STRUCTURAL AND FUNCTIONAL CHARACTERIZATION OF THE PANAMANIAN GOLDEN FROG (ATELOPUS ZETEKI) SPERMATOZOA – IMPACT OF MEDIUM OSMOLALITY AND CRYOPRESERVATION ON MOTILITY AND CELL VIABILITY

Abstract

The Panamanian Golden Frog (Atelopus zeteki, Anura: Bufonidae), an endemic species from Panama, is critically endangered and believed to be extinct from the wild. Infectious disease, habitat destruction and pet trade are among the major causes of the populations decline. Conservation initiatives in Panama and the US have established captive breeding programs for the species but there is still much to be learned about its reproductive physiology to manage and sustain populations using Assisted Reproductive Technologies (ARTs). The main objectives of this dissertation were: 1) characterize the general sperm parameters and assess the effect of hormonal stimulation on motility, forward progressive motility, DNA integrity, sperm morphology and seasonality; 2) identify the effect of extracellular conditions, mainly dilution and temperature, on motility, forward progressive motility, duration of motility, morphology and DNA integrity; and 3) evaluate the effect of cryoprotectants on motility, forward progressive motility and sperm DNA integrity before and after freezing. Results demonstrated that: 1) A. zeteki sperm morphology is similar to that described in other Bufonids; the species successfully produces high quality spermatozoa when stimulated with intraperitoneal injection of Amphiplex, GnRH and hCG; hormonal stimulation is not detrimental to the motility parameters, cell morphology or DNA integrity; and, there is no seasonal effect on the response to the hormonal stimulation. These results indicate that the use of hormone treatments can be included in captive breeding programs to safely collect good quality sperm; 2) dilution of spermic urine in water highly reduces sperm motility, forward progressive motility, DNA and morphological integrity while storage of spermic urine at 4 °C preserves sperm quality for at least 46 min after collection; and 3) some recovery of viable A. zeteki spermatozoa after cryopreservation can be achieved by equilibrating the ARS-diluted samples for 5 min at 4 °C in CPA3-REY, using step-wise cooling before plunging the samples in LN2. Collective results offer missing information on the reproductive biology of the male A. zeteki and lead to the application of ARTs for the captive management of this charismatic but critically endangered species