Gamete Collection, Artificial Fertilization and Captive-Rearing of Eggs in a Terrestrial-Breeding Anuran with Parental Care: IAlytes obstetricans/I

Amphibians are the most threatened vertebrate class on Earth, and assisted reproductive technologies are essential tools to complement other conservation efforts. We established initial assisted reproductive technologies for Alytes obstetricans, a terrestrial breeding anuran whose males carry the egg strings twined around their hind limbs until hatching. Non-lethal gamete collection by injection of hormones was performed and allowed the collection of hormonally-induced sperm and eggs. However, poor response to hormonal stimulation of spermiation was obtained (range 17 to 50% of treated males). These gametes were combined and fertilization took place and completed embryo development in 10% of eggs. In addition, two methods for captive-rearing of embryos were tested: keeping the eggs together (mimicking natural arrangement) or splitting the eggs (to reduce fungal propagation) on moist paper. When eggs were separated, all clutches exhibited embryo survival (ranging from 27 to 61%) while only 8% of clutches presented embryo survival when eggs were kept together. This method for captive-rearing of eggs could be implemented in captive breeding programs for different species of Alytes. Although successful, sperm collection and artificial fertilization should be optimized to contribute to the management of wild and captive populations of this and related species. 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 sur