Theses and Dissertations

Issuing Body

Mississippi State University


Willard, Scott T.

Committee Member

Kouba, Andrew J.

Committee Member

Ryan, Peter L.

Committee Member

Feugang, Jean M.

Committee Member

Holt, William V.

Date of Degree


Document Type

Dissertation - Open Access


Animal Physiology

Degree Name

Doctor of Philosophy (Ph.D)


College of Agriculture and Life Sciences


Animal Physiology Program


An alarming number of anuran (frog and toad) species are facing the threat of extinction in the wild. In efforts to address this conservation crisis, captive breeding programs are rapidly being established at zoos and research institutions worldwide. However, the captive management of anurans can be challenging, as their reproduction is a tightly regulated hormonal response to environmental stimuli, often unknown or absent in captivity. Consequently, ex-situ breeding efforts tend to be greatly hindered by a paucity of knowledge in anuran reproductive physiology and, for many species on the brink of extinction, time is running out. Assisted reproductive technologies (ARTs), such as exogenous hormone induction of gamete release, artificial fertilization for population augmentation, and cryopreservation for the long-term storage of genetics, have the potential to greatly enhance captive breeding efforts in lieu of natural breeding. Broadly, research aims were to develop assisted reproductive technologies for captive populations of the declining Southern Rocky Mountain boreal toad (Anaxyrus boreas boreas), the critically endangered Mississippi Gopher Frog (Lithobates captio sevosa) and Puerto Rican crested toad (Peltophryne lemur). Specific objectives were to a) trial the efficacy of exogenous hormone treatments on sperm release in male target species by characterizing spermiation response across time; b) investigate methods for increasing sperm longevity through cold-storage and cryopreservation techniques; c) ascertain motility recovery rates and functional capacity of cold-stored and frozen-thawed spermatozoa through artificial fertilization techniques, and; d) apply successfully developed ARTs to determine the feasibility of genetically linking in-situ and ex-situ populations of A. b. boreas, through artificial fertilization of male and female gametes from wild and captive toads, respectively. Research outcomes from this study include the successful development of exogenous hormone protocols, spermiation profiles and sperm cryopreservation techniques for all target species. Additionally, these studies enabled validation of an alternative method for increasing genetic diversity in captive anurans through in-situ-ex-situ gamete linkage. Overall, this research emphasizes the potential value of assisted reproductive technologies as conservation tools for supporting the recovery of endangered frog and toad species worldwide.