Freeze tolerance and cryoprotection of erythrocytes from Dryophytes chrysoscelis

Date of Award


Degree Name

M.S. in Biology


Department of Biology


Advisor: Carissa Krane


Dryophytes chrysoscelis (Cope's gray treefrog) is a freeze-tolerant anuran that survives the freezing of its extracellular fluids. Treefrogs accumulate glycerol and urea in the plasma during cold acclimation and at the onset of freezing. It is hypothesized that glycerol and urea function as cryoprotectants by minimizing osmotically induced cell damage during freezing and thawing; and therefore, the post-freeze viability of red blood cells (RBCs) would improve when frozen in phosphate buffered saline (PBS) containing glycerol or urea. In the present study, erythrocytes were obtained from warm (22░C) and cold-acclimated (4░C) frogs and suspended in PBS. RBCs were frozen in PBS that ranged in osmolarity: PBS at 230 mM is hypoosmotic/hypotonic, 280 mM PBS is isosmotic/isotonic, and PBS was made hyperosmotic/hypertonic by the addition of 150 mM solutes. Post-freeze viability was determined with a hemolysis assay. Viability of erythrocytes from warm frogs was 31.6▒4.9% in 230 mM PBS but was enhanced to 59.4▒8.7% and 72.9▒4.3%, respectively, when cells were frozen with glycerol (p<0.05) or urea (p<0.001). Post-freeze viability of cells from warm-acclimated frogs improved from 18.9▒1.3% to 47.4▒5.2% with the addition of urea to 280 mM PBS (p<0.01). RBCs from cold-acclimated frogs had 45.8▒3.4% viability when frozen in 280 mM PBS which improved to 71.6▒8.9% or 71.9▒1.6%, respectively, when frozen with glycerol (p<0.01) or urea (p<0.001). The viability of RBCs from cold-acclimated frogs was not different when cells were frozen with glycerol, 71.6▒8.9%, or left unfrozen (0░C), 88.4▒3.5% (p>0.05). There was also no difference in viability between cells from cold-acclimated frogs that were frozen with urea, 71.9▒1.6%, or left unfrozen, 86.7▒3.8% (p>0.05). These data suggest that glycerol and urea are part of a complex cryoprotectant system in D. chrysoscelis.


Biology, Physiology, Copes gray treefrog, freeze tolerance, cryoprotection

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Copyright © 2018, author