Challenges in the development of sperm cryopreservation protocols for snakes

Abstract

Snake populations are declining worldwide, but research devoted to the development of sperm cryopreservation techniques for this taxon is very limited. Spermatozoa were collected postmortem from snakes of four squamate families (Elapidae, Colubridae, Viperidae and Pythonidae). Viability assessment was performed before and after cryopreservation. Spermatozoa were extended in TES and Tris (TEST) yolk buffer with 12% dimethylsulfoxide (DMSO) or 12% glycerol and frozen at a rate of 0.3°Cmin−1. The sperm quality index (SQI), representing three viability parameters (motility, plasma membrane and acrosome integrity), was determined. Despite some species differences, glycerol was a more effective cryoprotectant for Colubridae, whereas DMSO provided greater cryoprotection for spermatozoa of members of the other three families.

Cryopreservation of Sperm from an Endangered Snake with Tests of Post-Thaw Incubation in Caffeine

“…We then explored how this CPA control base mixture could be improved by the addition of energy sources, antioxidants, and non-membrane permeating CPAs and by varying the concentration and combination of permeating CPAs. These experimental tests were comprised of three categories ( Table 1 ): (1) the addition of non-permeable CPAs, amino acids, or energy source to the CPA control base (CPA ID #3–8); (2) the addition of glycerol below toxicity levels to the CPA control base (CPA ID #9–12) or the addition of glycerol at a cryoprotectant concentration (CPA ID #13–17) [ 25 , 29 , 31 ] in both Lake’s and TEST egg yolk buffers; and (3) a comparative test of the CPA control mix of 10% DMF + 2% methanol using TEST egg yolk buffer (CPA ID #2, Table 1 ).…”

“…This may be due in part to the filiform shape of reptilian sperm [ 26 ], which might make them more vulnerable to cryo-injury [ 27 ]. However, several recent studies have produced encouraging results [ 28 , 29 , 30 ]. The diversity of reproductive systems found across reptilia suggests that cryopreservation might need to be tailored for specific taxonomic groups or mode of reproduction.…”

“…Therefore, we need to conduct tests on a broad range of species to better understand if general principles exist and to identify common aspects of sperm cryopreservation methods in reptiles. For example, a recent comparative study of several species of snake found that the cryoprotective agent (CPA) mixture that provided the best recovery of post-thaw sperm motility varied by family [ 29 ], while an investigation of the toxicity of commonly used CPAs to freshly diluted sperm from two closely related species differed markedly in their responses [ 31 ]. Comparative studies that identify broadly applicable cryopreservation methods are indeed of significant scientific value, but the seemingly taxon-specific findings of recent studies suggest that initial efforts to develop cryopreservation methods should focus on species that are currently threatened with extinction.…”

Common goals, different stages: the state of the ARTs for reptile and amphibian conservation

“…The other two new reports of sperm cryopreservation in reptiles in this issue of RFD advanced our understanding of snake sperm cryopreservation (Young et al 2021(Young et al , 2022, adding to one prior published study reviewed in Mengden et al (1980) and another also recently published outside of this special issue (Sandfoss et al 2021). Taken together, this literature, with all the other reptile cryopreservation literature, confirms the focus of cryopreservation technology development in reptiles has been restricted to sperm, fulfilling the prediction by Clulow and Clulow (2016) that 'sperm cryopreservation will be the ART that yields the most progress in the near future' for reptiles.…”

“…(2) development of non-lethal methods of sperm collection that lead to good quality and quantities of sperm for successful cryopreservation; and (3) development of AI with cryopreserved sperm. We note many sperm cryopreservation protocols developed to date have used sperm collected postmortem, due to the large quantities and quality of sperm obtained and the ease of protocol development this confers (Young et al 2017(Young et al , 2021(Young et al , 2022Campbell et al 2021aCampbell et al , 2021bSandfoss et al 2021). However, encouragingly, two recent studies have looked at sperm collected non-lethally, including the first study on tuatara (Lamar et al 2021;Hobbs et al 2022).…”