Seasonal change in the capacity for supercooling by neonatal painted turtles.

Hatchlings of the North American painted turtle (Chrysemys picta) typically spend their first winter of life inside the shallow, subterranean nest where they completed incubation the preceding summer. This facet of their natural history commonly causes neonates in northerly populations to be exposed in mid-winter to ice and cold, which many animals survive by remaining unfrozen and supercooled. We measured the limit of supercooling in samples of turtles taken shortly after hatching and in other samples after 2 months of acclimation (or acclimatization) to a reduced temperature in the laboratory or field. Animals initially had only a limited capacity for supercooling, but they acquired an ability to undergo deeper supercooling during the course of acclimation. The gut of most turtles was packed with particles of soil and eggshell shortly after hatching, but not after acclimation. Thus, the relatively high limit of supercooling for turtles in the days immediately after hatching may have resulted from the ingestion of soil (and associated nucleating agents) by the animals as they were freeing themselves from their eggshell, whereas the relatively low limit of supercooling attained by acclimated turtles may have resulted from their purging their gut of its contents. Parallels may, therefore, exist between the natural-history strategy expressed by hatchling painted turtles and that expressed by numerous terrestrial arthropods that withstand the cold of winter by sustaining a state of supercooling.

The role of the integument as a barrier to penetration of ice into overwintering hatchlings of the painted turtle (Chrysemys picta).

Hatchlings of the North American painted turtle (Chrysemys picta) spend their first winter of life inside a shallow, subterranean hibernaculum (the natal nest) where they may be exposed for extended periods to ice and cold. Hatchlings seemingly survive exposure to such conditions by becoming supercooled (i.e., by remaining unfrozen at temperatures below the equilibrium freezing point for body fluids), so we investigated the role of their integument in preventing ice from penetrating into body compartments from surrounding soil. We first showed that hatchlings whose epidermis has been damaged are more likely to be penetrated by growing crystals of ice than are turtles whose cutaneous barrier is intact. We next studied integument from a forelimb by light microscopy and discovered that the basal part of the alpha-keratin layer of the epidermis contains a dense layer of lipid. Skin from the forelimb of other neonatal turtles lacks such a layer of lipid in the epidermis, and these other turtles also are highly susceptible to inoculative freezing. Moreover, epidermis from the neck of hatchling painted turtles lacks the lipid layer, and this region of the skin is readily penetrated by growing crystals of ice. We therefore conclude that the resistance to inoculation imposed by skin on the limbs of hatchling painted turtles results from the presence of lipids in the alpha-keratin layer of the epidermis. Neonates apparently are able to avoid freezing during winter by drawing much of the body inside the shell, leaving only the ice-resistant integument of the limbs exposed to ice in the environment. The combination of behavior and skin morphology enables overwintering hatchlings to exploit an adaptive strategy based on supercooling.

Accumulation of lactate by supercooled hatchlings of the painted turtle (Chrysemys picta): implications for overwinter survival.

Hatchlings of the North American painted turtle (Chrysemys picta) typically spend their first winter of life inside the shallow, subterranean nest where they completed embryogenesis the preceding summer. Neonates at northern localities consequently may be exposed during winter to subzero temperatures and frozen soil. Hatchlings apparently survive exposure to such conditions by supercooling, but the physiological consequences of this adaptive strategy have not been examined. We measured lactate in hatchling painted turtles after exposure to each of three temperatures (0 degree C, -4 degrees C, and -8 degrees C) for three time periods (5 days, 15 days, and 25 days) to determine the extent to which overwintering hatchlings might rely on anaerobic metabolism to regenerate ATP. Whole-body lactate increased with increasing duration of exposure and decreasing temperature, and the highest levels were associated with the group that experienced the highest mortality. These results indicate that animals may develop a considerable lactic acidosis during a winter in which temperatures fall below 0 degree C for weeks or months and that accumulation of lactate may contribute to mortality of overwintering animals.

Availability of water affects organ growth in prenatal and neonatal snapping turtles (Chelydra serpentina).

We manipulated the amount of water that was available to prenatal and neonatal snapping turtles (Chelydra serpentina) in order to assess the impact of water on growth by different organs in these animals. Three treatments were used: (1) turtles that completed their incubation on a wet substrate, (2) turtles that completed their incubation on a dry substrate, and (3) turtles that spent a few days in water after completing incubation on a dry substrate. Turtles hatching on a dry substrate (treatment 2) were smaller than animals in the other two treatments (which did not differ in size), so data for mass of different organs were adjusted by ANCOVA to remove effects of body size. Scaled masses of liver, stomach, lungs, kidneys, and small intestine did not differ between turtles emerging in wet environments and those hatching in dry environments, but hearts of turtles hatching in dry settings were substantially larger than those of animals hatching in wet ones. Thus, the mass of most organs in turtles developing in wet and dry environments scaled to body size, whereas the heart was hypertrophied in embryos developing in dry environments. Turtles that spent a few days in water after hatching from eggs in dry environments grew rapidly in size, and the increase in body size was accompanied by disproportionately rapid growth in the liver, stomach, lungs, kidneys, and small intestine. The heart did not increase in size during this period, despite the substantial increase in body mass over that at hatching. The enlarged heart of turtles hatching on dry substrates may have been caused by a circulatory hypovolemia late in incubation; the rapid growth of organs other than the heart when these animals were placed in water may reflect a release from constraints on growth once circulatory volume was restored.

Evaluation of a protocol for studying the chick chorioallantoic membrane in vitro.

Explants of eggshell with and without the chorioallantoic membrane were taken from fertile chicken eggs on day 16 of incubation and exposed in vitro to inhibitors (acetazolamide and benzolamide) of carbonic anhydrase to determine if enzyme inhibition affected release of calcium from the shell. A separate experiment examined the effect of the metabolic poison dinitrophenol (DNP) on release of calcium from explants. Explants with the chorioallantois in situ released more calcium than those lacking the epithelium, but neither the enzyme inhibitors nor DNP affected release of calcium. The lack of effect of the enzyme inhibitors could indicate that activity of carbonic anhydrase is not as important to the release of calcium from the eggshell as has been assumed. However, the absence of an effect of DNP instead indicates that release of calcium mediated by the chorioallantois in vitro simply lacks physiological relevance. Thus, results of this investigation raise doubts that the mechanism underlying release of calcium from the eggshell can be assessed in vitro.

Hatchling painted turtles (Chrysemys picta) survive only brief freezing of their bodily fluids.

Neonatal painted turtles (Chrysemys picta) spend their first winter inside the shallow, subterranean nest cavity where they completed embryogenesis. Consequently, hatchlings at high latitudes may be exposed to ice and cold during the winter. This study was undertaken to determine how long hatchlings withstand freezing at temperatures slightly below 0 degree C because tolerance for freezing has been proposed to be the key to survival by overwintering animals. A thermocouple was glued to the carapace of each hatchling. The animal was dipped in water to provide a site of nucleation of ice and was then placed into a glass jar that was partially immersed in a circulating bath at -2 degrees C. Carapace temperature was monitored throughout the procedure. When a freezing exotherm was detected, timing of the freezing event began. Animals were maintained in a frozen state for 12-48 h prior to being warmed to room temperature. Of the 39 hatchlings, 22 did not survive, and mortality increased as the duration of freezing increased. Logistic regression indicates that no turtle would have survived in a frozen state for more than 54 h. These results indicate that hatchlings can survive only brief exposure to freezing of the body fluids. Thus, hatchlings cannot tolerate freezing during prolonged periods of cold.

The effect of calcium-regulating hormones on transport of calcium across the chorioallantoic membrane of the chicken embryo.

The hormonal form of vitamin D3 (1,25(OH)2D3), parathyroid hormone (PTH), or appropriate vehicle were injected into the yolk sac of eggs of domestic fowl on days 16 and 17 of incubation. The chorioallantoic membrane (CAM) and overlying inner shell membrane were removed from eggs on day 18 and mounted in a Ussing-type apparatus. Transport of calcium was assessed by monitoring movements of radiolabeled calcium. Transport of calcium from the chorionic aspect of the CAM to the allantoic aspect increased considerably with time for all treatment groups except the one receiving PTH. "Back-flux" of calcium (movement of calcium from the allantoic aspect to the chorionic) was negligible for all treatment groups at all sampling periods. PTH treatment did not affect flux of calcium from allantois to chorion but reduced flux from chorion to allantois considerably. The underlying cause of this effect has not been identified. The hormonal form of vitamin D3 did not affect flux of calcium in either direction. These data raise the possibility that control of calcium transport by the CAM may not be the primary function of the vitamin D hormone.

Cold tolerance in hatchling painted turtles (Chrysemys picta): supercooling or tolerance for freezing?

We studied tolerance for cold in hatchling painted turtles (Chrysemys picta) from Lake Metigoshe, Bottineau County, North Dakota, to determine whether neonates in populations near the northern limit of distribution rely on a tolerance for freezing or on a capacity for supercooling to survive their first winter of life. We placed hatchlings individually into artificial hibernacula constructed in jars of damp, loamy sand and then cooled the jars to approximately -0.45 degrees C, which was below the equilibrium freezing point for water held by the sand but above that for body fluids of the neonatal turtles. A piece of ice next was placed on the surface of the sand in each jar to induce freezing of the soil water. After the soil water had frozen to an equilibrium, the temperature in the jars was lowered by 1 degrees C/d to minima averaging -2.5 degrees C, -4.5 degrees C, -6.5 degrees C, and -10.5 degrees C in different treatments. These temperatures were maintained for varying periods, so that animals in each treatment were exposed to temperatures below the equilibrium freezing point for their body fluids for a total of 11 d. Thirty of 32 hatchlings survived exposure to -2.5 degrees C; 24 of 32 survived at -4.5 degrees C; 14 of 32 withstood -6.5 degrees C; and 7 of 32 tolerated -10.5 degrees C. Freezing exotherms were detected in temperature profiles for turtles that succumbed but not in those for hatchlings that survived. Thus, the ability of hatchlings to withstand subzero temperatures for extended periods apparently requires that they avoid freezing. Although other workers contend that tolerance for freezing is the key to survival over winter by hatchling painted turtles from the region of Lake Metigoshe, our findings indicate that neonates rely primarily on their ability to remain unfrozen and supercooled.

Mobilization of shell calcium by the chick chorioallantoic membrane in vitro.

Two explants of shell were removed from each of several fertile eggs of domestic fowl at different times during incubation. The chorioallantoic membrane (CAM) was removed from one of the explants (SHELL ONLY) and was left in situ on the other (SHELL+CAM). Explants were cultured for 24, 48 or 96 h at 37 degrees C and 5% CO2 in air in individual Petri dishes containing Dulbecco's modified Eagle's medium, bovine serum albumin, penicillin and streptomycin. Both SHELL+CAM and SHELL ONLY explants released calcium into the culture medium, but the former released considerably more calcium than the latter. More calcium was released by SHELL+CAM explants taken from older eggs than from younger ones, but the age of the donor eggs did not affect release of calcium by SHELL ONLY explants. In addition, release of calcium by SHELL+CAM explants exceeded that shown by SHELL ONLY explants for multiple 24 h intervals. However, the capacity for sustained release of calcium by SHELL+CAM explants declined with age and maturity of the CAM. Manipulations that lead to the death of the CAM abolish the capacity for SHELL+CAM explants to release more calcium than SHELL ONLY explants. Differential release of calcium by SHELL+CAM explants was not attributable to calcium present in the CAM at the onset of culture or to non-specific degradation of the shell by intracellular constituents released as a result of the death of the CAM. Taken in concert, these results indicate that the CAM mobilizes calcium from the eggshell during in vitro culture.

Water loss from eggs of domestic fowl and calcium status of hatchlings.

Water balance in eggs of domestic fowl was manipulated by drilling holes (each 3 mm in diameter) through the calcareous layer into the air cell on day 10 of incubation. Water loss between days 0 and 18 averaged 6 g for eggs in the control group (no hole) but increased to 8, 12, and 15 g for eggs with 1, 2, or 3 holes, respectively. Hatching success was 79-87% for eggs with 0-2 holes through the eggshell but only 43% for eggs with three holes. Live mass of hatchlings declined as the number of holes drilled in the eggshell increased, but dry mass of carcasses was unaffected by the treatments. The quantity of Ca2+, Mg2+, and phosphorus in residual yolks and yolk-free carcasses of hatchlings was not influenced by the amount of water lost from eggs. Plasma Ca2+ and Mg2+ were elevated in hatchlings from eggs with high rates of water loss. The inability to regulate plasma Ca2+ may be a negative consequence of excessive water loss and could contribute to increased mortality of embryos.

Variation during development in the response of chicken embryos to calcitriol administered via slow-release pellets.

Embryonic chickens were exposed to 0, 30, or 300 pg of calcitriol per day via slow-release pellets implanted adjacent to the chorioallantoic membrane. Pellets were placed in eggs on Days 10 and 15, and eggs were sampled on Days 12 and 17, respectively. The hormone induced high mortality among embryos receiving pellets on Day 10, but not among those whose treatment was begun on Day 15. Embryos receiving hormone were hypercalcemic and hypophosphatemic on both Day 12 and Day 17, but the concentration of magnesium in plasma was not affected. Size of embryos sampled on Day 12 was not affected by hormone treatment, but embryos sampled on Day 17 showed a dose-related reduction in size. Yolk-free carcasses of the embryos sampled on Day 17 also showed dose-dependent reductions in phosphorus and magnesium, but calcium content of carcasses on Day 17 was unaffected by treatment. These results indicate that both younger and older embryos respond to very small quantities of calcitriol administered via slow-release pellets. The absence of sustained hypercalcemia in earlier studies that used this protocol to dispense hormone was not caused by exposing older embryos to subthreshold quantities of calcitriol. The fact that reduced body size in late embryos receiving calcitriol was not accompanied by a reduction also in calcium content may mean that embryos deposit calcium in the carcass in an effort to deal with the extreme hypercalcemia induced by calcitriol.

Use of slow-release pellets to administer calcitriol to avian embryos: effects on plasma calcium, magnesium, and phosphorus.

Calcitriol (1,25-dihydroxycholecalciferol) was administered to embryos of domestic fowl by slow-release pellets inserted into eggs on Day 10 of incubation. Mortality among embryos receiving 10 pg/day was no different from that of controls, but mortality among embryos receiving 100-1000 pg/day was elevated in later stages of incubation. Concentrations of calcium in plasma did not vary over the course of incubation among surviving embryos receiving carrier, but concentrations of magnesium and inorganic phosphorus declined with time. All doses of calcitriol elicited increases in plasma Ca and Mg on Day 12, and embryos may have responded also with a dose-dependent hypophosphatemia. The increases in plasma Ca and Mg were not sustained for the remainder of incubation, but the hypophosphatemia seemingly was of longer duration. This study demonstrates a new procedure for administering hormones to avian embryos and reveals that embryonic chickens respond in the expected manner to very small quantities of calcitriol.

Mobilization of calcium, phosphorus, and magnesium by embryonic alligators (Alligator mississippiensis).

The yolk of an alligator (Alligator mississippiensis) egg contains approximately 115 mg of Ca, 180 mg of P, and 17 mg of Mg at oviposition. This compartment is the primary (or sole) source of P and Mg for the forming embryo. Both of these elements are depleted rapidly from the yolk once the embryo enters the growth phase of development. The yolk also is an important source of Ca for the embryo, particularly during the first two-thirds of incubation. During the last trimester of development, however, the embryo supplements Ca from the yolk with Ca mobilized from the eggshell. Indeed, more Ca is withdrawn from the eggshell during the last 2 wk of incubation than can be used by the embryo in skeletogenesis. The excess Ca is stored in the yolk, thereby causing net transfer of Ca to shift from withdrawal from the yolk to deposition in the yolk. Consequently, the residual yolk in the hatchling alligator contains a substantial reserve of Ca to support skeletal growth during the neonatal period. The pattern of mobilization and deposition of Ca during embryogenesis in alligators is similar to that characterizing avian embryos, but is distinct from that characterizing embryos of all other oviparous reptiles.

Control of metabolism and growth in embryonic turtles: a test of the urea hypothesis.

We performed two experiments to determine (1) whether the metabolism and growth of embryonic snapping turtles (Chelydra serpentina) incubating in wet and dry environments are correlated inversely with the concentration of urea inside their eggs, and (2) whether urea accumulating inside eggs might be the cause of reductions in metabolism and growth by embryos. Eggs in the first experiment were incubated in different hydric environments to induce different patterns of net water exchange between the eggs and their surroundings. Turtles hatching from eggs that were in positive water balance had larger carcasses, smaller residual yolks and lower concentrations of urea in their blood than animals emerging from eggs that were in negative water balance. Thus, we confirmed the existence of correlations among water exchange by eggs, concentrations of urea in fluid compartments inside eggs, and metabolism and growth of embryos. In the second experiment, eggs were injected with solutions of urea at the mid-point of incubation to induce different levels of uremia in developing embryos. The injection protocol induced variation in the concentration of urea in blood of hatchlings similar to that observed in the first experiment for turtles hatching in wet and dry environments. However, the injection protocol did not induce variation in size of hatchlings or in mass of their residual yolk. Thus, the reduction in metabolism and growth of chelonian embryos developing in dry environments does not result from an inhibition of intermediary metabolism caused by urea, and the 'urea hypothesis' for control of metabolism cannot be accepted in its present form.

Calcium regulation in the embryonic chick. III. Calcium and phosphate in serum and allantoic fluid of normal and shell-less embryos.

Calcium metabolism of chicken embryos was profoundly affected by incubation in shell-less culture, but phosphate metabolism was largely undisturbed. Shell-less embryos exhibited hypocalcemia and hypocalciuria relative to normal embryos but had similar levels of phosphate in serum and allantoic fluid. The concentration of calcium in allantoic fluid declined during incubation in both groups, owing largely to accompanying increases in allantoic volume, but total amounts of calcium in the allantois did not vary with time. Both normal and shell-less embryos maintained higher concentrations of calcium in serum than in allantoic fluid, with shell-less embryos maintaining a larger gradient between serum and allantoic compartments. In contrast, serum and allantoic concentrations of inorganic phosphate increased over time in both normal and shell-less embryos, and both groups maintained generally higher concentrations of inorganic phosphate in the allantoic sac than in serum. Treatment of embryos with parathyroid hormone had no effect on calcium and phosphate metabolism. Embryos maintained in shell-less culture grew more slowly than those incubated normally and consequently had a dry mass about half that of normal embryos on day 18. Shell-less embryos also exhibited abnormalities in fluid balance, which were reflected in their inability to maintain normal allantoic volume and in their higher relative hydration compared to embryos incubated in ovo.

Calcium mobilization, water balance, and growth in embryos of the agamid lizard Amphibolurus barbatus.

Embryos of the agamid lizard Amphibolurus barbatus are at developmental stage 29 of Dufaure and Hubert at the time of oviposition. Mobilization of calcium and other nutrients from the yolk proceeds slowly for the first half of incubation, during which time embryonic growth also proceeds slowly. During the second half of incubation, however, embryos withdraw calcium and other nutrients from the yolk very rapidly, and growth rates are correspondingly high. Approximately 60% of the calcium used by developing embryos is obtained from the yolk, but fully 40% of their requirement is met by calcium mobilized from the eggshell. Very little calcium remains in residual yolk of hatchlings, so this yolk must be used in maintenance metabolism rather than in growth of neonates. No dichotomy exists among oviparous, amniotic vertebrates with respect to sources of calcium used by developing embryos, but one does exist with respect to patterns of mobilization of this element. Whereas calcium is extracted from yolk of embryonic reptiles throughout incubation, it actually is deposited in yolk of embryonic birds after the midpoint in development.

Calcium metabolism in embryos of the oviparous snake Coluber constrictor.

Total calcium in embryos of an oviparous, colubrid snake (Coluber constrictor L.) rises rapidly during the last half of incubation as the embryos increase in size. Although most of this calcium is drawn from stores in the yolk, hatchlings contain more calcium than was present in yolk of eggs at oviposition. Because shells from eggs incubated to hatching contain less calcium than do shells from freshly-laid eggs, the extra calcium appears to be drawn from the eggshell. Indeed, approximately 20% of the calcium required for development in this snake is obtained from the eggshell, with the remainder coming from the yolk. Thus, embryos of oviparous snakes, like embryonic chelonians, crocodilians and birds, withdraw calcium from their eggshells and do not rely exclusively on calcium supplied in their yolk for support of growth and development.

Patterns of Nitrogen Excretion by Embryonic Softshell Turtles (Trionyx spiniferus) Developing in Cleidoic Eggs.

Embryonic softshell turtles develop inside eggs exchanging little, if any, liquid water with the nest environment. Despite the consequent restriction on availability of water to support development, embryos convert most of the ammonia released in catabolism of proteins into soluble urea rather than insoluble urate, and thereby commit a portion of their limited reserve of water to serve as solvent for metabolic wastes. This finding is contrary to expectations from prior studies of cleidoic eggs.

Possible adaptive value of water exchanges in flexible-shelled eggs of turtles.

Use of energy reserves by embryos of common snapping turtles (Chelydra serpentina) is related to the hydric conditions to which eggs are exposed during incubation and to the net exchanges of water through the eggshells. Embryos developing inside eggs with a relatively favorable water balance use more of their energy reserves metabolically and grow larger before hatching than embryos inside eggs with less favorable water exchanges.