Toxicity Of Cryoprotective Agents To Semen From Two Closely Related Snake Species: The Endangered Louisiana Pinesnake (Pituophis Ruthveni) And Bullsnake (Pituophis Cantenifer)

Cryopreservation of sperm is an important tool for the conservation of threatened species. Many species of reptile are under considerable threat of extinction and there has been limited investigation of sperm cryopreservation in this taxonomic group. We performed a comparative test of toxicity to sperm of six commonly used cryoprotective agents (CPAs) at three concentrations (5%, 10%, 20%) from the Louisiana pinesnake, Pituophis ruthveni (n = 11), and the closely related bullsnake, Pituophis cantenifer (n = 8). Our objective was to determine the general toxicity of CPAs for cryopreservation in snakes and the cryoprotective ability of CPAs for sperm from the endangered Louisiana pinesnake. We conducted three experiments to: 1) evaluate the short-term in vitro toxicity of common CPAs in two closely related snake species, 2) determine the effectiveness of cryoprotectants for freezing and thawing semen in the Louisiana pinesnake, and 3) test the possible reduction in toxic effects of individual CPAs on semen of the Louisiana pinesnake by combining two of them. We used measures of motility including total motility, forward motility, and forward progressive motility index to characterize toxic effects and cryoprotective ability of each CPA. The results of our three experiments provide several important findings: 1) sperm of the bullsnake and Louisiana pinesnake responded differently to CPAs, 2) few CPAs provided any cryoprotection, as measured by percent recovered motility, in Louisiana pinesnakes, and 3) using mixtures of CPAs did not reduce toxicity relative to the best performing CPA on its own. Motility was best maintained at a concentration of 5% for CPAs tested; however, cryoprotection was best achieved with glycerol at 20% followed by DMA and DMF at 10%. These results provide further insight into the challenges faced by researchers attempting to cryopreserve sperm from snakes. Further comparative studies are required to determine the generality of cryopreservation methods in reptiles and suggest caution should be taken when developing cryopreservation protocols across species, particularly in snakes. All CPAs tested in this study were permeating CPAs and showed a significant acute toxic effect on motility at concentrations that provided cryoprotection. Future work in snakes might consider additional avenues of cryoprotection and combinations of multiple approaches.