Year-end Sale % OFF 
Abstract
Gene banking is arguably the best method available to prevent the loss of genetic diversity caused by declines in wild populations, when the causes of decline cannot be halted or reversed. For one of the most impacted vertebrate groups, the amphibians, gene banking technologies have advanced considerably, and gametes from the male line can be banked successfully for many species. However, cryopreserving the female germ line remains challenging, with attempts at cryopreserving oocytes unsuccessful due to their large size and yolk content. One possible solution is to target cryopreservation of early embryos that contain the maternal germ line, but consist of smaller cells. Here, we investigate the short term incubation, cryoprotectant tolerance, and cryopreservation of dissociated early embryonic cells from gastrulae and neurulae of the Striped Marsh Frog, Limnodynastes peronii. Embryos were dissociated and cells were incubated for up to 24 hours in various media. Viability of both gastrula and neurula cells remained high (means up to 40–60%) over 24 hours of incubation in all media, although viability was maintained at a higher level in Ca2+-free Simplified Amphibian Ringer; low speed centrifugation did not reduce cell viability. Tolerance of dissociated embryonic cells was tested for two cryoprotectants, glycerol and dimethyl sulphoxide; dissociated cells of both gastrulae and neurulae were highly tolerant to both—indeed, cell viability over 24 hours was higher in media containing low-to-medium concentrations than in equivalent cryoprotectant-free media. Viability over 24 hours was lower in concentrations of cryoprotectant higher than 10%. Live cells were recovered following cryopreservation of both gastrula and neurula cells, but only at low rates. Optimal cryodiluents were identified for gastrula and neurula cells. This is the first report of a slow cooling protocol for cryopreservation of amphibian embryonic cells, and sets future research directions for cryopreserving amphibian maternal germ lines.
Highlights
There is a growing interest in cryobanking amphibian cells and tissues as a conservation tool [1,2,3,4,5,6] to address the catastrophic collapse in amphibian biodiversity that has been well documented since 1990 [7,8,9,10,11,12,13]
The mean viability of dissociated cells taken from gastrulae (Fig. 2a) were high and within a narrow range; mean viability for neurula cells at 0.5 hr was highest in Ca2+-free Simplified Amphibian Ringer (SAR)/EDTA (83.0%, Fig. 2b), but ranged lower in other media (58.5 to 74.8%)
The data indicated that the recovery of viable cells after dissociation was generally higher in gastrulae than neurulae, but not in all equivalent treatments at equivalent times; in cells of both embryonic stages there was a significant decline over 24 hour of culture (Fig. 2)
Summary
The most obvious target for cryopreservation is the embryo; this is not likely to be achieved in the near term, given the failure of progress in cryopreserving fish oocytes and embryos, which have similar structural and biochemical properties such as large cell size and high yolk composition [14,15,16,17,18] This is unfortunate, as external fertilisation and development is the norm in anurans, and the resumption of development of cryopreserved embryos would complete a simple procedure to store, retrieve and restore the diploid genome (mitochondrial and nuclear) without the need for additional complicated post thaw procedures such as implantation. There is one report of the generation of nuclear transfer embryos in an anuran species from cryopreserved blastomeres [36]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
