Reproduction Technology in Frog Conservation

In still declining and threatened frog (and fish) species the most promising approach for conservation is provided by a combination of captive breeding and the use of Reproduction Technologies including cryopreservation. Captive breeding is needed to provide oocytes (unfertilised eggs) from the same species or closely related species. Cryopreservation of sperm and nuclei is needed to provide security and genetic diversity. Even in moderately sized captive breeding programs genetic diversity will inevitably diminish with time. At some stage loss of genetic diversity (inbreeding depression) will result in animals which cannot survive in the natural environment.

The ability of cryopreservation to provide viable sperm has been shown in a wide variety of amphibians. With cryopreservation sperm, or other cells used to provide nuclei for cloning, should remain viable for more than 10,000 years. Even in species where some fertilising ability of the sperm is lost the use of sperm injection results in fertilisation.

Sperm for cryopreservation can be obtained from male frogs in the field with non-invasive methods such as the application of hormones in skin creams. Donor cells for the sampling of nuclei can be sampled from very small fin snips from tadpoles. Therefore, there are no ethical or practical reasons preventing the banking of the genome from frog (or fish) species.

However, even if we preserve sperm or cells we cannot preserve eggs or embryos, nor are we likely to in the future. So how are these sperm or nuclei to be used to generate a tadpole. For the foreseeable future oocytes must be available. Oocytes can then be fertilised directly by cryopreserved sperm or fertilised then used for cloning.

For reasons of compatibility it is optimal to have available the oocytes of the same species that provided the sperm or nuclei. However, other methods may have not successfully maintained the threatened species and oocytes are not available. Even in this case species can be generated from cryopreserved sperm (1,2) or nuclei (2).

1) If viable animals can be obtained by inter species crosses, back crossing between cryopreserved sperm and the foreign oocytes will re-establish the species.

2) If oocytes from females of a related species are available androgenesis could be used to generate males.

3) In the case of cloning from nuclei there is probably less chance of incompatibility between species than with direct crossing, due to the replacement of all of the genetic material during the cloning procedure. Cloning with the cryopreserved nuclei of frogs is an established technique.

There are institutions such as State or National Museums which have secure facilities for the storage of cryopreserved material. The distribution of samples between institutions greatly increases the security of storage.