1. Resurrection of an Extinct SpeciesThe Effects of Activators and Cryoprotectants And its Future Applications

2. Eleutherodactylus Eleutherodactylus in Greek means “free fingers” Largest monophyletic vertebrate Direct Development Exhibit parental care

3. Why Eleutherodactylus? Eleutherodactylus species have been experiencing a decline Thus, creating a need for ecological remediation via resurrecting an extinct species

4. Why Eleutherodactylus coqui? Eleutherodactylus coqui native to Puerto Rico Eleutherodactlus eneidae is extinct and native to Puerto Rico E. coqui enucleated egg and UV irradiated sperm for egg activation and sperm from E. eneidae

6. Six Areas of Focus Induction of Ovulation Sperm Activation Sperm Freezing and Thawing In Vitro Fertilization UV Irradiatian of Sperm Sperm Decondensation

7. Induction of Ovulation Initial problems with ovulation –Obtaining sufficient female frog eggs Initial problems corrected v –Use of estrogen related hormones via IP injection

8. Sperm Activation Adequate amount of sperm available Sperm must be active Sperm extraction –Sacrifice 5 frogs –Removed testes and extracted sperm

9. Preparation of Sperm Samples Each sample placed in isotonic buffer solution (ESB) representing the control Each sample from ESB preparedin 3mM concentrations of 5 different activators –Theophylline –Pentoxyfylline –Jelly coat –2-deoxyadenosine –Caffeine

10. Preparation of Sperm Samples cont. 100 sperm were counted in each sample –Moving sperm vs. nonmoving sperm

11. Sperm Activators vs. Control

12. Sperm Freezing and Thawing The control consisted of –50ul ESB/sperm + 50ul ESB 4 cryoprotectans samples were obtained using extracted sperm –A). 50ul ESB/sperm + 50ul FBS/glycerol –B). 50ul ESB/sperm + 50ul FBS/sucrose –C). 50ul ESB/sperm + 50ul FBS/DMSO –D). 50ul ESB/sperm + 50ul FBS

13. Sperm Freezing and Thawing cont. Cryoprotectant sperm samples frozen in -80C freezer Sperm samples prepared on slides Two dyes were applied –SYBER 14 –Propidium iodide The purpose of these dyes were to assess intact vs. ruptured membranes

14. Cryoprotectants vs. Control SYBER 14 –Intact membranes fluoresces green Propidium iodide –Ruptured membranes fluoresces red

15. In Vitro Fertilization Induced ovulated eggs and extracted sperm were used in the in vitro fertilization experiment AMP B antifungal was used to prevent bacterial growth and infection One egg was successfully fertilized

18. Sperm Nuclear Decondensation Importance in initiating development Sperm placed in egg cytoplasm decondensed A DNA specific fluorescent dye was used to observe decondensation

19. Future Applications Resurrection of an extinct species via in vitro fertilization E. coqui host enucleated egg injected with UV irradiated sperm Decondensed sperm from E. eneidae injected into host egg and sperm Ecological remediation of an extinct frog species due –Human population growth –Environmental altercation

20. Scott F. Michael John Carlson S. Blair Hedges Shawn F. Vincent Acknowledgements