Assay of inert gas contamination in studies of hydrostatic pressure effects.

In hyperbaric tissue studies it is common to use gas pressure as a means for increasing the hydrostatic pressure of the preparation. When this is done, one must take the necessary precautions to avoid gas contamination of the tissue. In the present study, we demonstrate a) methods for calculating the gas contamination using particular materials in the perfusion system, and b) a technique for isolating the fluid environment from contamination by the compressing gas in a flow-through tissue chamber. The present work is exemplified with a specific apparatus, but the general principle is applicable to other studies.

The acoustocardiogram: a noninvasive method for measuring heart rate of avian embryos in ovo.

A method is presented for measuring the heart rate of avian eggs noninvasively during the last half of incubation. The technique involves briefly placing an egg in tightly sealed vessel containing an inexpensive condenser microphone. The amplified output of the microphone, termed the acoustocardiogram (ACG), is nearly sinusoidal in shape and synchronous with the electrocardiogram. The ACG can also be obtained by mounting the microphone directly on the shell with Plasticine. The method offers advantages over previously described techniques in simplicity, low cost, and noninvasiveness.

Metabolic responses to gradual cooling in chicken eggs treated with thiourea and oxygen.

1. Late prenatal chicken embryos in eggs injected with saline showed a feeble homeothermic metabolic response to gradual cooling. This response was absent in thiourea-treated eggs. This suggests that the incipient homeothermic metabolic response before paranatal life may be attributed to thyroid development. 2. The compensatory metabolic response disappeared in embryos exposed to a hypoxic environment, while it was augmented in eggs in pure O2, decreasing as ambient temperature fell. 3. These results may indicate that the homeothermic metabolic response in late embryos is O2-conductance-limited and power-limited as previously suggested.

Diffusive resistance of avian eggshell pores.

Resistance to gas diffusion through the avian eggshell resides in the microscopic pores which penetrate the shell. We calculated the resistance to water vapor diffusion of individual pores in the shells of 23 species of avian eggs, based on measurements of pore dimensions taken from drawings of 321 pore casts published by Tyler (1962, 1964, 1965) and Tyler and Simkiss (1959). Diffusive resistances were calculated from Fick's first law, using a 100-segment model of each pore. In addition, we added 2 series resistances, calculated from Stefan's law, to account for boundary layer resistances at the inner and outer pore apertures. Convective resistances for the same 100-segment model were computed from Poiseuille's law. A special, symmetrically branching model is presented for the diffusive resistance of the branched pores of ostrich eggshells, based on the drawings of Tyler and Simkiss (1959). The total aperture resistance was less than 6.2% of total pore resistance, while the outside aperture effect was on average only 1.5%. The calculated average pore conductance for all species was 5.4 micrograms (day Torr)-1, about three times higher than the average value of 1.6 micrograms (day Torr)-1 obtained by dividing measured shell conductance by the number of pores (Ar and Rahn, 1985). A possible explanation for this discrepancy is advanced. However, it is to be noted that in spite of the discrepancy, both calculated and functional values of pore conductance appear to be independent of egg mass.

Regional differences in diffusive conductance/perfusion ratio in the shell of the hen's egg.

Are both gas exchange and gas tensions uniform in different regions of the developing hen's egg? To answer this question we measured the O2 uptake and CO2 production of the whole egg, and at the same time the O2 and CO2 tensions of the air cell. The gas exchange ratio (R) of the whole egg differed from R calculated from air cell PO2 and PCO2 values, in agreement with the findings of Visschedijk [Br. Poultry Sci. 9:173-184 (1968)], who measured gas exchange separately over both the air cell region and the remainder of the egg. We constructed a diffusive shell conductance/perfusion (G/Q) line on the O2-CO2 diagram from a blood nomogram for the chick embryo in late development [Olszowka et al., Fed. Proc. 46:512 (1987)], and used this to analyze our results. The G/Q ratio for the area of shell over the air cell differs from that for the remainder of the egg. Our analysis permits us to calculate, for each area, the regional shell conductance, blood flow, and O2 and CO2 tensions in the gas spaces between the shell and the chorioallantoic capillaries.

Cooling rates of living and killed chicken and quail eggs in air and in helium-oxygen gas mixture.

1. In a helium atmosphere, heat is dissipated from a surface 3.5 times faster than it is in air. Eggs in a helium-oxygen atmosphere cool only 1.4 times faster than they cool in air. This signifies that internal resistance to heat flow is a significant factor in the cooling rates of eggs. 2. Heat flow occurs inside an egg in two ways: by conduction through the tissues and in flowing blood. Killing an embryo stops the latter, but not the former. Eggs cool more slowly after they have been killed, signifying that blood flow can be an important component in an egg's internal flows of heat. 3. Blood flow should be a relatively more important component of heat flow in large eggs than in small eggs. The difference in conductance between living and killed eggs is larger in 60 g chicken eggs than it is in 10 g quail eggs.

Diffusion-induced convective gas flow through the pores of the eggshell.

Although gas exchange across the avian eggshell has been treated as a simple diffusion process heretofore, the nonequimolar nature of diffusive fluxes engenders a convective flow and thus causes an overpressure (delta Ph) within the shell relative to the surrounding atmosphere. The convective flow must be taken into account in assessing the driving forces and corresponding flows of the gases involved. Using the nonmetabolizing hen's egg as a model, we describe the interactions of diffusion and convection as they affect water vapor and inert gas exchange through the pores. A typical infertile hen's egg containing water vapor at 50 torr and immersed in dry air (the situation in a desiccator at 38 degrees C) will lose about 900 cm3 d-1 of water vapor by a combination of diffusion and convection. Diffusion is the predominant process, accounting by calculation for 96% of total water loss as vapor. The remaining 4% of water vapor exits by convection, which also carriers with it 480 cm3 d-1 of air. We measured delta Ph caused by this total convective flow across the shell in air, He-O2, and He, and found values of about 1, 4, and 8 mm H2O, respectively. A theoretical treatment of gas flow through pores based on Fick's and Poiseuille's laws yields delta Ph values somewhat lower than those measured. Possible reasons for the discrepancy are discussed, together with implications of our findings for gas exchange and conductance measurements in metabolizing eggs.

Influence of eggshell pore shape on gas diffusion.

We describe the influence of pore shape in the hen's eggshell on calculated resistance to diffusion. These calculations are based on measurements of 70 pore casts, pictures of which were published by Tyler (In: Recent Advances in Food Sciences, Butterworth, London, Vol. 1, 1962). Single-pore resistance was estimated from Fick's first law as the summed resistances of 100 cylindrical segments in series and the calculated boundary layer resistance at the pore apertures. In addition, we constructed an average pore profile from all measurements. The calculated average single pore water vapor conductance (3.2 micrograms X day-1 X torr-1) is about twice the measured literature value. The presence of organic material in the pore openings is discussed as one of several possible reasons for this discrepancy. Average diameter should not be used to calculate conductance of trumpet-shaped pores. The narrow part of the pores plays a dominant role in total pore conductance, and pore shape must be taken into account when estimating conductance from pore dimensions.

Avian eggs: barriers to the exchange of heat and mass.

Measured boundary-layer conductance to heat exchange for bird eggs varies with egg mass to the 0.53 power. Calculations based on the Nusselt-Reynolds relationship for a sphere and the thermal properties of air indicate that the conductance of the boundary layer to heat and to mass at any wind speed other than still air should scale with mass to the 0.53 power. Although the boundary layer contributes little to the total barrier to mass flux between bird eggs and their environment, we show that it is the major barrier to the exchange of heat. From these observations we infer that birds incubating eggs in natural nests can alter only the gradient affecting mass flux between their eggs and the environment while having the capability to change both the gradient and conductance affecting heat flux.

Pores and gas exchange of avian eggs: a review.

Pores of the avian eggshell provide the only communicating channels for the exchange of molecules between the developing embryo and the external environment. These trumpet-shaped ducts are microscopic. Their number and effective pore radius increase with egg mass from ca. 300 and 3 microns in 1 gm eggs to about 30,000 and 13 microns, respectively, in 500 gm eggs (Ar and Rahn, Respir. Physiol. 61:1-20, 1985). The total volume of all the gases that diffuse through these pores is large. For example, up to the time when internal pipping takes place, when pulmonary ventilation is initiated, about 20 liters (O2 + CO2 + water vapor) have passed through 10,000 pores of an 80 gm egg. The rules governing this exchange of gases are best described by Fick's first law rather than by Stefan's law, which describes the transfer of gases across stomata of plants. Data are presented for those species for which the water vapor conductance, the oxygen flux at the preinternal pipping stage, the rate of incubation water loss, and the number of pores in the egg have been established. These conductances and flux rates are directly proportional to the number of pores. These relationships suggest that, in spite of differences in egg mass and incubation time, in most birds the average conductance of individual pores is similar, namely, 1.5 microgram (d X torr)-1 and that the average O2 and water vapor flux per pore are 60 and 54 microliters X d-1, respectively. These values are similar to the estimates previously presented (Ar and Rahn, '85).

Transport by gas-phase diffusion: lessons learned from the hen's egg.

Diffusive gas transport obeys laws which differ from those of convective transport. Diffusive gas transport can be described as an elite transport system because it not only distinguishes between O2, CO2, and water vapor molecules, but it is also influenced by barometric pressure and the presence of particular inert gas species. In the presence of air binary diffusion coefficients are applicable, but in the presence of He or SF6 effective diffusion coefficients must be used. By contrast, convective transport is an egalitarian transport system which conveys O2, CO2, and water vapor without discrimination at any altitude or in the presence of any inert gas mixture. Experiments in progress offer the opportunity to delineate for the first time precise diffusion-perfusion ratios and their effects upon the gas-space O2 and CO2 tensions.

Changes in eggshell conductance after transfer of hens from an altitude of 3,800 to 1,200 m.

Hens acclimated to an altitude of 3,800 m (PB 480 Torr) were transferred to 1,200 m (PB 657 Torr). Eggs were collected before departure and daily after the transfer so that changes in eggshell conductance could be studied. Over the next 2 mo eggshell conductance increased 30%, presumably to compensate for the 37% reduction (from 657 to 480 Torr) in gas diffusivity at the lower altitude. Measurements of shell thickness and number of pores in the shell allow one to calculate that most of the change in total pore area occurred by an increase in cross-sectional area of individual pores.

Estimation of gas-phase diffusivities in hyperbaric environments.

Diffusion of a particular gas in a mixture of three or more gases depends on diffusion characteristics and concentrations of the other gases and also on environmental pressure. 1) Estimates of gas-phase diffusivities in hyperbaric environments can be calculated from binary coefficients by the Wilke equation. Sample calculations show that addition of carbon dioxide and water to inspired gas has very little effect on diffusivity of oxygen but that neglect of lesser components of a mixture, such as the nitrogen in "trimix" or the helium in crude neon, would lead to errors of 10% to 20%. 2) It is not possible to match a compressed air environment with a helium-oxygen or a helium-oxygen-nitrogen environment for both density and diffusivity. Diffusivities of oxygen and carbon dioxide in a helium mixture can be less than half the values in compressed air having the same density. In a plot of diffusivity vs. gas density, most useful mixtures are included in a hyperbolic-shaped band; diffusivity falls to below 25% of the room air value when density is 5 times normal.

Embryonic oxygen consumption and growth of Laysan and black-footed albatross.

The constraints placed on diffusive gas exchange by the eggshell and the adaptive features of embryonic respiration and metabolism in large Laysan and black-footed albatross eggs (300 g) during prolonged incubation (65 days) were examined in naturally incubated eggs on Sand Island, Midway, in the Northwestern Hawaiian Islands. A low eggshell gas conductance and slow growth rate were associated with a relatively low oxygen consumption (MO2) throughout incubation. Just prior to internal pipping (IP) of the inner shell membrane and penetration of the air space, the MO2 (pre-IP MO2) was approximately 1,250 ml O2 (STPD).day-1 for both species, resulting in air cell O2 and CO2 tensions of 106 and 40 Torr, respectively. During the 4- to 5-day pipping-to-hatching interval, O2 uptake increases rapidly as pulmonary respiration is initiated. Hatchling O2 consumption averaged 3,700 ml O2 (STPD).day-1 or about three times the pre-IP MO2. Data support the hypothesis that embryonic metabolism among Procellariiformes is related to the extent to which the incubation period deviates from the expected value based on initial egg mass.

Method for separation of cells and suspending medium at high pressure.

We describe a pressure chamber designed for measuring fluxes and equilibrium distributions of substances between cells in suspension and the extracellular medium at pressures up to 400 ATA. The pressure chamber contains a filtration apparatus capable of taking four separate samples of cell-free medium from a cell suspension at timed intervals, without necessity of decompression. In addition, a high pressure injection system makes possible the addition to a cell suspension of precise quantities of agents such as ionophores or metabolic inhibitors, for observations of their effects on cell functions at pressure. The chamber of filtration apparatus functioned successfully in preliminary experiments designed to measure the equilibrium distribution of 36Cl- in human erythrocytes at high pressure.

Hana kai ii: a 17-day dry saturation dive at 18.6 ATA. III. Body fluid balance.

Comprehensive studies on body fluid balance on 5 divers were conducted during the Hana Kai II dive (17 days at 18.6 ATA and 7 days of decompression). Daily urine flow increased from about 2000 ml at 1 ATA to 2600 ml at 18.6 ATA, at 31 degrees C. This diuresis was accompanied by a reduction in urine osmolality (from 650 to 500 mOsm) and a slight increase in osmolal clearance. Endogenous creatinine clearance remained at about 173 ml/min throughout the dive. Despite such a sustained diuresis, neither daily water intake nor total body water volume changed significantly. The plasma renin activity changed little, while both plasma aldosterone concentration and urinary aldosterone excretion increased significantly during the first week at 18.6 ATA. The plasma prolactin concentration showed a significant decrease during the first 3 days at 18.6 ATA. The daily excretion of antidiuretic hormone (ADH) decreased significantly (by 40%) 4 days after compression and remained low throughout the rest of the dive. Insensible waterloss at 18.6 ATA was 35% lower than that at 1 ATA. It is suggested that the observed hyperbaric diuresis is due primarily to suppression of ADH as a result of suppression of insensible water loss.

Diffusion of water vapor in binary and ternary gas mixtures at increased pressures.

Diffusion of water vapor was measured at 25 degrees C in the binary systems water vapor-He and water vapor-N2 at approximately 1, 4 10, 20 and 50 atm. Diffusion was also measured in the ternary system water vapor-He-O2 in both 2%O2-98%He, and at 1 and 20 atm. At 1 atm, comparison of binary diffusion coefficients (delta) shows that water vapor diffuses 3.3 times more readily in He than in N2. Values of delta at elevated pressures were reduced in proportion to 1/p, the absolute pressure, in qualitative agreement with Chapman-Enskog theory. However, the reduction in Delta was less than predicted at pressure above 4 atm in water vapor-N2 and above 20 atm in water vapor-He. The deviations from theory are more pronounced in N2 than in He, and become larger at higher pressures. When 26% O2 is present in the ternary system water vapor-He-O2, diffusion of water vapor is reduced to about 60% of its rate in pure He. In contrast, 2% O2 in He has little effect on diffusion of water vapor.

Bubble formation within decompressed hen's eggs.

Decompression sickness follows a reduction in ambient pressure and is a result of bubble formation in blood or tissues. The origin of such bubbles is the subject of considerable controversy, and a number of mechanisms have been proposed to account for them. In testing these mechanisms, freshly-laid hen's eggs provide a particularly intriguing model--namely, an intact biological system in which bubbles form readily and many of the proposed processes are excluded.