Initial Characterization of Male Southern Stingray (Hypanus americanus) Reproductive Parameters and Preliminary Investigation of Sperm Cryopreservation

SIMPLE SUMMARY: Understanding the reproductive biology of a species is critical for the development of biobanks and assisted reproductive techniques to aid in the genetic management of isolated populations. Male southern stingray (Hypanus americanus) reproductive examinations were opportunistically conducted in March and June. Semen and plasma were collected to characterize ejaculate parameters and to investigate the effect of plasma total testosterone on semen quality. Semen was used for preliminary sperm cryopreservation studies. Changes in semen quality were observed with changes in plasma testosterone concentrations and body conditions. Southern stingray spermatozoa were highly sensitive to cooling rates with slower rates, producing a higher post-thaw survival. ABSTRACT: This study investigated the reproductive biology and sperm cryopreservation of ex situ southern stingrays (Hypanus americanus) by semen collection and characterization and the development and validation of an enzyme-linked immunoassay for plasma total testosterone. Semen was collected in March and June using a manual massage technique, and the ejaculates were assessed for volume, pH, osmolarity, motility, status (0–5 scale: 0 = no forward progression, 5 = rapid linear progression) and total sperm count. Semen was extended in Hank’s elasmobranch ringer solution containing 10% DMSO, 10% glycerol or 5% glycerol with 5% N-methylformamide and cryopreserved using a conventional freezing method (~−50 °C/min) or a modified slow freezing method (~−3 °C/min). Body condition was scored from 1–5 and was noted to be low in March (1.93 ± 0.07) due to feeding practices and increased by June (2.93 ± 0.05) after dietary corrections were made. A concomitant increase (p < 0.05) in plasma total testosterone concentration and sperm motility was noted between March (8.0 ± 7.2 ng/mL, 5.71 ± 2.77%) and June (97.3 ± 11.3 ng/mL, 51.4 ± 14.3%). Samples cryopreserved using a modified slow freeze method (~−3 °C/min) had higher post-thaw motility and plasma membrane integrity than conventionally cryopreserved samples. Data indicate that southern stingray sperm morphometrics adheres to those of other elasmobranch species and that a slow cooling rate may be an avenue of research to improve southern stingray sperm survival during cryopreservation.