Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE (Open Access)

Fertilization potential of cold-stored Fowler’s toad (Anaxyrus fowleri) spermatozoa: temporal changes in sperm motility based on temperature and osmolality

Lucia Arregui https://orcid.org/0000-0002-4355-0155 A C , Andy J. Kouba B C , Jennifer M. Germano C D , Laura Barrios E , Marian Moore C and Carrie K. Kouba https://orcid.org/0000-0001-8466-6077 F *
+ Author Affiliations
- Author Affiliations

A Department of Biology, Universidad Autónoma de Madrid, Madrid 28049, Spain.

B Wildlife, Fisheries and Aquaculture, Mississippi State University, Mississippi State, MS 39762, USA.

C Conservation and Research Department, Memphis Zoological Society, Memphis, TN 38112, USA.

D New Zealand Department of Conservation, Hamilton, New Zealand.

E Department of Statistics, CTI, Consejo Superior Investigaciones Científicas, 28006 Madrid, Spain.

F Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS 39762, USA.

* Correspondence to: ckv7@msstate.edu

Handling Editor: John Clulow

Reproduction, Fertility and Development 34(5) 461-469 https://doi.org/10.1071/RD21037
Published online: 1 November 2021

© 2022 The Author(s) (or their employer(s)). Published by CSIRO Publishing. This is an open access article distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND)

Abstract

Asynchrony of gamete release is problematic in amphibian captive breeding programs but can be overcome by short-term storage of spermatozoa. Hormonally induced sperm from the model species Anaxyrus fowleri were used to determine storage conditions for optimal fertilisation capacity. Sperm motility was measured over time, as a function of storage temperature (4°C or 22°C) and solution osmolality (7–40 mOsm/kg). Sperm at 40 mOsm/kg (spermic urine) stored at 4°C exhibited higher motility compared to 22°C. Also, sperm stored at 40 mOsm/kg retained higher motility compared to sperm stored below 15 mOsm/kg at both temperatures. Under optimal storage conditions (40 mOsm and 4°C) a 30% decrease in sperm motility occurred within 24 h, however, subsequent loss of sperm motility was lower (<10%/day) for days 2–8 thereafter. Sperm samples stored for 1–8 days under optimal conditions were tested for fertilising capacity by conducting in vitro fertilisation trials. Sperm stored for 8 days yielded 48% neurula development, similar to sperm stored for 1 day, which produced 60% neurula development. Overall, sperm stored for up to 8 days at 4°C as spermic urine retained fertilising capacity and thus can be used to circumvent asynchronous gamete release in assisted breeding efforts for amphibians.

Keywords: amphibian, anuran, bufonid, captive breeding, embryo, human chorionic gonadotrophin, in vitro fertilisation, reproduction.


References

Alavi, SMH, and Cosson, J (2005). Sperm motility in fishes. (I). Effects of temperature and pH: a review. Cell Biology International 29, 101–110.
Sperm motility in fishes. (I). Effects of temperature and pH: a review.Crossref | GoogleScholarGoogle Scholar |

Alavi, SMH, and Cosson, J (2006). Sperm motility in fishes. (II). Effects of ions and osmolality: a review. Cell Biology International 30, 1–14.
Sperm motility in fishes. (II). Effects of ions and osmolality: a review.Crossref | GoogleScholarGoogle Scholar |

Arregui, L, Diaz-Diaz, S, Alonso-López, E, and Kouba, AJ (2019). Hormonal induction of spermiation in a Eurasian bufonid (Epidalea calamita. Reproductive Biology and Endocrinology 17, 92.
Hormonal induction of spermiation in a Eurasian bufonid (Epidalea calamita.Crossref | GoogleScholarGoogle Scholar | 31711511PubMed |

Arregui, L, Bóveda, P, Gosálvez, J, and Kouba, AJ (2020). Effect of seasonality on hormonally induced sperm in Epidalea calamita (Amphibia, Anura, Bufonidae) and its refrigerated and cryopreservated storage. Aquaculture 529, 735677.
Effect of seasonality on hormonally induced sperm in Epidalea calamita (Amphibia, Anura, Bufonidae) and its refrigerated and cryopreservated storage.Crossref | GoogleScholarGoogle Scholar |

Browne, RK, Clulow, J, Mahony, M, and Clark, A (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, RK, Clulow, J, and Mahony, M (2001). Short-term storage of cane toad (Bufo marinus) gametes. Reproduction 121, 167–173.
Short-term storage of cane toad (Bufo marinus) gametes.Crossref | GoogleScholarGoogle Scholar | 11226040PubMed |

Browne, RK, Clulow, J, and Mahony, M (2002a). The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. CryoLetters 23, 129–136.
| 12050781PubMed |

Browne, RK, Davis, J, Pomering, M, and Clulow, J (2002b). Storage of cane toad (Bufo marinus) sperm for 6 days at 0°C with subsequent cryopreservation. Reproduction, Fertility and Development 14, 267–273.
Storage of cane toad (Bufo marinus) sperm for 6 days at 0°C with subsequent cryopreservation.Crossref | GoogleScholarGoogle Scholar |

Browne, RK, Silla, AJ, Upton, R, Della-Togna, G, Marcec-Greaves, R, Shishova, NV, Uteshev, VK, Proaño, B, Pérez, OD, Mansour, N, Kaurova, SA, Gakhova, EN, Cosson, J, Dyzuba, B, Kramarova, LI, 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 |

Byrne, PG, Dunne, C, Munn, AJ, and Silla, AJ (2015). Environmental osmolality influences sperm motility activation in an anuran amphibian. Journal of Evolutionary Biology 28, 521–534.
Environmental osmolality influences sperm motility activation in an anuran amphibian.Crossref | GoogleScholarGoogle Scholar | 25586700PubMed |

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. Reproduction, Fertility and Development 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, Kouba A (2014) Amphibian declines in the twenty-first century: why we need assisted reproductive technologies. In ‘Reproductive sciences in animal conservation. Vol. 753.’ (Eds WV Holt, JL Brown, P Comizzoli) pp. 275–316. (Springer: New York, NY, USA)
| Crossref |

Costanzo, JP, Mugnano, JA, Wehrheim, HM, and Lee, RE (1998). Osmotic and freezing tolerance in spermatozoa of freeze-tolerant and -intolerant frogs. American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 275, R713–R719.

Della Togna, G, Gratwicke, B, Evans, M, Augustine, L, Chia, H, Bronikowski, E, Murphy, JB, and Comizzoli, P (2018). Influence of extracellular environment on the motility and structural properties of spermatozoa collected from hormonally stimulated Panamanian Golden Frog (Atelopus zeteki. Theriogenology 108, 153–160.
Influence of extracellular environment on the motility and structural properties of spermatozoa collected from hormonally stimulated Panamanian Golden Frog (Atelopus zeteki.Crossref | GoogleScholarGoogle Scholar | 29216539PubMed |

Della Togna, G, Howell, LG, Clulow, J, Langhorne, CJ, Marcec-Greaves, R, and Calatayud, NE (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 |

Duellman WE, Trueb L (1986) ‘Biology of amphibians’. (The Johns Hoppkins University Press)

Figiel, CR (2013). Cryopreservation of sperm from the axolotl Ambystoma mexicanum: implications for conservation. Herpetological Conservation and Biology 8, 748.

Figiel, CR (2020). Cold storage of sperm from the Axolotl, Ambystoma mexicanum. Herpetological Conservation and Biology 15, 367–371.

Gosner, KL (1960). A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica 16, 183–190.

Guy, EL, Gillis, AB, Kouba, AJ, Barber, D, Poole, V, Marcec-Greaves, RM, and Kouba, CK (2020). Sperm collection and cryopreservation for threatened newt species. Cryobiology 94, 80–88.
Sperm collection and cryopreservation for threatened newt species.Crossref | GoogleScholarGoogle Scholar | 32437677PubMed |

Hettyey, A, and Roberts, JD (2007). Sperm traits in the quacking frog (Crinia georgiana), a species with plastic alternative mating tactics. Behavioral Ecology and Sociobiology 61, 1303–1310.
Sperm traits in the quacking frog (Crinia georgiana), a species with plastic alternative mating tactics.Crossref | GoogleScholarGoogle Scholar |

Hinkson, KM, Baecher, JA, 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 |

Inoda, T, and Morisawa, M (1987). Effect of osmolality on the initiation of sperm motility in Xenopus laevis. Comparative biochemistry and physiology. A, Comparative Physiology 88, 539–542.
Effect of osmolality on the initiation of sperm motility in Xenopus laevis.Crossref | GoogleScholarGoogle Scholar | 2892629PubMed |

IUCN (2020) The IUCN red list of threatened species. Summary tables. Version 2020-1. http://www.iucnredlist.org

Kouba, AJ, and Vance, CK (2009). Applied reproductive technologies and genetic resource banking for amphibian conservation. Reproduction, Fertility and Development 21, 719–737.
Applied reproductive technologies and genetic resource banking for amphibian conservation.Crossref | GoogleScholarGoogle Scholar |

Kouba, AJ, Vance, CK, Frommeyer, MA, and Roth, TL (2003). Structural and functional aspects of Bufo americanus spermatozoa: effects of inactivation and reactivation. Journal of Experimental Zoology 295A, 172–182.
Structural and functional aspects of Bufo americanus spermatozoa: effects of inactivation and reactivation.Crossref | GoogleScholarGoogle Scholar |

Kouba, A, Willis, E, Vance, C, Hasenstab, S, Reichling, S, Krebs, J, Linhoff, L, Snoza, M, Langhorne, C, and Germano, J (2011). Development of assisted reproduction technologies for the endangered Mississippi Gopher frog (Rana sevosa) and sperm transfer for in vitro fertilization. Reproduction, Fertility and Development 24, 170–170.
Development of assisted reproduction technologies for the endangered Mississippi Gopher frog (Rana sevosa) and sperm transfer for in vitro fertilization.Crossref | GoogleScholarGoogle Scholar |

Kouba A, Vance C, Calatayud N, Rowlison T, Langhorne C, Willard S (2012) Assisted reproductive technologies (ART) for amphibians. In ‘Amphibian husbandry resource guide’. (Eds VA Poole, S Grow). (AZA’s Amphibian Taxon Advisory Group)

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

Langhorne, C, Calatayud, NE, Kouba, CK, Willard, ST, Smith, T, Ryan, PL, and Kouba, AJ (2021). Efficacy of hormone stimulation on sperm production in an alpine amphibian (Anaxyrus boreas boreas) and the impact of short-term storage on sperm quality. Zoology 146, 125912.
Efficacy of hormone stimulation on sperm production in an alpine amphibian (Anaxyrus boreas boreas) and the impact of short-term storage on sperm quality.Crossref | GoogleScholarGoogle Scholar | 33743452PubMed |

Mansour, N, Lahnsteiner, F, and Patzner, RA (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 |

Marcec, R, Langhorne, C, Vance, C, Kouba, A, and Willard, S (2014). C-1013: cryopreservation of spermic milt in the model species Ambystoma tigrinum (Tiger salamander) for application in endangered salamanders. Cryobiology 69, 515.
C-1013: cryopreservation of spermic milt in the model species Ambystoma tigrinum (Tiger salamander) for application in endangered salamanders.Crossref | GoogleScholarGoogle Scholar |

Peng, L-Y, Xiao, Y-M, and Liu, Y (2011). Effect of cryopreservation and short-term storage of Chinese Giant salamander sperm. Acta Hydrobiologica Sinica 35, 325–332.
Effect of cryopreservation and short-term storage of Chinese Giant salamander sperm.Crossref | GoogleScholarGoogle Scholar |

Sherman, C, Uller, T, Wapstra, E, and Olsson, M (2008). Within-population variation in ejaculate characteristics in a prolonged breeder, Peron’s tree frog, Litoria peronii. Naturwissenschaften 95, 1055–1061.
Within-population variation in ejaculate characteristics in a prolonged breeder, Peron’s tree frog, Litoria peronii.Crossref | GoogleScholarGoogle Scholar | 18618091PubMed |

Shishova, NR, Uteshev, VK, Kaurova, SA, Browne, RK, and Gakhova, EN (2011). Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species. Theriogenology 75, 202–232.
Cryopreservation of hormonally induced sperm for the conservation of threatened amphibians with Rana temporaria as a model research species.Crossref | GoogleScholarGoogle Scholar |

Shishova, NR, Uteshev, VK, Sirota, NP, Kuznetsova, EA, Kaurova, SA, Browne, RK, and Gakhova, EN (2013). The quality and fertility of sperm collected from European common frog (Rana temporaria) carcasses refrigerated for up to 7 days. Zoo Biology 32, 400–406.
The quality and fertility of sperm collected from European common frog (Rana temporaria) carcasses refrigerated for up to 7 days.Crossref | GoogleScholarGoogle Scholar |

Silla, AJ, Keogh, LM, and Byrne, PG (2015). Antibiotics and oxygen availability affect the short-term storage of spermatozoa from the critically endangered booroolong frog, Litoria booroolongensis. Reproduction, Fertility and Development 27, 1147–115.
Antibiotics and oxygen availability affect the short-term storage of spermatozoa from the critically endangered booroolong frog, Litoria booroolongensis.Crossref | GoogleScholarGoogle Scholar |

Unger, S, Mathis, A, and Wilkinson, R (2013). A comparison of sperm health in declining and stable populations of Hellbenders (Cryptobranchus alleganiensis alleganiensis and C.a. bishopi). The American Midland Naturalist 170, 382–392.
A comparison of sperm health in declining and stable populations of Hellbenders (Cryptobranchus alleganiensis alleganiensis and C.a. bishopi).Crossref | GoogleScholarGoogle Scholar |

Uteshev, VK, Shishova, NR, Kaurova, SA, Manokhin, AA, and Gakhova, EN (2013). Collection and cryopreservation of hormonally induced sperm of pool frog (Pelophylax lessonae. Russian Journal of Herpetology 20, 105–109.

Uteshev, VK, Kaurova, SA, Shishova, NV, Stolyarov, SD, Browne, RK, and Gakhova, EN (2015). In vitro fertilization with hormonally induced sperm and eggs from sharp-ribbed newts Pleurodeles waltl. Russian Journal of Herpetology 22, 35–40.

Waggener, WL, and Carroll, EJ (1998). Spermatozoon structure and motility in the anuran Lepidobatrachus laevis. Development, Growth & Differentiation 40, 27–34.
Spermatozoon structure and motility in the anuran Lepidobatrachus laevis.Crossref | GoogleScholarGoogle Scholar |

Wells KD (2007) ‘The ecology and behaviour of amphibians’. (The University of Chicago Press: Chicago, IL, USA and London, UK)

Wolf, DP, and Hedrick, JL (1971). A molecular approach to fertilization: II. Viability and artificial fertilization of Xenopus laevis gametes. Developmental Biology 25, 348–359.
A molecular approach to fertilization: II. Viability and artificial fertilization of Xenopus laevis gametes.Crossref | GoogleScholarGoogle Scholar | 5567826PubMed |