Cryopreservation of Sperm from Endangered Pallid Sturgeon

Abstract

We sought to develop sperm cryopreservation methods for the pallid sturgeon Scaphirhynchus albus, a federally listed endangered species. Males were injected with synthetic luteinizing hormone releasing hormone at 50 μg/kg of body weight. After 24 h, sperm were collected, diluted at a ratio of 1:4 (sperm : extender) with Hanks' balanced salt solution (HBSS; diluted to 100 milliosmoles/kg), and kept refrigerated until use. Methanol was used as a cryoprotectant at concentrations of 5, 10, and 15% (volume per volume) and was mixed 1:1 with HBSS before the experiment to reduce effects of initial mixing. Sperm were mixed with the cryoprotectant, loaded into 0.5-mL straws, packed into goblets (5 straws/goblet), and placed in the lower position on aluminum canes. Motility was estimated before freezing to determine the effects of cryoprotectant toxicity; there was no significant difference in motility at the concentrations tested (P = 0.4828). After a 2-min equilibration period, the canes were lowered into a nitrogen vapor shipping dewar. The cooling rate of −22°C/min was recorded by thermocouples inserted into 0.5-mL straws filled with extender and cryoprotectant. After 1 year of storage in liquid nitrogen, straws were thawed in a 40°C water bath for 9 s and motility was estimated. Postthaw motility did not differ among the cryoprotectants tested (P = 0.4880). Each sample was used to fertilize approximately 150 eggs, which were incubated at 21°C. Sperm that was cryopreserved with 5% or 10% methanol produced eggs with significantly higher hatch rates did sperm cryopreserved with 15% methanol (P < 0.0001). The development of techniques for cryopreserved sperm of pallid sturgeon allows for the creation of germplasm repositories that will aid in the recovery of this endangered species.