Insights on sperm assays and cryopreservation in six Neotropical pit vipers

Breeding snakes in captivity has become more and more relevant due not only to the growing interest on their venoms but also to the increasing number of endangered species worldwide. Unfortunately, studies on the formation of germplasm banks for these reptiles do not follow the same pace, and literature on sperm cryopreservation remains in its infancy when compared to other taxa. Herein, we first validated a sperm-egg binding assay (using chicken egg perivitelline membrane - EPM) and some nonfluorescent staining techniques for semen analysis of two pit viper genera (Bothrops and Crotalus), and then we investigated the protective effects of dimethylacetamide (DMA), dimethylformamide (DMF), and dimethylsulfoxide (DMSO) at different concentrations (3, 6 and 12%) throughout the freezing process in five species of lancehead and one of rattlesnake. Our validation process showed high correlations among sperm functional tests (including sperm-binding to EPM) and motion parameters. A total of 166 fresh ejaculates were acquired from 233 collection attempts, and 63.9% of these samples exhibited minimal motility for freezing (≥20%). During cryopreservation we observed that post-thaw motility and quality was improved by higher levels of cryoprotectants (CPA), regardless the CPA type. Lower concentrations of CPA were less harmful to sperm motility and progressive motility following the equilibrium phase, but were ineffective in protecting these cells from the freeze-thaw cycle. Likewise, higher CPA concentrations increased post-thaw integrity of the acrosome and plasma membrane for most species, except for rattlesnakes in which only 12% DMSO produced better outcomes.