. G., Frankham, R., Rodger, J. C., Witt, R. R., Clulow, S., Upton, R. M. O., and Clulow, J. (2020). Integrating biobanking minimises inbreeding and produces significant cost benefits for a threatened frog captive breeding programme. Conserv. Lett. , .
| Integrating biobanking minimises inbreeding and produces significant cost benefits for a threatened frog captive breeding programme.Crossref | GoogleScholarGoogle Scholar |
Khosla, K., Wang, Y., Hagedorn, M., Qin, Z., and Bischof, J. (2017). Gold Nanorod Induced Warming of Embryos from the Cryogenic State Enhances Viability. ACS Nano 11, 7869–7878.
| Gold Nanorod Induced Warming of Embryos from the Cryogenic State Enhances Viability.Crossref | GoogleScholarGoogle Scholar | 28702993PubMed |
Langhorne, C. J., Calatayud, N. E., Kouba, A. J., Feugang, J. M., Vance, C. K., and Willard, S. T. (2013). 026 Cryoconservation: successful sperm cryopreservation and develop-mental outcomes using endangered North American amphibians. Cryobiology 67, 405.
| 026 Cryoconservation: successful sperm cryopreservation and develop-mental outcomes using endangered North American amphibians.Crossref | GoogleScholarGoogle Scholar |
Lawson, B., Clulow, S., Mahony, M. J., and Clulow, J. (2013). Towards gene banking amphibian maternal germ lines: Short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii. PLoS One 8, e60760.
| Towards gene banking amphibian maternal germ lines: Short-term incubation, cryoprotectant tolerance and cryopreservation of embryonic cells of the frog, Limnodynastes peronii.Crossref | GoogleScholarGoogle Scholar | 23577155PubMed |
Lenth, R., Singmann, H., Love, J., Buerkner, P., and Herve, M. (2018) Emmeans: estimated marginal means, aka least-squares means. In ‘R package version 1.2.2’. Available at https://CRAN.R-project.org/package=emmeans [verified 10 March 2021].
Luyet, B. J., and Hodapp, E. L. (1938). Revival of Frog’s Spermatozoa Vitrified in Liquid Air. Proc. Soc. Exp. Biol. Med. 39, 433–434.
| Revival of Frog’s Spermatozoa Vitrified in Liquid Air.Crossref | GoogleScholarGoogle Scholar |
Mahony, M. J., Hamer, A. J., Pickett, E. J., McKenzie, D. J., Stockwell, M. P., Garnham, J. I., Keely, C. C., Deboo, M. L., O’Meara, J., and Pollard, C. J. (2013). Identifying conservation and research priorities in the face of uncertainty: a review of the threatened bell frog complex in eastern Australia. Herpetol. Conserv. Biol. 8, 519–538.
Mansour, N., Lahnsteiner, F., and Patzner, R. A. (2009). Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm. Theriogenology 72, 1221–1228.
| Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm.Crossref | GoogleScholarGoogle Scholar | 19766299PubMed |
Mansour, N., Lahnsteiner, F., and Patzner, R. A. (2010). Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria. Theriogenology 74, 724–732.
| Motility and cryopreservation of spermatozoa of European common frog, Rana temporaria.Crossref | GoogleScholarGoogle Scholar | 20537698PubMed |
Michael, S. F., and Jones, C. (2004). Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui. Cryobiology 48, 90–94.
| Cryopreservation of spermatozoa of the terrestrial Puerto Rican frog, Eleutherodactylus coqui.Crossref | GoogleScholarGoogle Scholar | 14969686PubMed |
Morrow, S., Gosálvez, J., López-Fernández, C., Arroyo, F., Holt, W. V., and Guille, M. J. (2017). Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa. Reprod. Fertil. Dev. 29, 1556–1566.
| Effects of freezing and activation on membrane quality and DNA damage in Xenopus tropicalis and Xenopus laevis spermatozoa.Crossref | GoogleScholarGoogle Scholar | 27692061PubMed |
Mugnano, J. A., Costanzo, J. P., Beesley, S. G., and 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.
O’Hanlon, S. J., Rieux, A., Farrer, R. A., Rosa, G. M., Waldman, B., Bataille, A., Kosch, T. A., Murray, K. A., Brankovics, B., Fumagalli, M., Martin, M. D., Wales, N., Alvarado-Rybak, M., Bates, K. A., Berger, L., Böll, S., Brookes, L., Clare, F., Courtois, E. A., Cunningham, A. A., Doherty-Bone, T. M., Ghosh, P., Gower, D. J., Hintz, W. E., Höglund, J., Jenkinson, T. S., Lin, C.-F., Laurila, A., Loyau, A., Martel, A., Meurling, S., Miaud, C., Minting, P., Pasmans, F., Schmeller, D. S., Schmidt, B. R., Shelton, J. M. G., Skerratt, L. F., Smith, F., Soto-Azat, C., Spagnoletti, M., Tessa, G., Toledo, L. F., Valenzuela-Sánchez, A., Verster, R., Vörös, J., Webb, R. J., Wierzbicki, C., Wombwell, E., Zamudio, K. R., Aanensen, D. M., James, T. Y., Gilbert, M. T. P., Weldon, C., Bosch, J., Balloux, F., Garner, T. W. J., and Fisher, M. C. (2018). Recent Asian origin of chytrid fungi causing global amphibian declines. Science 360, 621–627.
| Recent Asian origin of chytrid fungi causing global amphibian declines.Crossref | GoogleScholarGoogle Scholar | 29748278PubMed |
Pearl, E., Morrow, S., Noble, A., Lerebours, A., Horb, M., and Guille, M. (2017). An optimized method for cryogenic storage of Xenopus sperm to maximise the effectiveness of research using genetically altered frogs. Theriogenology 92, 149–155.
| An optimized method for cryogenic storage of Xenopus sperm to maximise the effectiveness of research using genetically altered frogs.Crossref | GoogleScholarGoogle Scholar | 28237331PubMed |
Proaño, B., and Pérez, O. D. (2017). In vitro fertilizations with cryopreserved sperm of Rhinella marina (Anura: Bufonidae) in Ecuador. Amphib. Reptile Conserv. 11, 1–6.
Ralls, K., Sunnucks, P., Lacy, R. C., and Frankham, R. (2020). Genetic rescue: A critique of the evidence supports maximizing genetic diversity rather than minimizing the introduction of putatively harmful genetic variation. Biol. Conserv. 251, 108784.
| Genetic rescue: A critique of the evidence supports maximizing genetic diversity rather than minimizing the introduction of putatively harmful genetic variation.Crossref | GoogleScholarGoogle Scholar |
Sargent, M. G., and Mohun, T. J. (2005). Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis. Genesis 41, 41–46.
| Cryopreservation of sperm of Xenopus laevis and Xenopus tropicalis.Crossref | GoogleScholarGoogle Scholar | 15645449PubMed |
Scheele, B. C., Pasmans, F., Skerratt, L. F., Berger, L., Martel, A., Beukema, W., Acevedo, A. A., Burrowes, P. A., Carvalho, T., Catenazzi, A., De la Riva, I., Fisher, M. C., Flechas, S. V., Foster, C. N., Frías-Álvarez, P., Garner, T. W. J., Gratwicke, B., Guayasamin, J. M., Hirschfeld, M., Kolby, J. E., Kosch, T. A., La Marca, E., Lindenmayer, D. B., Lips, K. R., Longo, A. V., Maneyro, R., McDonald, C. A., Mendelson, J., Palacios-Rodriguez, P., Parra-Olea, G., Richards-Zawacki, C. L., Rödel, M.-O., Rovito, S. M., Soto-Azat, C., Toledo, L. F., Voyles, J., Weldon, C., Whitfield, S. M., Wilkinson, M., Zamudio, K. R., and Canessa, S. (2019). Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity. Science 363, 1459–1463.
| Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity.Crossref | GoogleScholarGoogle Scholar | 30923224PubMed |
Shishova, N. R., Uteshev, V. K., Kaurova, S. A., Browne, R. K., and Gakhova, E. N. (2011). Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species. Theriogenology 75, 220–232.
| Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species.Crossref | GoogleScholarGoogle Scholar | 21040966PubMed |
Stockwell, M., Clulow, S., Clulow, J., and Mahony, M. (2008). The impact of the Amphibian Chytrid Fungus Batrachochytrium dendrobatidis on a Green and Golden Bell Frog Litoria aurea reintroduction program at the Hunter Wetlands Centre Australia in the Hunter Region of NSW. Aust. J. Agric. Res. 34, 379–386.
Stuart, S. N., Chanson, J. S., Cox, N. A., Young, B. E., Rodrigues, A. S. L., Fishman, D. L., and Waller, R. W. (2004). Status and trends of amphibian declines and extinctions worldwide. Science 306, 1783–1786.
| Status and trends of amphibian declines and extinctions worldwide.Crossref | GoogleScholarGoogle Scholar | 15486254PubMed |
Takamune, K. (1987). Detection of acrosome-reacted toad sperm based on specific lectin binding to the inner acrosomal membrane. Gamete Res. 18, 215–223.
| Detection of acrosome-reacted toad sperm based on specific lectin binding to the inner acrosomal membrane.Crossref | GoogleScholarGoogle Scholar | 3507373PubMed |
Ueda, Y., Yoshizaki, N., and Iwao, Y. (2002). Acrosome reaction in sperm of the frog, Xenopus laevis: Its detection and induction by oviductal pars recta secretion. Dev. Biol. 243, 55–64.
| Acrosome reaction in sperm of the frog, Xenopus laevis: Its detection and induction by oviductal pars recta secretion.Crossref | GoogleScholarGoogle Scholar | 11846477PubMed |
Upton, R., Clulow, S., Mahony, M. J., and Clulow, J. (2018). Generation of a sexually mature individual of the Eastern dwarf tree frog, Litoria fallax, from cryopreserved testicular macerates: proof of capacity of cryopreserved sperm derived offspring to complete development. Conserv. Physiol. 6, coy043.
| Generation of a sexually mature individual of the Eastern dwarf tree frog, Litoria fallax, from cryopreserved testicular macerates: proof of capacity of cryopreserved sperm derived offspring to complete development.Crossref | GoogleScholarGoogle Scholar | 30151196PubMed |
Uteshev, V. K., Shishova, N., Kaurova, S., Manokhin, A., and Gakhova, E. (2013). Collection and cryopreservation of hormonally induced sperm of pool frog (Pelophylax lessonae). Russ. J. Herpetol. 20, 105–109.
Zippel, K., Johnson, K., Gagliardo, R., Gibson, R., McFadden, M., Browne, R., Martinez, C., and Townsend, E. (2011). The Amphibian Ark: a global community for ex situ conservation of amphibians. Herpetol. Conserv. Biol. 6, 340–352.
