27 Comparison of cryoprotectants and their combinations in the optimisation of a sperm cryopreservation protocol in the Argentine black and white tegu (Salvator merianae)

Of the nearly 11 000 recognised squamate (lizards and snakes) species, at least one-third are threatened with extinction. A systematic semen banking effort is essential to preserve the genetic diversity of these species. Ten Argentine black and white tegu were captured from May 2013 to March 2014 in the Florida Everglades as part of an invasive species eradication program. Sperm collected from the vas deferens served as a model for the development of sperm cryopreservation protocols for related endangered lizards. Initial motility score (IMS, % motile × speed of progression²), plasma membrane integrity (IPL), and acrosome integrity (IAC) were recorded before freezing. Sperm was extended in TEST-Yolk buffer with 8%, 12%, or 16% dimethyl sulfoxide (DMSO) or glycerol (GLY), or 4:4%, 6:6%, 8:8% DMSO:GLY, and frozen in vials at 0.3°C min⁻¹ to −40°C before storing in liquid nitrogen. Three to four individual males were randomly assigned to each of the three cryopreservation treatment groups (3 concentrations each of DMSO, GLY, and DMSO:GLY). For each treatment, triplicate vials were thawed at 37°C for 90 s. Cryoprotectant (CPA) was removed by centrifugation and the sperm pellet was resuspended in M199 + HEPES. Sperm was evaluated at 37°C immediately following resuspension (T0) and at 60 min postincubation (T60). All data were expressed as a percentage of initial (%IMS, %IPL, and %IAC). Data for 8%, 12%, 16% DMSO and GLY (Young et al. 2017 Theriogenology 87, 55-63) were included for meta-data analysis and comparison with CPA combination treatments. The effects of CPA on %IMS, %IPL, and %IAC were analysed by ANOVA and Tukey’s honestly significant difference test. CPA did not affect %IMS at T0 (P = 0.1870), but by T60 sperm frozen in 8%, 12%, 16% DMSO demonstrated significantly greater %IMS than all other CPA treatments (P < 0.0001) except 4:4% DMSO:GLY. %IPL was significantly affected by CPA at T0 (P < 0.0001) and at T60 (P = 0.0107). Sperm frozen in 16% DMSO retained greater %IPL at T0 and T60, although the advantage was not significant. Overall, acrosome integrity was significantly affected by freeze method at T0 (P < 0.0001), and sperm frozen in 8% DMSO resulted in the greatest %IAC. Interestingly, as % GLY increased, %IAC decreased. To simplify these analyses and to determine the best overall CPA for this species, a sperm quality index (SQI) was calculated, giving equal weight to each of the three measured indicators of cryosurvival. The SQI analysis revealed that tegu sperm frozen in 8%, 12%, or 16% DMSO exhibited higher post-thaw viability at T0 (P < 0.0001) and T60 (P < 0.0001) than all other treatments. In contrast to our Burmese python study (Young et al. 2021 Reprod. Fertil. Dev. https://doi.org/10.1071/RD21023), these results suggest that combining DMSO and GLY does not provide greater cryoprotection of tegu sperm compared with DMSO alone. This study is the first to evaluate the combination of DMSO and GLY for the cryopreservation of lizard sperm.