92 Sperm Cryopreservation in the Burmese Python (Python bivittatus) as a Model for Endangered Snakes

Abstract

The Burmese python (Python bivittatus) is listed as vulnerable by the International Union for Conservation of Nature (IUCN). Released pet Burmese pythons have detrimental effects on fauna native to southern Florida and are responsible for localised declines of several species in some parts of the Everglades National Park (IUCN, 2012; 10.2305/IUCN.UK.2012-1.RLTS.T193451A2237271.en). As part of an invasive species monitoring program, Burmese pythons were captured in the Florida Everglades and used as a model for the development of sperm cryopreservation protocols for endangered snakes. Sperm was collected by flushing the vas deferens postmortem and initial motility score (IMS; % motile × speed of progression²), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded before cryopreservation. Sperm was extended in TEST-yolk buffer with final dimethyl sulfoxide (DMSO) or glycerol (GLY) concentrations of 8, 12, or 16%, or combinations of DMSO and GLY with final concentrations of 4:4, 6:6, or 8:8%. Sperm in 500 µL of extender was frozen in vials at 0.3°C/min to –40°C before storage in liquid nitrogen. For each treatment, triplicate vials from each of 3 males were thawed at 37°C for 90 s. Cryoprotectant was removed by centrifugation and the sperm pellet was resuspended in TCM-199+HEPES. Sperm was evaluated at 22°C immediately following resuspension (T0) and at 60 (T60) minutes. All data were expressed as a percentage of initial (%IMS, %IPL, and %IAC). The effects of freeze method on %IMS, %IPL and %IAC were analysed by ANOVA and Tukey’s HSD test. Freeze method significantly affected %IMS at T0 (P = 0.0004) and T60 (P = 0.0001), with sperm frozen in the 6%DMSO:6%GLY and 4%DMSO:4%GLY treatments resulting in the highest %IMS at both T0 (19.4% and 17.7%, respectively) and T60 (26.7% and 14.4%, respectively). Regardless of cryoprotectant concentrations, sperm frozen in a combination of DMSO and GLY exhibited significantly higher %IMS than all treatments of DMSO or GLY alone (P < 0.0001 at T0 and T60). The %IPL was significantly affected by freeze method at T0 (P < 0.0001) and T60 (P = 0.0266). Sperm frozen in 8%DMSO:8%GLY and 6%DMSO:6%GLY retained greater %IPL at both T0 (69.1% and 65.7%, respectively) and T60 (47.8% and 49.9%, respectively). Acrosome integrity was significantly affected by freeze method at T0 (P < 0.0001) and sperm frozen in 8% DMSO resulted in the greatest %IAC (56.4%). In addition, all DMSO and DMSO:GLY treatments preserved a significantly greater proportion of intact acrosomes than GLY alone (P < 0.0001). To simplify these analyses and to determine the best overall freeze method for this species, a sperm quality index (SQI) was calculated, giving equal weight to each of the 3 measured indicators of cryosurvival. The SQI analysis revealed that Burmese python sperm frozen at 0.3°C/min in either 6%DMSO:6%GLY or 4%DMSO:4%GLY exhibited significantly higher post-thaw viability at T0 and T60 than all other treatments. This study represents the first comparative, comprehensive attempt to develop a sperm cryopreservation protocol for any snake species.