Sperm cryopreservation in an Australian skink (

Abstract

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.

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

“…In the only other known test of Lake's solution as a diluent for cryopreservation in snakes, Zacarotti et al [23], found no post-thaw motility; however, Lake's was used in combination with the poorly performing CPA dimethyl sulfoxide (DMSO) at a low concentration (2 and 4%) [31]. We found that the extender mixture containing TEST yolk plus glycerol [29,67] conferred a similar recovery of viability as the CPA control mixture containing Lake's buffer with 10% DMF, 2% methanol, and 5% egg. We are not aware of any cryopreservation study that has used this combination of extender and CPA mixture to cryopreserve sperm of a species of snake other than the Louisiana pinesnake.…”

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

“…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). We also believe that further studies on fundamental reptile reproductive ecology and physiology, along with testing fundamental hypotheses around cryopreserving reptile reproductive cells and tissues (Campbell et al 2021a) will aid with these conservation and research goals.…”

“…The small number of published lizard protocols stands in stark contrast to the more than 6000 species of lizard worldwide that constitute half of all extant reptile species (Böhm et al 2013). The study by Hobbs et al (2022) extends the range of cryoprotectant and extender formulations available for lizards while recognising interactions between cooling rates and cryoprotectant type. The study by Campbell et al (2021b) demonstrates it is possible to cryopreserve sperm of the yellow-spotted monitor lizard Varanus panoptes after 4 days of cold-storage and still recover acceptable levels of motility (~30%) and plasma membrane integrity (~50%).…”

“…Cryopreservation studies of reproductive cells and tissues in the reptile literature exclusively deal with the cryopreservation of sperm (Clulow and Clulow 2016), but the total number of cryopreservation studies remains low. For example, two new protocols for sperm cryopreservation in lizards (Campbell et al 2021b;Hobbs et al 2022) almost doubles the number of studies of sperm cryopreservation published for lizards worldwide, but only raises the number from three to five (Young et al 2017;Campbell et al 2020Campbell et al , 2021a. This compares to at least 26 reported studies on sperm cryopreservation for more than 35 species of amphibians prior to the RFD special issue (Clulow and Clulow 2016;Browne et al 2019;Clulow et al 2019).…”