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 × 106 cells/mL) followed by 4 µg/gbw GnRH-A (2.65 ± 0.21 × 106 cells/mL). Other stimulation protocols and doses induced sperm production, but at lower levels (ranging from 1.34 to 1.70 × 106 cells/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.