Semen cryopreservation to conserve amphibian biodiversity

Amphibians are a unique group of vertebrates containing over 6.000 known species worldwide. Nearly one-third of the worlds amphibians are threatened. Currently, several attempts are applying to conserve and protect these declines in amphibians. Semen cryopreservation followed by androgenesis ultimately can counteract further endangerment and protect these amphibian species. In this project, the African clawed frog, Xenopus tropicalis, and axolotl, Ambystoma mexicanum, have been selected as models representative for Anura and Caudata, respectively. Success of semen cryopreservation will be tested and standardized in aspect of semen diluent, cryoprotectants, freezing and thawing rates. For a viability control, sperm motility will be measured with computer assisting motility analysis, percent of dead sperm cells, sperm acrosome integrity and fertility tests have to be conducted. For androgenesis, UV will be used to destruct the egg genome. The doses UV and the exposure time which required for destruction of egg genome will be established for both amphibian species. Then, the eggs are fertilized with the cryopreserved sperm. Restoration of the diploid state can be done by applying heat shock in which the exact temperature and exposure time will be tested. Heterozygous androgenesis, in which the diploid state is created through fusion of chromosome sets of two spermatozoa, will be also applied. Additionally, the inter- specific androgenic, in which frozen spermatozoa of Xenopus tropicalis, and, Ambystoma mexicanum can fertilize the genetic- destroyed eggs from Xenopus laevis and Ambystoma tigrinum will be tested. This is for the base of fertilization of a sperm from the threatened or endangered amphibian with an egg from other related available amphibians. The success of androgenesis experiments will be determined in the bases of tadpole survival rates and genetic compositions by the microsatellite technique.

Amphibians are a unique group of vertebrates containing over 6.000 known species worldwide. Nearly one-third (32%) of the world`s amphibians are threatened, representing 1.856 species, and about 168 species are believed to have gone. Additionally, at least 2.469 (43%) species are declining in population, indicating that the number of threatened species will continue to rise. The exact causes of the recent amphibian declines are not known, but there are several hypothesized causes which act either separately or synergistically. These causes include factors general to the overall biodiversity crisis as habitat destruction, alteration and fragmentation, introduced species, over- exploitation, climate change, increased UV radiation, chemical contaminants with biologically active substances, and the emerging infectious disease e.g. Chytridiomycosis which leads to up 100% mortality in many amphibian populations. Semen cryopreservation and application of genetic manipulation of the preserved spermatozoa have a high impact as a short-term action for conservation of the genomes of endangered and threatened amphibian species. Therefore, in the present project semen cryopreservation followed by genetic manipulation and gene banking was tried to counteract further endangerment of amphibian species. We have worked mainly on 2 models, the African clawed frog, Xenopus laevis, as model representative for Anura and axolotl, Ambystoma mexicanum, as model for Caudata. We could successfully establish a complete protocol to cryopreserve the sperm of X. laevis and to assess the post-thaw motility by computer assessing sperm motility programme, viability by a flourecence staining and fertility. In this protocoal we established the successful diluents, extender, freezing and thawing steps. Additionally, we established another successful protocol to activate and cryopreserve the sperm of the native common European frog, Rana temporaria. From these experiments, we realized that for each anuran species, a special cryopreservation protocol in aspect of semen diluents and used cryoprotectants is needed for each species and a single protocol for all frogs is unpractical. For the axolotl, A. mexicanum, we could success for the first time to collect fertile semen and eggs from these animals without sacrificing them. Also, a complete in-vitro fertilization protocol was successfully established. Additionally semen collection protocol was also successfully tested in some native urodele amphibians as fire salamander, Salamanadra salamandra, crested newt, Triturus cristatus and common newt, Lissotriton vulgaris. Also, sperm fine structure, motility behaviour, cryopreservation, and trials to apply the androgenesis technique in axolotl were conducted. Finally, a study was conducted on the motility activation and prolongation and metabolism of the urine spermatozoa in marsh frog, Pelophylax ridibundus. The results of this project have a great value in understanding the reproductive biology of amphibians and open new fields for further research and conservation methods.