25 Biobanking the veiled chameleon (Chamaeleo calyptratus); investigation of sperm cryopreservation protocols

Of the 202 recognised chameleon species, 37% are threatened with extinction and another 18% are near threatened. The veiled chameleon’s threatened status (Chamaeleo calyptratus) is listed as “least concern” by the International Union for Conservation of Nature. Released pet veiled chameleons have established breeding populations in South Florida and may contribute to the decline of native species on which they feed. As part of an invasive species monitoring program, 10 male chameleons were killed between September 2021 and July 2022 and served as a model for the development of sperm cryopreservation protocols for endangered chameleons. Sperm was collected from the vas deferens postmortem and plasma membrane integrity (IPL; live sperm), acrosome integrity (IAC), and initial motility score (IMS; % motile × speed of progression²) were recorded before cryopreservation. Sperm was extended in TEST-yolk buffer with dimethyl sulfoxide (DMSO) or a combination of DMSO and glycerol (GLY) and frozen at four different rates (Table 1) before liquid nitrogen storage. For each of the 15 treatments, 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 (T60). All data were expressed as a percentage of initial (% IPL, % IAC, and % IMS). The effect of treatment on % IPL, % IAC, and % IMS was analysed by ANOVA and Tukey’s HSD test. % IPL could not be accurately assessed because the majority of sperm took up the stain regardless of motility scores, indicating live sperm. Acrosome integrity was significantly affected by treatment at T0 (P < 0.0399); however, % IAC remained high in all treatments (88–100%). Treatment did not affect % IMS at T0 (P = 0.4071) or at T60 (P = 0.1216). Regardless of CPA, freeze rate affected % IMS at T60 (P = 0.0215), with the slowest freeze rate, retaining significantly higher motility scores than the fastest. Table 1 depicts % IMS at T60, as motility scores were highest at this time and more accurately reflect the time lapse between thawing and the ultimate goal of artificial insemination. Although not significant, the % IMS analysis revealed that veiled chameleon sperm frozen in 12% DMSO at 0.1°C min⁻¹ exhibited higher % IMS at T60 than all other treatments. While the number of available chameleons and low sperm volumes from these small reptiles limited this preliminary study, it represents the first attempt to develop a sperm cryopreservation protocol for any chameleon species.

Table 1. Initial sperm motility score (IMS) (% motile × speed of progression²) at 60 minutes post-cryopreservation¹