The terrestrial wood frog, Rana sylvatica, can tolerate sub-freezing environmental temperatures because of biochemical adaptations that protect cells from freezing. As frogs freeze, extracellular water is lost to ice crystals, and intracellular glycogen is converted into glucose. Both these events raise the intracellular solute concentration, thereby lowering the freezing point of the cell and preventing intracellular ice crystal formation. The physiological costs of these responses are not well known, and Irwin and colleagues have investigated the impact of the protective responses on locomotor endurance.
In the spring, thawed frogs can travel more than 400 m from their over-wintering sites to breeding ponds. As their breeding success depends on how quickly the frogs get to the pond, there's a lot at stake if the amphibians arrive too late or too exhausted to mate. To investigate the impact of freezing on locomotor endurance, frogs frozen for 36 h, were thawed and allowed to recover for 24, 48 or 96 h at 4°C. Then, the frogs were warmed to 15°C and placed on a treadmill where they walked until exhausted.
Despite the fact that thawed frogs displayed normal behavioural patterns,their endurance was significantly reduced relative to frogs that had been cooled to 0°C but not frozen. After 1 day of recovery, frozen frogs had about half the endurance of cooled frogs. After 4 days of recovery, both the thawed and cooled frogs' endurance increased, although frozen frogs only had the stamina that cooled frogs had recovered after 1 or 2 days. Increased endurance in the cooled frogs could be related to deleterious effects of cold exposure on both groups of frogs.
Because locomotor endurance may be limited by oxygen transport, metabolic efficiency and energy reserves, the team sampled blood and tissue from frozen,thawed and cooled frogs to see whether freezing had affected any of the processes. These samples were used to assess levels of erythrocyte damage during freezing (plasma hemoglobin), to assess the energetic poise of frogs during each stage of the thaw process (lactate accumulation) and to examine energy reserves (glycogen and glucose available for ATP generation).
Frozen frogs had elevated plasma hemoglobin, suggesting that extracellular ice crystals had lysed erythrocytes. However, within 24 h of thawing,hemoglobin was no longer detectable in plasma samples, and hematocrit was at prefreeze levels, indicating that blood water content had returned to normal.
Metabolite levels changed dramatically as the amphibians froze, and the responses were most pronounced in liver tissue. As the frogs froze, hepatic glycogen levels plummeted 95% and rose to just 17% of control levels after two days post-thaw. Hepatic glucose levels rose more than 5-fold during the freezing process and remained elevated after thawing. These observations demonstrate that the frozen frogs had dramatically lower energy reserves than control frogs, as glycogen degraded to glucose during the freezing process was only partially replenished by 48 h post-thaw. Hepatic lactate levels rose as the frogs froze but returned to control levels within a day of recovery,suggesting that in resting frogs oxygen delivery to tissues was adequate to maintain aerobic poise.
There was no clear indication that levels of tissue glycogen or glucose were correlated with locomotor endurance. Lactate levels increased in both cooled and frozen frogs after exercise to exhaustion, suggesting exhausted frogs were experiencing physiological hypoxia. Thus, endurance may be better correlated with oxygen delivery to metabolically active tissues than to tissue energy reserves.
Future studies with frogs acclimatized to different seasons and acclimated to different nutritional states may help resolve the roles of oxygen transport and delivery and tissue metabolites in locomotor endurance in thawed frogs.