Innovations in reproductive technologies could save reptile species from extinction
In this extract from Conservation Physiology, Lachlan Campbell et al. address the lacking research on reproductive technologies for non-mammalian species that can aid conservation efforts and even reverse species extinction. The authors look to the Australian lizard Varanus panoptes to investigate and develop an optimised protocol for reptile sperm cryopreservation.
The collection and storage of genomes through sperm cryopreservation, combined with associated assisted reproductive technologies (ARTs) for restoring animals and genes using the genetic material, could play a large role in threatened species conservation (Browne et al., 2011; Clulow and Clulow, 2016; Howard et al., 2015). Despite the potential to apply these technologies to all vertebrate taxa, reptiles are a relatively neglected taxon, with few successful sperm cryopreservation protocols developed for this group despite some attempts and typically much lower rates of successful post-thaw recovery (Browne, et al., 2011; Clulow and Clulow, 2016; Molinia et al., 2010). This is important because up to 25% of reptilian species are threatened with extinction globally and a further 25% are classed as data deficient (Böhm et al., 2013). Indeed, some reptile orders such as the Testudines and Crocodilia may be as high as 50% threatened with extinction globally (Böhm, et al., 2013; Grigg and Kirshner, 2015).
Given the limited number of reports and species investigated across the reptiles, whose major lineages have evolved very different reproductive systems, there is a strong imperative to understand general principles and shared aspects of sperm cryopreservation protocols in one or more model reptile species. Ideally, these studies would serve as a prelude for the development of optimized protocols within and between reptilian groups. Lizards are one of the most diverse reptile groups, yet sadly, of the known approximately 6100 lizard species globally, the International Union for Conservation of Nature (IUCN) has classified at least one third as threatened with extinction (Böhm, et al., 2013; Gibbons et al., 2000). Given their significance and diversity within squamate reptiles, lizards are an appropriate group to focus on for the development of an optimized sperm cryopreservation protocol, notwithstanding the success reported for one species (Young et al., 2017). One such lizard that is currently suffering large population declines is the yellow-spotted monitor (Varanus panoptes), an Australian species that has been severely impacted by the presence of the invasive cane toad (Rhinella marina) (Doody et al., 2009; Doody et al., 2014; Doody et al., 2017). Affected populations decline through increased mortality rates resulting from lethal toxic ingestion when the toad is consumed as prey (Doody, et al., 2009; Doody, et al., 2017; Ujvari and Madsen, 2009). Since its introduction to Australia in 1935 (Doody, et al., 2019; Lever, 2006), the cane toad has caused population crashes of up to 97% in V. panoptes (Doody, et al., 2009) as well as extirpations of this and at least one other monitor species in some parts of northern Australia (Doody, et al., 2017), which in turn has resulted in rippling effects throughout ecosystems via trophic cascades (Doody, et al., 2017; Doody et al., 2015). Varanus panoptes is thus an ideal model species for the development of sperm cryostorage and ARTs, because it is abundant ahead of the toad front for conducting experiments, but is threatened behind the invasion vanguard and exerts a disproportionately large influence over its ecosystem, thus warranting urgent conservation attention.
The addition of caffeine could thus potentially be a novel and simple means by which to stimulate post-thaw motility of lizard sperm, although to the best of our knowledge this principle has never been tested.
Sperm cryopreservation involves a series of steps that must be optimized to minimize the cell damage associated with deep freezing (Dinnyes et al., 2007). Dimethyl sulfoxide (DMSO) and glycerol are perhaps the two most commonly investigated cryoprotectants for the spermatozoa of wildlife species, although the relative success of each often varies depending on the species (Comizzoli et al., 2012). Cryoprotective agents (CPAs) also become increasingly toxic as the concentration increases (Best, 2015), which can result from non-specific toxicity via water molecule interference with the cell membrane or specific toxicity derived from the CPA type and concentration (Fahy, 1986; Fahy, 2010). It is important to understand the interplay of these factors in order to develop appropriate conditions for sperm cryopreservation.
In addition to optimizing cryoprotectant, media and cooling rates as a part of the freeze-thaw protocol, other factors may aid in increasing the viability of sperm and thus improve their motility characteristics in preparation for ARTs such as in vitro fertilization (IVF) or artificial insemination. For example, the addition of the phosphodiesterase inhibitor caffeine has been shown to increase intracellular levels of the second messenger, cyclic-adenosine monophosphate (cAMP) (Pukazhenthi et al., 2005; Saragusty, 2012), resulting in a consequential increase in forward progressive motility in unfrozen epididymal sperm of the lizard, Lacerta vivipara (Depeiges and Dacheux, 1985). In a similar context, cryopreserved mammalian spermatozoa have also been shown to display elevated levels of post-thaw motility following incubation in caffeine (Mbizvo et al., 1993; Rota et al., 2018; Schill et al., 1979; Stachecki et al., 1994). The addition of caffeine could thus potentially be a novel and simple means by which to stimulate post-thaw motility of lizard sperm, although to the best of our knowledge this principle has never been tested. Therefore, optimizing cryopreservation protocols requires attention to the separate components of cryoprotectant, media and cooling/thawing rates, but also may be enhanced by additional post-thaw treatments.
Conservation Physiology is an online only, fully open access journal published on behalf of the Society for Experimental Biology. Biodiversity across the globe faces a growing number of threats associated with human activities. Conservation Physiology publishes research on all taxa (microbes, plants and animals) focused on understanding and predicting how organisms, populations, ecosystems and natural resources respond to environmental change and stressors. Lachlan Campbell, Shenae L Cafe, Rose Upton, J Sean Doody, Brett Nixon, John Clulow, Simon Clulow contributed to the above excerpt.
