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We monitored the resumption of physiological functions in frogs that were frozen at -2 to -3 degrees C for 24 h and thawed rapidly (at 23-25 degrees C) or slowly (at 6-8 degrees C). Bodily functions were restored sooner during fast thawing, but this did not enhance the survival of frogs. The first physiological parameter to return was cardiac function, but during the early stages of thawing heart rates were lower than heart rates of unfrozen frogs at comparable body temperatures. Heart rates increased thereafter in conjunction with the rise in frog body temperatures. Spontaneous breathing and hindleg reflexes resumed after cardiac function, but neither response was exhibited by all frogs after the conclusion of the observation periods (3-4 h). Finally, isolated gastrocnemius muscles that had undergone in vitro freezing showed no significant (P greater than 0.05) impairment of twitch and tetanic tensions even as soon as 1 h after the onset of thawing. Body systems thus vary in their rates of recovery after nonlethal freezing episodes. Furthermore, recovery of specific body systems corresponds to essential needs that must be met immediately after thawing, such as reperfusion of body tissues.

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Freeze tolerance in the frog Rana sylvatica is supported by nonanticipatory mobilization of cryoprotectant (glucose) and redistribution of organ water. Other freeze-tolerant frogs may manifest these responses but differences exist. For example, the gray treefrog (Hyla versicolor) accumulates mostly glycerol as opposed to glucose. The current study reports additional novel features about cryoprotection in H. versicolor. Frogs were acclimated to low temperature for 12 weeks and frozen for 3 days at -2.4 degrees C. Some frogs were then thawed at 3 degrees C for 4 hr. Calorimetry revealed that frozen frogs had 53.9% +/- 11.1% of their body water in ice, and all frogs recovered following this procedure. Plasma glucose was low prior to the onset of freezing (1.1 +/- 0.9 micromol/ml) and it was 20x higher in postfreeze frogs. Constituting nearly 30% of plasma solute, glycerol was 117.2 +/- 13.6 micromol/ml prior to freezing and it remained equally high in postfreeze frogs. Liver water content was moderately lower in frozen frogs when compared to controls (62.9% +/- 3.7% vs. 68.6% +/- 1.7%), whereas postfreeze frogs excessively hydrated their livers (75.7% +/- 2.1%). Less-pronounced changes were seen in muscle water content. H. versicolor can mobilize its major cryoprotectant, glycerol, in response to extended cold acclimation, which is unique in comparison to other freeze-tolerant frogs, and it experiences only moderate organ dehydration during freezing. This species conforms with other freeze-tolerant frogs, however, by mobilizing glucose as a direct response to tissue freezing.

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Freeze tolerance and ice formation were examined in a population of Rana sylvatica from southern Ohio following their emergence in February. Frogs were tolerant of freezing at −2.5 °C but did not survive freezing at −5.5 °C. The level of glucose in the blood of frogs frozen for 48 h at −2.5 °C was fivefold higher than in unfrozen frogs. Both interpopulational and seasonal factors appeared to modify freeze tolerance and ice accumulation in Rana sylvatica when compared with previous studies. The directly determined time course of ice formation roughly paralleled the duration of the exotherm in this species. Ice content of frogs was determined using calorimetry and integrated to the specific heat of wet and dry masses. The equilibrium ice content represented nearly two-thirds of the total water content of these frogs. Freezing beyond this level proved lethal to frogs.

Five species of frogs from North A n~e n c a survive extensive freezing of their body fluids to temperatures as low as 8°C for periods lasting at least 2 wk. These frogs hibernate in leaf litter where subzero temperatures commonly occur during the winter. The onset of freezing triggers Liver glycogenolysis and the production of high concentrations of glucose or glycerol (to 100 X normal) that functions as a cryoprotectant against freezing Injury. Concomitantly the release of the latent heat of crystallization as body water freezes promotes the continued function of the cardiovascular system for many hours and serves to distribute glucose throughout the body. The water content of major organs is reduced by 50 U/:> or more during the first 24 h of freezing, wlth the water being relocated and frozen in other body spaces. Organ dehydration functions to concentrate cryoprotectant and to reduce mechanical damage by ice during freezing. As freezing progresses, breathing, heart beat, and most other v~t a l functions cease, but reanimation occurs ~v~t h l n a few hours after thawing. The evolution of freeze tolerance in these animals illustrates the h~g h l y flexible capacities of frogs to adapt to stressful environments

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