{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,2,2]],"date-time":"2026-02-02T20:13:01Z","timestamp":1770063181504,"version":"3.49.0"},"reference-count":50,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2023,9,4]],"date-time":"2023-09-04T00:00:00Z","timestamp":1693785600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Organismo Aut\u00f3nomo Parques Nacionales of Spain","award":["2399\/2017"],"award-info":[{"award-number":["2399\/2017"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Animals"],"abstract":"<jats:p>The genus Alytes consists of six primitive terrestrial-breeding species (five of them endangered or present in restricted distributions) with unique male parental care. Alytes obstetricans was used as a model for the development of assisted reproductive technologies for gamete collection, artificial fertilization and captive-rearing of embryos. Sperm collection was achieved with human chorionic gonadotropin (hCG), but only 17 to 50% of males responded to the hormone. In addition, an effect of captivity on sperm quality was observed. A combination of gonadotropin-releasing hormone agonist (GnRHa) and hCG was tested for induction of ovulation, and 100% of females responded to the treatment. Recently collected sperm was used in artificial fertilization, and 10% of eggs showed complete embryo development. To design a method for captive-rearing of embryos, natural clutches (n = 13) were removed from males and divided into two sub-clutches. One was kept with all eggs together and in the other, the egg string was cut and eggs were split individually. All sub-clutches with eggs kept separated presented embryo survival (ranging from 27 to 61%) that hatched normally, while only one sub-clutch with embryos maintained together had some embryo survival (36%). These results may contribute to the management of this and related species.<\/jats:p>","DOI":"10.3390\/ani13172802","type":"journal-article","created":{"date-parts":[[2023,9,4]],"date-time":"2023-09-04T02:26:12Z","timestamp":1693794372000},"page":"2802","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Gamete Collection, Artificial Fertilization and Captive-Rearing of Eggs in a Terrestrial-Breeding Anuran with Parental Care: Alytes obstetricans"],"prefix":"10.3390","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4355-0155","authenticated-orcid":false,"given":"Luc\u00eda","family":"Arregui","sequence":"first","affiliation":[{"name":"Centro de Investigaci\u00f3n, Seguimiento y Evaluaci\u00f3n, Parque Nacional de la Sierra de Guadarrama, Ctra. M-604, Km 27.6, Rascafria, 28740 Madrid, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0099-7934","authenticated-orcid":false,"given":"Jaime","family":"Bosch","sequence":"additional","affiliation":[{"name":"Centro de Investigaci\u00f3n, Seguimiento y Evaluaci\u00f3n, Parque Nacional de la Sierra de Guadarrama, Ctra. M-604, Km 27.6, Rascafria, 28740 Madrid, Spain"},{"name":"IMIB-Biodiversity Research Institute, University of Oviedo-CSIC-Principality of Asturias, C\/Gonzalo Guti\u00e9rrez Quir\u00f3n S\/N, Mieres, 33600 Asturias, Spain"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,9,4]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Silla, A.J., Kouba, A.J., and Heatwole, H. (2022). Reproductive Technologies and Biobanking for the Conservation of Amphibians, CSIRO Publishing.","DOI":"10.1071\/9781486313341"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"1557","DOI":"10.1016\/j.cub.2019.04.005","article-title":"Phylogenetic and trait-based prediction of extinction risk for data-deficient amphibians","volume":"29","author":"Freckleton","year":"2019","journal-title":"Curr. Biol."},{"key":"ref_3","first-page":"691","article-title":"Global Declines of Amphibians","volume":"Volume 3","author":"Levin","year":"2013","journal-title":"Encyclopedia of Biodiversity"},{"key":"ref_4","unstructured":"IUCN (2022, January 03). The IUCN Red List of Threatened Species. Available online: http:\/\/www.iucnredlist.org."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"105","DOI":"10.1163\/15685381-20191312","article-title":"Hybrid zone genomics supports candidate species in Iberian Alytes obstetricans","volume":"41","author":"Dufresnes","year":"2020","journal-title":"Amphib.-Reptil."},{"key":"ref_6","unstructured":"Michaels, C.J., Bosch, J., Jervis, P.A., M\u00e1rquez, R., and Rosa, G.M. (2023). Midwife Toads. The Genus Alytes. History, Ecology, Systematics, Captive Breeding, Edition Chimaira."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"331","DOI":"10.1016\/S0006-3207(00)00132-4","article-title":"Evidence of a chytrid fungus infection involved in the decline of the common midwife toad (Alytes obstetricans) in protected areas of central Spain","volume":"97","author":"Bosch","year":"2001","journal-title":"Biol. Conserv."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"72","DOI":"10.1080\/21564574.2021.1998235","article-title":"Fatal chytridiomycosis in the Moroccan midwife toad Alytes maurus and potential distribution of Batrachochytrium dendrobatidis across Morocco","volume":"71","author":"Bosch","year":"2022","journal-title":"Afr. J. Herpetol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"645491","DOI":"10.3389\/fvets.2021.645491","article-title":"Long-Term Monitoring of Amphibian Populations of a National Park in Northern Spain Reveals Negative Persisting Effects of Ranavirus, but Not Batrachochytrium dendrobatidis","volume":"8","author":"Bosch","year":"2021","journal-title":"Front. Vet. Sci."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"2586","DOI":"10.1016\/j.cub.2014.09.028","article-title":"Collapse of Amphibian Communities Due to an Introduced Ranavirus","volume":"24","author":"Price","year":"2014","journal-title":"Curr. Biol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"636","DOI":"10.1007\/BF00222519","article-title":"Amphibians: Suitable candidates for breeding-release programmes","volume":"4","author":"Bloxam","year":"1995","journal-title":"Biodivers. Conserv."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Serra, J.M., Griffiths, R.A., Bosch, J., Beebee, T., Schmidt, B., Tejedo, M., Lizana, M., Mart\u00ednez-Solano, I., Salvador, A., and Garc\u00eda-Paris, M. (2023, July 28). Alytes muletensis. The IUCN Red List of Threatened Species. 2009. Available online: http:\/\/dx.doi.org\/10.2305\/IUCN.UK.2009.RLTS.T977A13099129.en.","DOI":"10.2305\/IUCN.UK.2009.RLTS.T977A13099129.en"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"20150874","DOI":"10.1098\/rsbl.2015.0874","article-title":"Successful elimination of a lethal wildlife infectious disease in nature","volume":"11","author":"Bosch","year":"2015","journal-title":"Biol. Lett."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"8","DOI":"10.1186\/1742-9994-8-8","article-title":"Mitigating amphibian disease: Strategies to maintain wild populations and control chytridiomycosis","volume":"8","author":"Woodhams","year":"2011","journal-title":"Front. Zool."},{"key":"ref_15","unstructured":"Wells, E., Garcia-Alonso, D., Gon\u00c7alo, M.R., Garcia, G., and Tapley, B. (2015). Amphibian Taxon Advisory Group Best Practice Gudelines for Midwife toads (Alytes sp.). Version 1, EAZA."},{"key":"ref_16","unstructured":"Araujo, M.B., Guilhaumon, F., Rodrigues Neto, D., Pozo Ortego, I., and Gomez Calmaestra, R. (2011). Impactos, Vulnerabilidad y Adaptaci\u00f3n de la Biodiversidad Espa\u00f1ola Frente al Cambio Climatico, Fauna de Vertebrados."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"125","DOI":"10.1111\/j.1469-7998.1991.tb03806.x","article-title":"The courtship and mating of the Iberian midwife toad Alytes cisternasii (Amphibia: Anura: Discoglossidae)","volume":"225","author":"Marquez","year":"1991","journal-title":"J. Zool."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1111\/j.1748-1090.1989.tb03251.x","article-title":"Breeding the Mallorcan midwife toad Alytes muletensis in captivity","volume":"28","author":"Tonge","year":"1989","journal-title":"Int. Zoo Yearb."},{"key":"ref_19","first-page":"99","article-title":"Nuevo estatus taxon\u00f3mico para las poblaciones ibero-mediterraneas de Alytes obstetricans (Anura: Discoglossidae)","volume":"15","year":"2001","journal-title":"Rev. Esp. Herp."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"214","DOI":"10.1016\/j.theriogenology.2008.09.055","article-title":"Artificial fertilization for amphibian conservation: Current knowledge and future considerations","volume":"71","author":"Kouba","year":"2009","journal-title":"Theriogenology"},{"key":"ref_21","first-page":"413","article-title":"A revised classification of the amphibian reproductive modes","volume":"57","author":"Haddad","year":"2021","journal-title":"Salamandra Geman J. Herpetol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Duellman, W.E., and Trueb, L. (1986). Biology of Amphibians, The Johns Hoppkins University Press.","DOI":"10.2307\/1445022"},{"key":"ref_23","first-page":"5","article-title":"Biolog\u00eda de la reproduccion de Alytes cisternasii Bosc\u00e1, 1879","volume":"6","year":"1979","journal-title":"Do\u00f1ana Acta Vertebr."},{"key":"ref_24","first-page":"283","article-title":"Male Reproductive Success in Two Midwife Toads, Alytes obstetricans and A. cisternasii","volume":"32","year":"1993","journal-title":"Behav. Ecol. Sociobiol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"96","DOI":"10.1002\/jez.a.254","article-title":"Effect of human gonadotropins on spermiation and androgen biosynthesis in the testis of the toad Bufo arenarum (Amphibia, Anura)","volume":"305 PtA","author":"Pozzi","year":"2006","journal-title":"J. Exp. Zool. Part A Comp. Exp. Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"36","DOI":"10.1186\/1477-7827-8-36","article-title":"Hormonal induction of spawning in 4 species of frogs by coinjection with a gonadotropin-releasing hormone agonist and a dopamine antagonist","volume":"8","author":"Trudeau","year":"2010","journal-title":"Reprod. Biol. Endocrinol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"3837","DOI":"10.1016\/S0021-9258(19)41474-9","article-title":"Human chorionic gonadotropin. V. Tissue specificity of binding and partial characterization of soluble human chorionic gonadotropin-receptor complexes","volume":"250","author":"Bellisario","year":"1975","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1002\/aja.1001200203","article-title":"The mechanism of spermiation in the toad","volume":"120","author":"Burgos","year":"1967","journal-title":"Am. J. Anat."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"185","DOI":"10.1016\/0016-6480(69)90237-8","article-title":"Effect of HCG on the enzymic activity of toad testes","volume":"13","author":"Russo","year":"1969","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"59","DOI":"10.1186\/1477-7827-10-59","article-title":"A comparison of human chorionic gonadotropin and luteinizing hormone releasing hormone on the induction of spermiation and amplexus in the American toad (Anaxyrus americanus)","volume":"10","author":"Kouba","year":"2012","journal-title":"Reprod. Biol. Endocrinol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"coy080","DOI":"10.1093\/conphys\/coy080","article-title":"Hormone-induced sperm-release in the critically endangered Booroolong frog (Litoria booroolongensis): Effects of gonadotropin-releasing hormone and human chorionic gonadotropin","volume":"7","author":"Silla","year":"2019","journal-title":"Conserv. Physiol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"128","DOI":"10.1016\/j.ygcen.2012.08.009","article-title":"Investigating patterns in the spermiation response of eight Australian frogs administered human chorionic gonadotropin (hCG) and luteinizing hormone-releasing hormone (LHRHa)","volume":"179","author":"Silla","year":"2012","journal-title":"Gen. Comp. Endocrinol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"447","DOI":"10.1071\/RD21061","article-title":"Hormone treatment does not reliably induce spermiation or mating in Hamilton\u2019s frog from the archaic leiopelmatid lineage","volume":"34","author":"Germano","year":"2022","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_34","first-page":"e609","article-title":"Topical application of hormone gonadotropin-releasing hormone (GnRH-A) simulates reproduction in the endangered Texas blind salamander (Eurycea rathbuni)","volume":"4","author":"Anderson","year":"2021","journal-title":"Conserv. Sci. Pract."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"coaa104","DOI":"10.1093\/conphys\/coaa104","article-title":"The effect of injection and topical application of hCG and GnRH agonist to induce sperm-release in the roseate frog, Geocrinia rosea","volume":"8","author":"Silla","year":"2020","journal-title":"Conserv. Physiol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1002\/zoo.1007","article-title":"Non-invasive treatments luteinizing hormone-releasing hormone for inducing spermiation in American (Bufo americanus) and Gulf Coast (Bufo valliceps) toads","volume":"20","author":"Rowson","year":"2001","journal-title":"Zoo Biol."},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Julien, A.R., Kouba, A.J., Kabelik, D., Feugang, J.M., Willard, S.T., and Kouba, C.K. (2019). Nasal administration of gonadotropin releasing hormone (GnRH) elicits sperm production in Fowler\u2019s toads (Anaxyrus fowleri). BMC Zool., 4.","DOI":"10.1186\/s40850-019-0040-2"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1071\/RD02079","article-title":"Effect of sperm concentration, medium osmolality and oocyte storage on artificial fertilisation success in a myobatrachid frog (Limnodynastes tasmaniensis)","volume":"16","author":"Edwards","year":"2004","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_39","first-page":"333","article-title":"Amphibian sperm: Phylogeny and fertilization environment","volume":"166","author":"Wilson","year":"1995","journal-title":"Mem. Mus. Natl. D\u2019histoire Nat."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e12864","DOI":"10.1111\/csp2.12864","article-title":"Effects of wild, semi-captive, and captive management on male Chiricahua leopard frog sperm quality with implications for conservation breeding programs","volume":"5","author":"Julien","year":"2023","journal-title":"Conserv. Sci. Pract."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1016\/j.theriogenology.2019.03.035","article-title":"Sperm collection and storage for the sustainable management of amphibian biodiversity","volume":"133","author":"Browne","year":"2019","journal-title":"Theriogenology"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Ogielska, M. (2009). Reproduction of Amphibians, Science Publishers.","DOI":"10.1201\/9781482280135"},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Bl\u00fcm, V. (1986). Vertebrate Reproduction. A Textbook, Springer.","DOI":"10.1007\/978-3-642-71074-2"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Lombardi, J. (1998). Comparative Vertebrate Reproduction, Springer.","DOI":"10.1007\/978-1-4615-4937-6"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"163","DOI":"10.1086\/590221","article-title":"The Physical Movement of Urine from the Kidneys to the Urinary Bladder and Bladder Compliance in Two Anurans","volume":"82","author":"Martin","year":"2009","journal-title":"Physiol. Biochem. Zool. Ecol. Evol. Approaches"},{"key":"ref_46","first-page":"183","article-title":"A simplified table for staging anuran embryos and larvae with notes on identification","volume":"16","author":"Gosner","year":"1960","journal-title":"Herpetologica"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"1134","DOI":"10.1071\/RD12223","article-title":"Artificial fertilisation in a terrestrial toadlet (Pseudophryne guentheri): Effect of medium osmolality, sperm concentration and gamete storage","volume":"25","author":"Silla","year":"2013","journal-title":"Reprod. Fertil. Dev."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"339","DOI":"10.1006\/cryo.1998.2129","article-title":"Successful recovery of motility and fertility of cryopreserved Cane toad (Bufo marinus) sperm","volume":"37","author":"Browne","year":"1998","journal-title":"Cryobiology"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"481","DOI":"10.1242\/jeb.62.2.481","article-title":"Sperm concentration and fertilization rate in Bufo arenarum (Amphibia: Anura)","volume":"62","author":"Cabada","year":"1975","journal-title":"J. Exp. Biol."},{"key":"ref_50","unstructured":"Birkhead, T.R., and M\u00f6ller, A.P. (1998). Sperm Competition and Sexual Selection, Academic Press."}],"container-title":["Animals"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2615\/13\/17\/2802\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:45:56Z","timestamp":1760129156000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2615\/13\/17\/2802"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,9,4]]},"references-count":50,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2023,9]]}},"alternative-id":["ani13172802"],"URL":"https:\/\/doi.org\/10.3390\/ani13172802","relation":{},"ISSN":["2076-2615"],"issn-type":[{"value":"2076-2615","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,9,4]]}}}