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First published online October 21, 2005
Journal of Experimental Biology 208, 4079-4089 (2005)
Published by The Company of Biologists 2005
doi: 10.1242/jeb.01859

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Cryoprotection by urea in a terrestrially hibernating frog

Jon P. Costanzo^* and Richard E. Lee, Jr

Department of Zoology, Miami University, Oxford, OH 45056, USA

View larger version (23K): [in a new window]   Fig. 1. Seasonal variation in soil moisture (A) and its relationship to body water content (B), plasma osmolality (C) and plasma osmolyte concentrations (D) in R. sylvatica inhabiting a semi-natural, outdoor enclosure. Data presented in A are weekly means ± S.E.M. of values recorded at 6-h intervals. Data presented in B-D are means ± S.E.M.(N=3-6 frogs per group); all response variables varied significantly (ANOVA, P<0.0001). In D, `+' denotes each day that frogs were likely to be frozen, given that Te of a hibernating frog model was -0.4°C, the approximate equilibrium freezing/melting point of R. sylvatica tissues.

View larger version (18K): [in a new window]   Fig. 2. Cryoinjury to R. sylvatica erythrocytes frozen at -4 or -6°C in PBS (no urea) or PBS containing 40 mmol l-1 urea or 80 mmol l-1 urea. Damage was assessed as the percentage of total LDH leaking from cells and as the percentage of cells lysing. Within temperature-treatment groups, mean values (± S.E.M., N=6-7 replicates per group) identified by different letters differed significantly (repeated-measures ANOVA/Bonferroni; P<0.05).

View larger version (22K): [in a new window]   Fig. 3. Cryoinjury to R. sylvatica erythrocytes frozen at -4 or -6°C in PBS (no additive) or PBS containing 40 mmol l-1 urea, 40 mmol l-1 glucose or 40 mmol l-1 glycerol. Damage was assessed as the percentage of cells lysing. Within temperature-treatment groups, mean values (± S.E.M., N=7 replicates per group) identified by different letters differed significantly (repeated-measures ANOVA/Bonferroni; P<0.05).

View larger version (15K): [in a new window]   Fig. 4. Post-thaw viability of R. sylvatica organ samples incubated in PBS (no urea) or PBS containing 80 mmol l-1 urea and frozen at -4°C. Control samples were incubated in PBS but not frozen/thawed. Metabolic index is the percent reduction of indicator dye per milligram dry tissue per hour at 15°C. Mean values (± S.E.M., N=6-12 replicates per group) identified by different letters differed significantly (ANOVA/Bonferroni; P<0.05).

View larger version (14K): [in a new window]   Fig. 5. Cryoinjury to R. sylvatica organ samples incubated in PBS (no urea) or PBS containing 80 mmol l-1 urea and frozen at -4°C. Damage was assessed from LDH leakage from frozen/thawed samples; values were standardized to sample dry mass. Asterisk denotes that the mean value (± S.E.M., N=8 replicates per group) for urea-treated samples differed significantly (Wilcoxan signed-rank test; P<0.05) from the mean for samples tested in the absence of urea.