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Published in: Reproductive Biology and Endocrinology 1/2019

Open Access 01-12-2019 | Research

Hormonal induction of spermiation in a Eurasian bufonid (Epidalea calamita)

Authors: Lucía Arregui, Sergio Diaz-Diaz, Elia Alonso-López, Andrew J. Kouba

Published in: Reproductive Biology and Endocrinology | Issue 1/2019

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Abstract

Background

Amphibian diversity is declining at an alarming rate due to habitat loss, invasive species, climate change and diseases. Captive assurance colonies have been established for some species at risk; however, many species do not breed well in captivity and the development of assisted reproductive technologies (ART) is critical to help sustain genetic diversity. To date, the majority of the work has been accomplished in species from the American continent and Australia, and there is a need to address similar breeding challenges in Eurasian and African species of amphibians.

Methods

The aim of this study was to develop a hormone protocol for stimulation of spermiation in Epidalea calamita as a model for Eurasian bufonids. Hence, the effect on sperm production and quality of three doses of chorionic gonadotropin hormone (5, 10 and 15 IU hCG/g) over time (1 to 24 h) was evaluated. In addition, cold storage (at 5 °C) of sperm for 24 and 48 h and three frequencies for hormonal treatment (weekly, biweekly and monthly) were examined.

Results

Hormone concentrations of 10 or 15 IU of hCG induced spermiation in 100% of males and produced sperm of comparable quality, while 5 IU hCG stimulated spermiation in only 40% of males. Total motility peaked between 1 to 4 h post-treatment with 10 IU hCG, whereas treatment with 15 IU hCG peaked between 2 to 6 h. After 24 h of cold storage total motility dropped by 20% and forward motility dropped by 10% for both the 10 and 15 IU treatments. Weekly hormone administration resulted in higher variation between trials in all motility parameters and a lower overall Total Motility and Forward Movement. Furthermore, the effect of exogenous hormone treatment overlapped between the last two trials in the weekly frequency. Sperm concentration was higher in the first trial for all frequencies but showed no differences among other trials.

Conclusions

Overall, these results show that hormone concentration, time after treatment, frequency of hormone treatment and cold storage should be borne in mind when developing a hormone stimulation protocol for Eurasian amphibian species.
Literature
1.
go back to reference Hoffmann M, Hilton-Taylor C, Angulo A, Böhm M, Brooks TM, Butchart SHM, et al. The impact of conservation on the status of the world's vertebrates. Science. 2010;330:1503–9.CrossRefPubMed Hoffmann M, Hilton-Taylor C, Angulo A, Böhm M, Brooks TM, Butchart SHM, et al. The impact of conservation on the status of the world's vertebrates. Science. 2010;330:1503–9.CrossRefPubMed
3.
go back to reference Phillips K. Where have all the frogs and toads gone. Bioscience. 1990;40(6):422–4.CrossRef Phillips K. Where have all the frogs and toads gone. Bioscience. 1990;40(6):422–4.CrossRef
4.
go back to reference Kouba AJ, Vance CK. Applied reproductive technologies and genetic resource banking for amphibian conservation. Reprod Fertil Dev. 2009;21:719–37.PubMedCrossRef Kouba AJ, Vance CK. Applied reproductive technologies and genetic resource banking for amphibian conservation. Reprod Fertil Dev. 2009;21:719–37.PubMedCrossRef
5.
go back to reference Pukazhenthi B, Comizzoli P, Travis AJ, Wildt DE. Applications of emerging technologies to the study and conservation of threatened and endangered species. Reprod Fertil Dev. 2006;18:77–90.PubMedCrossRef Pukazhenthi B, Comizzoli P, Travis AJ, Wildt DE. Applications of emerging technologies to the study and conservation of threatened and endangered species. Reprod Fertil Dev. 2006;18:77–90.PubMedCrossRef
6.
go back to reference Holt WV, Brown JL, Comizzoli P. Reproductive sciences in animal conservation. New York: Springer Science+ Business Media; 2014. Holt WV, Brown JL, Comizzoli P. Reproductive sciences in animal conservation. New York: Springer Science+ Business Media; 2014.
7.
go back to reference Holt WV, Pickard AR, Rodger JC, Wildt DE. Reproductive science and integrated conservation. New York: Cambridge University Press; 2003. Holt WV, Pickard AR, Rodger JC, Wildt DE. Reproductive science and integrated conservation. New York: Cambridge University Press; 2003.
8.
go back to reference Wildt DE, Wemmer C. Sex and wildlife: the role of reproductive science in conservation. Biodivers Conserv. 1999;8:965–76.CrossRef Wildt DE, Wemmer C. Sex and wildlife: the role of reproductive science in conservation. Biodivers Conserv. 1999;8:965–76.CrossRef
9.
go back to reference Clulow J, Trudeau V, Kouba A. Amphibian declines in the twenty-first century: why we need assisted reproductive technologies. In: Holt WV, Brown JL, Comizzoli P, editors. Reproductive sciences in animal conservation, vol. 753. New York: Springer; 2014. p. 275–316. Advances in Experimental Medicine and Biology. Clulow J, Trudeau V, Kouba A. Amphibian declines in the twenty-first century: why we need assisted reproductive technologies. In: Holt WV, Brown JL, Comizzoli P, editors. Reproductive sciences in animal conservation, vol. 753. New York: Springer; 2014. p. 275–316. Advances in Experimental Medicine and Biology.
10.
go back to reference Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, et al. Status and trends of amphibian declines and extinctions worldwide. Science. 2004;306:1783–6.PubMedCrossRef Stuart SN, Chanson JS, Cox NA, Young BE, Rodrigues ASL, Fischman DL, et al. Status and trends of amphibian declines and extinctions worldwide. Science. 2004;306:1783–6.PubMedCrossRef
12.
go back to reference Browne R, Li H, Seratt J, Kouba A. Progesterone improves the number and quality of hormone induced fowler toad (Bufo fowleri) oocytes. Reprod Biol Endocrinol. 2006;4:3.PubMedPubMedCentralCrossRef Browne R, Li H, Seratt J, Kouba A. Progesterone improves the number and quality of hormone induced fowler toad (Bufo fowleri) oocytes. Reprod Biol Endocrinol. 2006;4:3.PubMedPubMedCentralCrossRef
13.
go back to reference Browne R, Seratt J, Vance C, Kouba A. Hormonal priming, induction of ovulation and in-vitro fertilization of the endangered Wyoming toad (Bufo baxteri). Reprod Biol Endocrinol. 2006;4:34.PubMedPubMedCentralCrossRef Browne R, Seratt J, Vance C, Kouba A. Hormonal priming, induction of ovulation and in-vitro fertilization of the endangered Wyoming toad (Bufo baxteri). Reprod Biol Endocrinol. 2006;4:34.PubMedPubMedCentralCrossRef
14.
go back to reference Browne RK, Clulow J, Mahony M, Clark A. Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology. 1998;37:339–45.PubMedCrossRef Browne RK, Clulow J, Mahony M, Clark A. Successful recovery of motility and fertility of cryopreserved cane toad (Bufo marinus) sperm. Cryobiology. 1998;37:339–45.PubMedCrossRef
15.
go back to reference Germano JM, Arregui L, Kouba AJ. Effects of aeration and antibiotics on short-term storage of Fowler's toad (Bufo fowleri) sperm. Aquaculture. 2013;396-399:20–4.CrossRef Germano JM, Arregui L, Kouba AJ. Effects of aeration and antibiotics on short-term storage of Fowler's toad (Bufo fowleri) sperm. Aquaculture. 2013;396-399:20–4.CrossRef
16.
go back to reference Kouba A, delBarco-Trillo J, Vance C, Milam C, Carr M. A comparison of human chorionic gonadotropin and luteinizing hormone releasing hormone on the induction of spermiation and amplexus in the American toad (Anaxyrus americanus). Reprod Biol Endocrinol. 2012;10:59.PubMedPubMedCentralCrossRef Kouba A, delBarco-Trillo J, Vance C, Milam C, Carr M. A comparison of human chorionic gonadotropin and luteinizing hormone releasing hormone on the induction of spermiation and amplexus in the American toad (Anaxyrus americanus). Reprod Biol Endocrinol. 2012;10:59.PubMedPubMedCentralCrossRef
17.
go back to reference Kouba AJ, Vance CK, Willis EL. Artificial fertilization for amphibian conservation: current knowledge and future considerations. Theriogenology. 2009;71:214–27.PubMedCrossRef Kouba AJ, Vance CK, Willis EL. Artificial fertilization for amphibian conservation: current knowledge and future considerations. Theriogenology. 2009;71:214–27.PubMedCrossRef
18.
go back to reference Silla AJ, Roberts JD. Investigating patterns in the spermiation response of eight Australian frogs administered human chorionic gonadotropin (hCG) and luteinizing hormone-releasing hormone (LHRHa). Gen Comp Endocrinol. 2012;179:128–36.PubMedCrossRef Silla AJ, Roberts JD. Investigating patterns in the spermiation response of eight Australian frogs administered human chorionic gonadotropin (hCG) and luteinizing hormone-releasing hormone (LHRHa). Gen Comp Endocrinol. 2012;179:128–36.PubMedCrossRef
19.
go back to reference Burgos MH, Vitale-Calpe R. The mechanism of spermiation in the toad. Am J Anat. 1967;120:227–51.CrossRef Burgos MH, Vitale-Calpe R. The mechanism of spermiation in the toad. Am J Anat. 1967;120:227–51.CrossRef
20.
go back to reference Obringer AR, O'Brien JK, Saunders RL, Yamamoto K, Kikuyama S, Roth TL. Characterization of the spermiation response, luteinizing hormone release and sperm quality in the American toad (Bufo americanus) and the endangered Wyoming toad (Bufo baxteri). Reprod Fertil Dev. 2000;12:51–8.PubMedCrossRef Obringer AR, O'Brien JK, Saunders RL, Yamamoto K, Kikuyama S, Roth TL. Characterization of the spermiation response, luteinizing hormone release and sperm quality in the American toad (Bufo americanus) and the endangered Wyoming toad (Bufo baxteri). Reprod Fertil Dev. 2000;12:51–8.PubMedCrossRef
21.
go back to reference Rowson AD, Obringer AR, Roth TL. Non-invasive treatments of luteinizing hormone-releasing hormone for inducing spermiation in American (Bufo americanus) and Gulf Coast (Bufo valliceps) toads. Zoo Biol. 2001;20:63–74.PubMedCrossRef Rowson AD, Obringer AR, Roth TL. Non-invasive treatments of luteinizing hormone-releasing hormone for inducing spermiation in American (Bufo americanus) and Gulf Coast (Bufo valliceps) toads. Zoo Biol. 2001;20:63–74.PubMedCrossRef
22.
go back to reference Iimori E, D'Occhio MJ, Lisle AT, Johnston SD. Testosterone secretion and pharmacological spermatozoal recovery in the cane toad (Bufo marinus). Anim Reprod Sci. 2005;90:163–73.PubMedCrossRef Iimori E, D'Occhio MJ, Lisle AT, Johnston SD. Testosterone secretion and pharmacological spermatozoal recovery in the cane toad (Bufo marinus). Anim Reprod Sci. 2005;90:163–73.PubMedCrossRef
23.
go back to reference Roth TL, Szymanski DC, Keyster ED. Effects of age, weight, hormones, and hibernation on breeding success in boreal toads (Bufo boreas boreas). Theriogenology. 2010;73:501–11.PubMedCrossRef Roth TL, Szymanski DC, Keyster ED. Effects of age, weight, hormones, and hibernation on breeding success in boreal toads (Bufo boreas boreas). Theriogenology. 2010;73:501–11.PubMedCrossRef
24.
go back to reference Kouba AJ, Vance CK, Frommeyer MA, Roth TL. Structural and functional aspects of Bufo americanus spermatozoa: effects of inactivation and reactivation. J Exp Zool. 2003;295A:172–82.CrossRef Kouba AJ, Vance CK, Frommeyer MA, Roth TL. Structural and functional aspects of Bufo americanus spermatozoa: effects of inactivation and reactivation. J Exp Zool. 2003;295A:172–82.CrossRef
25.
go back to reference Della Togna G, Trudeau VL, Gratwicke B, Evans M, Augustine L, Chia H, et al. Effects of hormonal stimulation on the concentration and quality of excreted spermatozoa in the critically endangered Panamanian golden frog (Atelopus zeteki). Theriogenology. 2017;91:27–35.PubMedCrossRef Della Togna G, Trudeau VL, Gratwicke B, Evans M, Augustine L, Chia H, et al. Effects of hormonal stimulation on the concentration and quality of excreted spermatozoa in the critically endangered Panamanian golden frog (Atelopus zeteki). Theriogenology. 2017;91:27–35.PubMedCrossRef
26.
go back to reference Uteshev V, Shishova N, Kaurova S, Browne R, Gakhova E. Hormonal induction of spermatozoa from amphibians with Rana temporaria and Bufo bufo as anuran models. Reprod Fertil Dev. 2012;24:599–607.PubMedCrossRef Uteshev V, Shishova N, Kaurova S, Browne R, Gakhova E. Hormonal induction of spermatozoa from amphibians with Rana temporaria and Bufo bufo as anuran models. Reprod Fertil Dev. 2012;24:599–607.PubMedCrossRef
27.
go back to reference Goncharov BF, Shubravy OI, Serbinova IA, Uteshev VK. The USSR programme for breeding amphibians, including rare and endangered species. Int Zoo Yearbook. 1989;28:10–21.CrossRef Goncharov BF, Shubravy OI, Serbinova IA, Uteshev VK. The USSR programme for breeding amphibians, including rare and endangered species. Int Zoo Yearbook. 1989;28:10–21.CrossRef
28.
go back to reference Browne RK, Clulow J, Mahony M. Short-term storage of cane toad (Bufo marinus) gametes. Reproduction. 2001;121:167–73.PubMedCrossRef Browne RK, Clulow J, Mahony M. Short-term storage of cane toad (Bufo marinus) gametes. Reproduction. 2001;121:167–73.PubMedCrossRef
29.
go back to reference Browne RK, Clulow J, Mahony M. The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. CryoLetters. 2002;23:129–36.PubMed Browne RK, Clulow J, Mahony M. The short-term storage and cryopreservation of spermatozoa from hylid and myobatrachid frogs. CryoLetters. 2002;23:129–36.PubMed
30.
go back to reference Silla AJ. Artificial fertilisation in a terrestrial toadlet (Pseudophryne guentheri): effect of medium osmolality, sperm concentration and gamete storage. Reprod Fertil Dev. 2013;25:1134–41.PubMedCrossRef Silla AJ. Artificial fertilisation in a terrestrial toadlet (Pseudophryne guentheri): effect of medium osmolality, sperm concentration and gamete storage. Reprod Fertil Dev. 2013;25:1134–41.PubMedCrossRef
31.
go back to reference Smith-Gill SJ, Berven KA. In vitro fertilization and assessment of male reproductive potential using mammalian gonadotropin-releasing hormone to induce spermiation in Rana sylvatica. Copeia. 1980;1980:723–8.CrossRef Smith-Gill SJ, Berven KA. In vitro fertilization and assessment of male reproductive potential using mammalian gonadotropin-releasing hormone to induce spermiation in Rana sylvatica. Copeia. 1980;1980:723–8.CrossRef
32.
go back to reference McKinnell RG, Picciano DJ, Krieg RE. Fertilization and development of frog eggs after repeated spermiation induced by human chorionic gonadotropin. Lab Anim Sci. 1976;26:932–5.PubMed McKinnell RG, Picciano DJ, Krieg RE. Fertilization and development of frog eggs after repeated spermiation induced by human chorionic gonadotropin. Lab Anim Sci. 1976;26:932–5.PubMed
33.
go back to reference McDonough CE, Martin MW, Vance CK, Cole JA, Kouba AJ. Frequency of exogenous hormone therapy impacts spermiation in male Fowler's toad (Bufo fowleri). Reprod Fertil Dev. 2015;28:995–1003.CrossRef McDonough CE, Martin MW, Vance CK, Cole JA, Kouba AJ. Frequency of exogenous hormone therapy impacts spermiation in male Fowler's toad (Bufo fowleri). Reprod Fertil Dev. 2015;28:995–1003.CrossRef
34.
go back to reference Roth TL, Obringer AR. Reproductive research and the worldwide amphibian extinction crisis. In: Holt WV, Pickard AR, Rodger JC, Wildt DE, editors. Reproductive sciences and integrated conservation. New York: Cambridge University Press; 2003. p. 359. Roth TL, Obringer AR. Reproductive research and the worldwide amphibian extinction crisis. In: Holt WV, Pickard AR, Rodger JC, Wildt DE, editors. Reproductive sciences and integrated conservation. New York: Cambridge University Press; 2003. p. 359.
35.
go back to reference Waggener WL, Carroll EJ. A method for hormonal induction of sperm release in anurans (eight species) and in vitro fertilization in Lepidobatrachus species. Develop Growth Differ. 1998;40:19–25.CrossRef Waggener WL, Carroll EJ. A method for hormonal induction of sperm release in anurans (eight species) and in vitro fertilization in Lepidobatrachus species. Develop Growth Differ. 1998;40:19–25.CrossRef
37.
go back to reference Denton JS, Hitchings SP, Beebee TJC, Gent A. A recovery program for the Natterjack toad (Bufo calamita) in Britain. Conserv Biol. 1997;11:1329–38.CrossRef Denton JS, Hitchings SP, Beebee TJC, Gent A. A recovery program for the Natterjack toad (Bufo calamita) in Britain. Conserv Biol. 1997;11:1329–38.CrossRef
38.
go back to reference Buckley J, Beebee TJC. Monitoring the conservation status of an endangered amphibian: the natterjack toad Bufo calamita in Britain. Anim Conserv. 2004;7:221–8.CrossRef Buckley J, Beebee TJC. Monitoring the conservation status of an endangered amphibian: the natterjack toad Bufo calamita in Britain. Anim Conserv. 2004;7:221–8.CrossRef
39.
go back to reference Allentoft ME, Siegismund HR, Briggs L, Andersen LW. Microsatellite analysis of the natterjack toad (Bufo calamita) in Denmark: populations are islands in a fragmented landscape. Conserv Genet. 2009;10:15–28.CrossRef Allentoft ME, Siegismund HR, Briggs L, Andersen LW. Microsatellite analysis of the natterjack toad (Bufo calamita) in Denmark: populations are islands in a fragmented landscape. Conserv Genet. 2009;10:15–28.CrossRef
40.
go back to reference Rogell B, Thörngren H, Palm S, Laurila A, Höglund J. Genetic structure in peripheral populations of the natterjack toad, Bufo calamita, as revealed by AFLP. Conserv Genet. 2010;11:173–81.CrossRef Rogell B, Thörngren H, Palm S, Laurila A, Höglund J. Genetic structure in peripheral populations of the natterjack toad, Bufo calamita, as revealed by AFLP. Conserv Genet. 2010;11:173–81.CrossRef
41.
go back to reference Lipke C, Meinecke-Tillmann S, Meinecke B. Induced spermiation and sperm morphology in a dendrobatid frog, Dendrobates auratus (Amphibia, Anura, Dendrobatidae). Salamandra. 2009;54:65–74. Lipke C, Meinecke-Tillmann S, Meinecke B. Induced spermiation and sperm morphology in a dendrobatid frog, Dendrobates auratus (Amphibia, Anura, Dendrobatidae). Salamandra. 2009;54:65–74.
42.
go back to reference Byrne PG, Silla AJ. Hormonal induction of gamete release and in-vitro fertilization in the critically endangered southern corroboree frog, Pseudophryne corroboree. Reprod Biol Endocrinol. 2010;8:144.PubMedPubMedCentralCrossRef Byrne PG, Silla AJ. Hormonal induction of gamete release and in-vitro fertilization in the critically endangered southern corroboree frog, Pseudophryne corroboree. Reprod Biol Endocrinol. 2010;8:144.PubMedPubMedCentralCrossRef
43.
go back to reference Russo J, Burgos MH. Effect of HCG on the enzymic activity of toad testes. Gen Comp Endocrinol. 1969;13:185–8.PubMedCrossRef Russo J, Burgos MH. Effect of HCG on the enzymic activity of toad testes. Gen Comp Endocrinol. 1969;13:185–8.PubMedCrossRef
44.
go back to reference Beebee TJC. A review of scientific information pertaining to the Natterjack toad Bufo calamita throughout its geographical range. Biol Conserv. 1979;16:107–34.CrossRef Beebee TJC. A review of scientific information pertaining to the Natterjack toad Bufo calamita throughout its geographical range. Biol Conserv. 1979;16:107–34.CrossRef
45.
go back to reference Silla AJ, Keogh LM, Byrne PG. Antibiotics and oxygen availability affect the short-term storage of spermatozoa from the critically endangered booroolong frog, Litoria booroolongensis. Reprod Fertil Dev. 2015;27:1147–53.PubMedCrossRef Silla AJ, Keogh LM, Byrne PG. Antibiotics and oxygen availability affect the short-term storage of spermatozoa from the critically endangered booroolong frog, Litoria booroolongensis. Reprod Fertil Dev. 2015;27:1147–53.PubMedCrossRef
46.
go back to reference McCourt RP. The effect of repeated injections of chorionic gonadotropin on the testes of the leopard frog (Rana pipiens schreber). Ohio J Sci. 1953;53:335–9. McCourt RP. The effect of repeated injections of chorionic gonadotropin on the testes of the leopard frog (Rana pipiens schreber). Ohio J Sci. 1953;53:335–9.
47.
go back to reference Thorborg JV. The use of Xenopus laevis, Bufo bufo, and Rana esculenta as test animals for gonadotrophic hormones. IV comparative investigations on the pregnancy reactions of Galli Mainini and Friedman. Acta Endocrinol. 1951;6:373–86.CrossRef Thorborg JV. The use of Xenopus laevis, Bufo bufo, and Rana esculenta as test animals for gonadotrophic hormones. IV comparative investigations on the pregnancy reactions of Galli Mainini and Friedman. Acta Endocrinol. 1951;6:373–86.CrossRef
Metadata
Title
Hormonal induction of spermiation in a Eurasian bufonid (Epidalea calamita)
Authors
Lucía Arregui
Sergio Diaz-Diaz
Elia Alonso-López
Andrew J. Kouba
Publication date
01-12-2019
Publisher
BioMed Central
Published in
Reproductive Biology and Endocrinology / Issue 1/2019
Electronic ISSN: 1477-7827
DOI
https://doi.org/10.1186/s12958-019-0537-0

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