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Assisted reproductive technologies for population and genetic management for threatened herpetofauna have grown substantially in the past decade. Here we describe experiments to optimise sperm cryopreservation in a model squamate, the eastern water skink Eulamprus quoyii. Small, concentrated volumes of highly motile spermatozoa were reliably collected from adult male E. quoyii by non-lethal ventral massage. Samples were used to: (1) test whether protein-rich diluents, namely Beltsville poultry semen extender (BPSE) and TES and Tris (TEST) yolk buffer (TYB), improve post-thaw quality metrics compared with Dulbecco's phosphate-buffered saline (DPBS); and (2) compare the efficacy of these diluents in combination with either 1.35 M glycerol or 1.35 M dimethyl sulfoxide (DMSO) at two freezing rates, fast (approximately –20°C min−1) versus slow (–6°C min−1). Glycerol and DMSO performed equally well in preserving spermatozoa under slow freezing rates. Under these conditions, the use of the complex diluents BPSE and TYB significantly improved post-thaw total motility compared with DPBS. Complex interactions occurred between cryodiluent type, cryoprotectant and freezing rate when testing fast versus slow freezing rates among treatment groups. Under slow freezing rates, DMSO was better at preserving membrane integrity and motility, regardless of diluent type, but successful fast freezing required complex diluents to support motility and membrane integrity, which has implications for implementation in a field setting. There is great scope to enhance genetic management within reptile conservation breeding programs by applying assisted reproductive technologies. We compared the efficacy of three sperm dilution media and two cryoprotectants to preserve sperm viability following cryopreservation by two methods. We found spermatozoa could survive freezing using either method provided the appropriate combination of buffer and cryoprotectant was used. This presents two alternative approaches worthy of further investigation for the cryopreservation of sperm from skinks both in the wild and ex situ. [ABSTRACT FROM AUTHOR]