Tissue glycogen and extracellular buffering limit the survival of red-eared slider turtles during anoxic submergence at 3 degrees C.

The goal of this study was to identify the factors that limit the survival of the red-eared slider turtle Trachemys scripta during long-term anoxic submergence at 3 degrees C. We measured blood acid-base status and tissue lactate and glycogen contents after 13, 29, and 44 d of submergence from ventricle, liver, carapace (lactate only), and four skeletal muscles. We also measured plasma Ca(2+), Mg(2+), Na(+), K(+), Cl(-), inorganic phosphate (P(i)), lactate, and glucose. After 44 d, one of the six remaining turtles died, while the other turtles were in poor condition and suffered from a severe acidemia (blood pH = 7.09 from 7.77) caused by lactic acidosis (plasma lactate 91.5 mmol L(-1)). An initial respiratory acidosis attenuated after 28 d. Lactate rose to similar concentrations in ventricle and skeletal muscle (39.3-46.1 micromol g(-1)). Liver accumulated the least lactate (21.8 micromol g(-1)), and carapace accumulated the most lactate (68.9 micromol g(-1)). Plasma Ca(2+) and Mg(2+) increased significantly throughout submergence to levels comparable to painted turtles at a similar estimated lactate load. Glycogen depletion was extensive in all tissues tested: by 83% in liver, by 90% in ventricle, and by 62%-88% in muscle. We estimate that the shell buffered 69.1% of the total lactate load, which is comparable to painted turtles. Compared with painted turtles, predive tissue glycogen contents and plasma HCO(-)(3) concentrations were low. We believe these differences contribute to the poorer tolerance to long-term anoxic submergence in red-eared slider turtles compared with painted turtles.

The physiology of hibernation in Canadian leopard frogs (Rana pipiens) and bullfrogs (Rana catesbeiana).

Canadian northern leopard frogs (Rana pipiens) and bullfrogs (Rana catesbeiana) were acclimated to 3 degrees C and submerged in anoxic (0-5 mmHg) and normoxic (Po(2) approximately 158 mmHg) water. Periodic measurements of blood Po(2), Pco(2), and pH were made on samples taken anaerobically from subsets of each species. Blood plasma was analyzed for [Na(+)], [K(+)], [Cl(-)], [lactate], [glucose], total calcium, total magnesium, and osmolality. Blood hematocrit was determined, and plasma bicarbonate concentration was calculated. Both species died within 4 d of anoxic submergence. Anoxia intolerance would rule out hibernation in mud, which is anoxic. Both species survived long periods of normoxic submergence (R. pipiens, 125 d; R. catesbeiana, 150 d) with minimal changes in acid-base and ionic status. We conclude that ranid frogs require a hibernaculum where the water has a high enough Po(2) to drive cutaneous diffusion, allowing the frogs to extract enough O(2) to maintain aerobic metabolism, but that an ability to tolerate anoxia for several days may still be ecologically meaningful.

The physiology of overwintering in a turtle that occupies multiple habitats, the common snapping turtle (Chelydra serpentina).

Common snapping turtles, Chelydra serpentina (Linnaeus), were submerged in anoxic and normoxic water at 3 degrees C. Periodic blood samples were taken, and PO(2), PCO(2), pH, [Na(+)], [K(+)], [Cl(-)], total Ca, total Mg, [lactate], [glucose], hematocrit, and osmolality were measured; weight gain was determined; and plasma [HCO(3)(-)] was calculated. Submergence in normoxic water caused a decrease in PCO(2) from 10.8 to 6.9 mmHg after 125 d, partially compensating a slight increase in lactate and allowing the turtles to maintain a constant pH. Submergence in anoxic water caused a rapid increase in lactate from 1.8 to 168.1 mmol/L after 100 d. Associated with the increased lactate were decreases in pH from 8.057 to 7.132 and in [HCO(3)(-)] from 51.5 to 4.9 mmol/L and increases in total Ca from 2.0 to 36.6 mmol/L, in total Mg from 1.8 to 12.1 mmol/L, and in [K(+)] from 3.08 to 8.45 mmol/L. We suggest that C. serpentina is tolerant of anoxic submergence and therefore is able to exploit habitats unavailable to some other species in northern latitudes.

Geographic variation of the physiological response to overwintering in the painted turtle (Chrysemys picta).

We compared the physiological responses of latitudinal pairings of painted turtles submerged in normoxic and anoxic water at 3 degrees C: western painted turtles (Chrysemys picta bellii) from Wisconsin (WI) versus southern painted turtles (Chrysemys picta dorsalis) from Louisiana (LA), Arkansas (AR), and Alabama (AL), and eastern painted turtles (Chrysemys picta picta) from Connecticut (CT) versus C. p. picta from Georgia (GA). Turtles in normoxic water accumulated lactate, with C. p. bellii accumulating less than (20 mmol/L) the other groups (44-47 mmol/L), but with relatively minor acid-base and ionic disturbances. Chrysemys picta bellii had the lowest rate of lactate accumulation over the first 50 d in anoxic water (1.8 mmol/d vs. 2.1 for AR C. p. dorsalis, 2.4 mmol/d for GA C. p. picta, and 2.5 mmol/d for CT C. p. picta after 50 d and 2.6 mmol/d for AL C. p. dorsalis after 46 d). Northern turtles in both groups survive longer in anoxia than their southern counterparts. The diminished viability in C. p. dorsalis versus C. p. bellii can be partially explained by an increased rate of lactate accumulation and a decreased buffering capacity, but for the CT and GA C. p. picta comparison, only buffering capacity differences are seen to influence survivability.

Comparative shell buffering properties correlate with anoxia tolerance in freshwater turtles.

Freshwater turtles as a group are more resistant to anoxia than other vertebrates, but some species, such as painted turtles, for reasons not fully understood, can remain anoxic at winter temperatures far longer than others. Because buffering of lactic acid by the shell of the painted turtle is crucial to its long-term anoxic survival, we have tested the hypothesis that previously described differences in anoxia tolerance of five species of North American freshwater turtles may be explained at least in part by differences in their shell composition and buffering capacity. All species tested have large mineralized shells. Shell comparisons included 1) total shell CO2 concentration, 2) volume of titrated acid required to hold incubating shell powder at pH 7.0 for 3 h (an indication of buffer release from shell), and 3) lactate concentration of shell samples incubated to equilibrium in a standard lactate solution. For each measurement, the more anoxia-tolerant species (painted turtle, Chrysemys picta; snapping turtle, Chelydra serpentina) had higher values than the less anoxia-tolerant species (musk turtle, Sternotherus odoratus; map turtle, Graptemys geographica; red-eared slider, Trachemys scripta). We suggest that greater concentrations of accessible CO2 (as carbonate or bicarbonate) in the more tolerant species enable these species, when acidotic, to release more buffer into the extracellular fluid and to take up more lactic acid into their shells. We conclude that the interspecific differences in shell composition and buffering can contribute to, but cannot explain fully, the variations observed in anoxia tolerance among freshwater turtles.

Physiology of hibernation in Rana pipiens: metabolic rate, critical oxygen tension, and the effects of hypoxia on several plasma variables.

Rates of O(2) consumption (.VO(2)) were determined for adult northern leopard frogs (Rana pipiens) submerged at 3 degrees C at water PO(2)s (P(w)O(2)) ranging from 0-160 mmHg. The critical O(2) tension (P(c)) was 36.4 mmHg. Hematocrit and blood levels of PO(2), glucose, lactate, pH, [Na(+)], [K(+)], and osmolality were determined for frogs submerged for two days. Above a P(w)O(2) of 50 mmHg, blood PO(2) ranged from 1-7 mmHg, which was sufficient to allow the frogs to function entirely aerobically. Plasma [lactate] increased as P(w)O(2) fell below 50 mmHg, the increase preceding significant changes in any other variable, and apparently preceding a fall in .VO(2). Most other variables showed little or no change from those of air-breathing control animals, even during anoxia. We present an analysis of the importance of a large decrease in P(c) in permitting frogs to successfully overwinter in icebound ponds and of the factors that contribute to that decrease.

Lactate accumulation, glycogen depletion, and shell composition of hatchling turtles during simulated aquatic hibernation.

We submerged hatchling western painted turtles Chrysemys picta Schneider, snapping turtles Chelydra serpentina L. and map turtles Graptemys geographica Le Sueur in normoxic and anoxic water at 3 degrees C. Periodically, turtles were removed and whole-body [lactate] and [glycogen] were measured along with relative shell mass, shell water, and shell ash. We analyzed the shell for [Na+], [K+], total calcium, total magnesium, Pi and total CO2. All three species were able to tolerate long-term submergence in normoxic water without accumulating any lactate, indicating sufficient extrapulmonary O2 extraction to remain aerobic even after 150 days. Survival in anoxic water was 15 days in map turtles, 30 days in snapping turtles, and 40 days in painted turtles. Survival of hatchlings was only about one third the life of their adult conspecifics in anoxic water. Much of the decrease in survival was attributable to a dramatically lower shell-bone content (44% ash in adult painted turtles vs. 3% ash in hatchlings of all three species) and a smaller buffer content of bone (1.3 mmol g(-1) CO2 in adult painted turtles vs. 0.13-0.23 mmol g(-1) CO2 in hatchlings of the three species). The reduced survivability of turtle hatchlings in anoxic water requires that hatchlings either avoid aquatic hibernacula that may become severely hypoxic or anoxic (snapping turtles), or overwinter terrestrially (painted turtles and map turtles).

Avenues of extrapulmonary oxygen uptake in western painted turtles (Chrysemys picta belli) at 10 degrees C.

The major avenues of extrapulmonary oxygen uptake were determined on submerged western painted turtles (Chrysemys picta bellii) at 10 degrees C by selectively blocking one or more potential pathways for exchange. Previous work indicated that the skin, the cloaca, and the buccopharyngeal cavity can all contribute significantly in various species of turtles. O(2) uptake was calculated from the rate of fall in water P(O(2)) in a closed chamber. Two series of experiments were conducted: in Series 1, each of the potential avenues was mechanically blocked either singly or in combination; in Series 2, active cloacal and buccal pumping were prevented pharmacologically using the paralytic agent rocuronium. In addition in Series 2, N(2)-breathing preceded submergence in some animals and in one set of Series 2 experiments arterial blood was sampled and analyzed for pH, lactate, P(O(2)), and P(CO(2)). Results in both Series 1 and Series 2 revealed that prevention of cloacal and/or buccopharyngeal exchange did not significantly affect total O(2) uptake. Interfering with skin diffusion in Series 1, however, significantly reduced O(2) uptake by 50%. N(2)-breathing prior to submergence in Series 2 did not affect O(2) uptake in paralyzed turtles but significantly increased uptake in unparalyzed turtles without catheters. Blood analysis revealed that all submerged turtles developed lactic acidosis, but the rate of rise in lactate was significantly lower in paralyzed animals. We conclude that passive diffusion through the integument is the principal avenue of aquatic O(2) uptake in this species.