title: Effects of hormonal stimulation on the concentration and quality of excreted spermatozoa in the critically endangered Panamanian golden frog (Atelopus zeteki).
abstract: Knowledge of basic gamete biology is critical to better protect and propagate endangered amphibian species and also to develop reproductive technologies combined with germplasm cryopreservation. The objectives of the study were to test different hormonal stimulations and then characterize the quantity and quality of Panamanian golden frog (Atelopus zeteki) spermatozoa. Following intraperitoneal injection of the gonadotropin-releasing hormone agonist (des-Gly10, D-Ala6, Pro-NHEt9--GnRH 1, 2 or 4 μg/g of body weight), human chorionic gonadotropin (hCG; 5 or 10 IU/gbw), or Amphiplex™ (0.4 μg/gbw GnRH-A + 10 μg/gbw metoclopramide hydrochloride), spermic urine samples from 29 males were collected at different time points (from 0.5 to 24 h post-injection) to analyze the concentration, motility, and morphology of the spermatozoa. Peak of sperm concentration was observed at 3.5 h post injection for all hormonal treatments. Amphiplex™ led to the highest sperm concentrations (4.45 ± 0.07 × 106cells/mL) followed by 4 μg/gbw GnRH-A (2.65 ± 0.21 × 106cells/mL). Other stimulation protocols and doses induced sperm production, but at lower levels (ranging from 1.34 to 1.70 × 106cells/mL). More than 60% of spermatozoa were motile following all treatments but the highest motility (>90%) was obtained from the 10 IU/gbw hCG treatment. Spermic urine samples obtained with all hormone treatments had higher pH (ranging from 7.1 to 7.8) than the urine alone (6.7-6.8). Spermatozoa were filiform and elongated with an apical acrosome, a mitochondrial sheath, a small midpiece and a long tail with an undulating membrane. More than 80% of cells were morphologically normal and 50-70% had intact DNA. These sperm characteristics were not influenced by hormonal treatments. This first comprehensive characterization of sperm samples following optimized hormonal stimulations in A. zeteki lays the foundation for more fundamental studies, reproductive technologies, and future preservation strategies.
authors.affiliation: Smithsonian Conservation Biology Institute, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC, 20008, USA.
authors.forename: Gina
authors.initials: G
authors.lastname: Della Togna
authors.affiliation: Department of Biology, University of Ottawa, 30 Marie Curie St., Ottawa, ON, K1N 6N5, Canada.
authors.forename: Vance L
authors.initials: VL
authors.lastname: Trudeau
authors.affiliation: Smithsonian Conservation Biology Institute, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC, 20008, USA.
authors.forename: Brian
authors.initials: B
authors.lastname: Gratwicke
authors.affiliation: Center for Animal Care Sciences, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC, 20008, USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Evans
authors.affiliation: Center for Animal Care Sciences, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC, 20008, USA.
authors.forename: Lauren
authors.initials: L
authors.lastname: Augustine
authors.affiliation: Smithsonian Conservation Biology Institute, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC, 20008, USA.
authors.forename: Han
authors.initials: H
authors.lastname: Chia
authors.affiliation: Center for Animal Care Sciences, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC, 20008, USA.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Bronikowski
authors.affiliation: Center for Animal Care Sciences, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC, 20008, USA.
authors.forename: James B
authors.initials: JB
authors.lastname: Murphy
authors.affiliation: Smithsonian Conservation Biology Institute, Smithsonian National Zoological Park, 3001 Connecticut Ave., NW, Washington, DC, 20008, USA. Electronic address: comizzolip@si.edu.
authors.forename: Pierre
authors.initials: P
authors.lastname: Comizzoli
mesh_terms: D000818:Animals; D002025:Bufonidae / Q000502:physiology*; D006063:Chorionic Gonadotropin / Q000494:pharmacology; D056727:Endangered Species; D007987:Gonadotropin-Releasing Hormone / Q000819:agonists* / Q000494:pharmacology; D008297:Male; D012098:Reproduction; D055101:Semen Analysis / Q000662:veterinary*; D053625:Sperm Retrieval / Q000662:veterinary; D013094:Spermatozoa / Q000166:cytology / Q000502:physiology
chemical_list: D006063:Chorionic Gonadotropin; D007987:Gonadotropin-Releasing Hormone
keywords: Atelopus zeteki; Hormones; Reproductive technologies; Spermatozoa