109 DEVELOPMENT OF A SPERM CRYOPRESERVATION PROTOCOL FOR THE ARGENTINE BLACK AND WHITE TEGU (TUPINAMBIS MERIANAE)

Abstract

Only 891 of the approximately 5600 lizard species have been evaluated by the International Union for Conservation of Nature (IUCN). Of those, at least one-third are threatened with extinction. However, there is no organised effort to preserve their genetic diversity through semen banking. As part of an invasive species monitoring program, Argentine black and white tegus were captured in the Florida Everglades. Following postmortem examination, sperm was collected by flushing the vas deferens and used 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 TES and Tris (TEST)-yolk buffer with a final glycerol or dimethyl sulfoxide (DMSO) concentration of 8, 12, or 16%, and frozen in vials at 0.3, 1, or 6.3°C min^–1. Vials were thawed at 37°C for 90 s. Cryoprotectant (CPA) was removed by centrifugation and resuspension of the sperm pellet in M199, at which time (T0) all variables were assessed and expressed as the percentage of initial (%IMS, %IPL, and %IAC). Statistical tests included multivariate ANOVA (MANOVA) and Student's t-test. Over all CPA concentrations and freeze methods, DMSO was significantly better than glycerol in maintaining %IMS (P = 0.01; 37.32 ± 3.5 and 25.44 ± 3.09, respectively) and %IAC (P < 0.01; 81.45 ± 3.45 and 22.99 ± 3.03, respectively). The 2 CPA were equally successful in protecting %IPL (P = 0.77; 56.61 ± 5.62 and 54.42 ± 4.93, respectively). The slowest freeze rate of 0.3°C min^–1 was more successful than 1 and 6.3°C min^–1 in preserving %IMS (P = 0.01; 37.85 ± 3.29, 26.03 ± 4.45, and 21.91 ± 4.45, respectively) and %IPL (P < 0.01; 77.43 ± 2.54, 27.99 ± 3.44, and 42.32 ± 3.44, respectively). The %IAC was not significantly affected by freeze rate (P = 0.14; 58.06 ± 6.89, 36.14 ± 9.33, and 42.99 ± 9.33, respectively). The interaction between CPA and freeze method affected %IMS (P < 0.01) and %IAC (P < 0.01), but did not affect %IPL (P = 0.28). All variables were affected (P < 0.05) by concentration of cryoprotectant as well as the interaction between freeze method and cryoprotectant concentration. 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. Table 1 depicts the product of %IMS, %IPL, and %IAC for each treatment. Because there were significant interactions between treatment parameters, each treatment was compared with all others (a–f). The SQI analysis revealed that tegu sperm frozen at 0.3°C min^–1 in 8% DMSO exhibited a significantly higher post-thaw viability compared with all other treatments.

Table 1.  Sperm quality index (SQI) of thawed tegu sperm