Elizabeth R. Evans



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Cope’s gray treefrog Dryophytes chrysoscelis is among a unique group of freeze tolerant vertebrates that can successfully freeze and thaw up to 70% of extracellular fluids. Previous studies have shown that the accumulation of cryoprotectants such as glycerol is vital to surviving the physiological stresses associated with freezing and thawing. Though D. chrysoscelis certainly experiences several freeze-thaw cycles annually in a natural setting, studies prior to this work have included one round of freeze-thaw in experimental protocols only. The objective of this study, therefore, is to document the post-freeze recovery of D. chrysoscelis with multiple rounds of freeze-thaw in a laboratory setting by observing several morphological and physiological factors associated with post-freeze recovery. Before freezing, animals were first cold acclimated and maintained at an environmental temperature of 5°C. Environmental temperature was lowered to -2.5°C over 7 days, at which point animals were inoculated with ice to initiate crystallization. After 24 hours in a frozen state, animals were permitted to thaw at 5°C, concluding a single freeze-thaw cycle. Of this cohort, a subset was selected for two additional rounds of freeze-thaw in which animals were rapidly cooled to -2.5°C and frozen over 24 hours then permitted to thaw at 5°C for 24 hours. Each thawing period was documented by observation and digital photography to characterize changes in skin color, respiratory function, neurological function, and muscular function associated with post-freeze recovery. Preliminary data suggests that post-freeze recovery of most morphological and behavioral factors observed were delayed in animals that had been repeatedly frozen and thawed compared to animals that had been frozen and thawed once. The trends observed in this study suggest that animals may experience increased physiological stresses with repeated freeze-thaw, providing novel and ecologically relevant insights into the natural freeze tolerance of D. chrysoscelis.

Publication Date


Project Designation

Graduate Research

Primary Advisor

Carissa M. Krane

Primary Advisor's Department



Stander Symposium project, College of Arts and Sciences

Post-freeze recovery is delayed with repeated freeze-thaw cycles in Cope's gray treefrog Dryophytes chrysoscelis