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Effects of ambient pressures, He and SF6 on O2 and CO2, transport en the avian egg
Respiration Physiology (1976) 2‘7, 53-64;
North-Holland Publishing Companv, Amsterdam
EFFECTS OF AMBIENT PRESSURES, He AND SF, ON 0, TRANSPORT IN THE AVIAN EGG’
AND CO,
B. DEW. ERASMUS and H. RAHN Department of Physiology, School of Medicine, State University of New Yorkat Buffalo, Buffalo, N. Y. 14214, U.S.A.
Abstract. The CO, and 0, tensions were determined in the air cells of l-4-16 day old chicken eggs before and after transfer to a 21 % 0, in He or SF, atmosphere. In the former gas mixture the air-cell Pco, (which reflects the arterialized blood Pco,) fell rapidly from 32 torr in air to 17 torr in He-O, attaining a new steady state in 24 h. In the SF,+, mixture Pco, rose from 36 torr in air to 66 torr in a similar period. A similar Pm, increase was also observed when eggs were compressed to 2 atm of air while exposure to 0.5 atm with a 40 % 0,-N, mixture decreased Pco2. Since gas transport across the eggshell is by gas phase diffusion, these findings can be explained by the changes in the diffusion coefficient of CO, which increase in the presence of He and decrease in SF,. Furthermore, the diffusion coefficient is also inversely related to the absolute pressure. Quantitative prediction of the changes of Po,, in the He and SF, mixture cannot be made, since the binary diffusion coefftcients are not necessarily applicable in a ternary gas mixture. However, effective diffusion coefficients in these multicomponent mixtures can be derived on the basis of the observed Pco, changes.
Ambient pressure Chicken eggs, air-cell Diffusion coefftcients
He, SF, gas mixture Metabolic gas exchange O,, CO, tension
The gas exchange of the avian egg during development differs from the usual transport systems in air-breathing animals because there is no convective flow between the environment and the capillaries of the chorioallantois. The metabolic exchange is carried out by diffusion of gases across the air-filled pores of the eggshell and the air-tilled spaces between the fibers of the outer and inner membrane (Wangensteen et al., 1970/71; Wangensteen and Rahn, 1970/71; Kutchai and Steen, 1971). During the normal course of development water vapor is lost to the environment which Accepted for publication 9 February 1976. ’ This study was supported in part by NIH Grant 5 PO1 HL 14414.
53
B. DEW. ERASMUS AND H. RAHN
54
produces an evergrowing air cell at the blunt end of the egg between the outer and inner membrane. This air cell composition can be conveniently sampled and reflects the CO, tension of the arterialized chorio-allantois blood returning to the embryo (Wangenst~n, 1972). Gas phase diffusion transport across the eggshell can be described as follows: the metabolic CO, elimination, &lco2, is proportional to the product of the diffusion coefficient, DcoI, and Pko2 of the air cell. The latter value represents the CO, difference across the shell if the CO, of the atmosphere is essentially zero. Thus: (1)
Qco2 = k Dcoz ..PAco2
where k = Ap/LRT and Ap represents the total pore area, L the length of the diffusion path or shell thickness, and RT the gas constant and temperature (Paganelli et al., 1975). Since the magnitude of the diffusion coefficient of a gas is not only inversely proportional to the absolute pressure but also modified by physical properties of the other gas (see Chapman-Enskog equation, Reid and Sherwood, t966), one would predict that for a given metabolic rate and a constant pore geometry of the shell, PAN-,*should be inversely related to any induced changes of the diffusion coefficient. To test this hypothesis the air cell Pcoz and Paz were measured in 14- to 16-day-old embryos incubated in air and compared with values after exposure to 0.5 and 2.0 atm or to 21 % 0, in He and SF,. Prelimina~ results were presented previously (Erasmus and Rahn, 1973; Rahn and Erasmus, 1973).
METHODS AND MATERIALS
White Leghorn chicken eggs were incubated at 37 “C and 60 % humidity in a Jamesway incubator for 13 to 16 days, at which time they were placed on a circular rack in the air-tight chamber sketched in fig. 1. Air temperature was maintained at 37.5 “C by a heat tape. To insure adequate gas circulation, the flow rate was monitored to maintain the incubator 0, above 20 % and the CO, below 0.3 %.
&f Gas k
Fig. 1. Schematic representation of the incubation chamber showing how the eggs were incubated with the various gas mixtures and how they were removed for sampling the air-cell gases.
co,
55
TRANSPORT IN EGGS
After an initial control period the air was replaced by 20.3 % 0, in sulfur hexafluoride (SF,) or by 20.7 % 0, in helium for periods up to four hours. Eggs were withdrawn from the bottom of the chamber into the acidulated water bath. The air cell space could, therefore, be sampled by syringe as previously described (Rahn et al., 1974) without exposure to air, a procedure particularly important in the heliumincubated eggs. Oxygen and carbon dioxide concentration were analyzed by Scholander 3 cc analyzer. To study the effect of a change in atmospheric pressure on diffusive gas exchange, the entire apparatus was moved into a large altitude or high pressure chamber. After a control period at sea level, pressure was changed to 0.5 or 2 ATA and gas space samples withdrawn at pressure at intervals up to four hours. Eggs were ventilated with air at 2 atm; however, at 0.5 atm 40% 0, in N, was used to prevent hypoxia. Since our theoretical analysis depended upon the assumption of a constant metabolic flux (eq. l), the oxygen consumption of individual eggs in the helium-oxygen and SF,-oxygen mixtures was measured using a modified Scholander technique (Scholander, 1949) in separate experiments.
Rt!!dts EFFECTSOF HE AND
SF, ON AIR-CELL COMPOSITION
Results of replacing the incubator air with the He or SF, mixture are shown in table 1 and illustrated in fig. 2A. After a 4-hour exposure in the He mixture the air cell Pm, was reduced to 16 torr compared with 32 torr in air. The 0, differences between ambient gas and air cell gas were similarly reduced. During the exposure to the SF, mixture, CO, tension increased above control values and the oxygen-gradient TABLE 1 Effects of SF, and He atmospheres on the 0, and CO, air-cell tensions (torr) in 16-day-old chicken eggs compared with their control values in air (mean values + SEM) Control
Age
20.3 % 0, in SF, 15 min
16days
CO, 0, n
35.7kO.8 107.8kl.l 43
45.1 k1.9 109.5 f 3.2 11
30 min 48.5 k2.6 93.9k2.1 13
1 hour
2 hours
4 hours
59.9k2.3 95.5 +2.8 4
65.4k2.2 72.9k2.4 12
64.3k3.6 76.7k3.8 21
15.OkO.9 129.3 f 1.9 14
17.3 kO.6 131.5k1.2 12
16.4+ 1.2 128.9& 1.6 12
20.7 % 0, in He 16days
CO, 0, n
32.0 +0.8 109.0+1.0 42
19.8k2.6 119.4k2.9 8
16.2+ 1.4 124.7k2.1 5
56
B. DEW.
EIUSMUS
140 -
1
AND
H. RAHN
I
1
ho2urs
3
Air 120 -
lOO-
80 -
torr
604020 -
0
0
1
4
Fig. 2A. Changes in air-cell 0, and CO, tensions after exposure to He-O, and SF&, Data from table 1, 16-day-old embryo.
torr
1
I
1
gas mixtures.
1
1 ATA
l-
Ml-
-
,__-_-““‘*’
0’ 8% / r’
0 2
I’
40 ,:
co
2
0
ATA
‘h ATA
Fig. 2B. Air-cell CO, tensions after exposure to 2 atm air and 0.5 atm 40% 0, in N,. Data from table 2. 16day-old embryo.
co,
TRANSPORT
IN EGGS
57
across the shell was greater. These trends were predicted from eq. (1) since the Dcoz and Do2 in He is larger and in SF, smaller than the respective diffusion coefficients in air. The halftime of the change in gas composition during exposure to He also was faster than during exposure to SF,, reflecting the large differences in densities of these two gas mixtures.
EFFECTS OF CHANGES IN AMBIENT PRFSSURB ON AIR-CELL
COMPOSITION
Table 2 and fig. 2B show the changes in gas tension after exposure to 2 atm in air and 0.5 atm of 40 y0 0, in N,, reflecting the change of the diffusion coeffkient of gases which are inversely proportional to the absolute pressure. Air-cell CO, tension increased from 34 to 59 torr at 2 atm in 16 day eggs and fell from 32 to 22 torr at 0.5 atm.
Effects of doubling
TABLE 2 or halving the ambient pressure on the 0,
and CO, air-cell tensions
(tort) (mean
values +_SEM)
Age
Control
2 ATA - air 1 hour
13 days
CO, 0, n
15 days
CO, 0, n
16 days
CO, 0, n
2 hours
25.2k2.1
44.1 k2.9
123.2* 1.4
260.0 k4.4
9
10
32.2f2.1
48.5k2.9
113.2k2.1
248.5 k5.9
7 34.Ok1.8 109.5fl.B 6
6 51.3*7.1
CO, 0, n
6
7
16 days
CO, 0, n
33.3kl.l
23.9kl.3
22.9 kO.9
85.Ok4.3
86.3 k4.6
32.4* 1.5 109.0*0.9 27
5 17.9* 1.2 115.9k2.7 12
6
242.8k4.3
112.8k1.6 I
4l.Ok4.4 258.6k4.4
59.2 f 3.2
242.8 f 8.9
1/2ATA-4O%G, 14 days
4 hours
6 21.8kl.O 93.8 k2.8 14
58
B. DEW.ERASMUS
EFFECT OF
AND H.RAHN
He AND SF, ON EMBRYONICOXYGEN
CONSUMPTION
One of our objectives was to calculate the change in diffusion coefficient on the basis of the observed Pco, changes (eq. 1) and to compare these values with published or theoretically derived diffusion coefficients. In order to do so it becomes imperative to know any changes in the metabolic rate that occurred when the gas mixture or the ambient pressure was altered. To test the effects of He and SF, mixtures on the metabolic rate the oxygen consumption of individual eggs was measured after 30-60 minutes incubation in the He-O, or SF,-0, mixtures. The results of these
TABLE3 0, consumption
of individual
eggs incubated
in O,-SF,
and O,-He
mixtures
20.3 % 0, in SF, Age
ire, (ml/min
Percentage
STPD)
change
(days) Air
O,-SF,
14 15
0.100 0.259
0.101 0.219
+1 -18
16
0.146
0.130
-12
16
0.181
0.173
16
0.247
0.199
0.247
-25
16
0.265
0.217
0.265
-22
17
0.168
0.131 Average
Air (post)
-5
-28 percentage
change
- 15.7
20.7 “/, 0, in He \iol (ml/min Air
srPD) O,-He
Air (post)
16
0.181
0.257
16
0.249
0.273
+9
16 16
0.305
0.332
0.374
0.370
+8 -1
16
0.243
0.315
16
0.253
0.292
16
0.254
0.343
16
0.235
0.308
16
0.350
0.350
0.310
17 17 17
0.280 0.260
0.320 0.310
0.280
+12
0.160
0.210
0.320 0.190
+16 +24
Average
+30
0.251
+23 +13
0.253
percentage
+26 +24 0
change
+ 15.3
for 30 min
co,
TRANSPORT
IN EGGS
59
experiments are shown in table 3. Incubation in SF&, resulted in a reduction in the &IO2averaging 15.7 % in 7 eggs. In two of these cases, the eggs were returned to air and I& restored to control levels. Conversely, incubation in He-O, mixture resulted in an average 15 % increase in the oxygen consumption of 12 eggs.
Discussion COMPARISON
OF CONVECTIVE
AND DIFFUSION TRANSPORT
To replenish the 0, deficit and to eliminate the metabolic CO, in a lung, fresh air must be transported from the environment to the exchange surface at regular intervals. This transport is achieved by convection. Not only does this require the expenditure of energy, but the ventilation is coordinated with the metabolic demands in such a manner that the CO, and 0, differences between the alveolar surface and the environment stay relatively constant. In the avian embryo similar 0, and CO, differences exist between the air spaces of the fibrous membranes inside the eggshell and the ambient environment. However, there is no convection and the 0, and CO, fluxes across the eggshell depend entirely upon the thermal motion of molecules which by random collision produce a net flow from areas of high concentrations to areas with low concentration. Since the pore geometry of the shell is fixed it becomes mandatory that the 0, and CO, differences across the diffusion barrier increase as metabolic demands increase during embryonic development. These have been described by Romijn and Roos (1938) and Wangensteen and Rahn (1970/71). We can express these two transport mechanisms by conventional equations. Considering CO, elimination, for example, we have for convective and diffusive transport:
(2)
(convection) h;lcol = [VA] * fico2 . P&o,
(3)
(diffusion)
h;rco2 =
1
*’ *LDco’* &oz [
. p&o,
where hjlGOl= CO, flux (ml.min-‘) (STPD); *A = alveolar ventilation (ml*min- ‘); PACO* = alveolar CO, or air cell CO, (torr); /Icol = l/864 = l/RT (ml*ml-‘*torr-‘); Ap = total pore area of shell (cm”); L = length of pore or shell thickness (cm); D co1 = diffusion coefficient of CO, in air (cm2 * min- ‘). The dimensions for the term in brackets in the equation are the same, ml*min- ‘, and Bco, is the transport coefficient for both transport systems and remains constant when different inert gases are introduced or the ambient pressure is changed (Piiper et al., 1971; Dejours, 1974). Thus in a typical convection system \i~ increases as h;lcol and keeps PACT,constant while it must increase in a diffusion transport system with a fvted pore geometry,
60
B. DEW. ERASMUS AND H. RAHN
Ap/L. Furthermore, a convection system carries all metabolic and inert gases with equal dispatch. For a given metabolic rate, alveolar ventilation and PO2 of the inspired gas, the ~~~~~remains constant when one exchanges other inert gases for N, or changes the ambient pressure or both. In this sense convection is a nondiscriminating or egalitarian transport system. On the other hand, diffusion transport discriminates between O,, CO, and other gases on the basis of their molecular weight and other physical properties. Furthermore, for a given pore geometry and metabolic gas flux, the partial pressure differences across the diffusion barrier will depend not only upon the ambient pressure but also the particular inert gas environment.
CHANGES IN co,
PRESSURES
While the changes in CO, tension resulting from changes in the gas composition or ambient pressure were in the direction expected, it is of interest to inquire how close they came to the prediction. There are two problems which confront us, namely the metabolic rate does not remain the same in the He and SF, mixture and must be corrected for. Secondly, the established or calculated binary diffusion coefficients for CO, in He and SF, are no longer applicable since we are dealing with multicomponent diffusion of 4 gases, CO, and water vapor in the presence of 21 % 0, in He or SF, (Chang et al., 1975). Since binary diffusion coefficients in these gases cannot be used to predict the ~~~~~changes, then it should be possible to calculate the correct multicomponent diffusion coefficient for CO, from the observed changes in PAcol.
EFFECT OF Pco2
CHANGES ON METABOLIC RATE
Upon exposure to He-O, we observed an average 15 % increase in oxygen consumption presumably due to the reduced CO, tensions and H+ ion concentration. Similar changes have been reported in man (Karetzky and Cain, 1970) and anesthetized dog (Cain, 1970). When dogs were hyperventilated and their Pcol reduced to one-half of normal, Cain observed an increase of about 14 % in their oxygen uptake. This value is similar to our increase of 15 % (table 3) when the CO, tension in He fell by 50 % (table 1). The mechanism for the increased metabolic rate is unknown but presumably triggered by the reduced H+ concentration. An increase in arterial Pco, in dogs gave less consistent results in decreasing oxygen uptake (Karetzky and Cain, 1970). In our observation exposure to SF, decreased oxygen uptake by 16 % (table 3) which presumably was attributable to the increase of Pco2 and H + concentration (table 1).
co,
TRANSPORTINEGGS
61
EFFECTSOFCHANGINGTHEAMBIENTPRESSURE
During exposure of eggs to 2 atm of air we need not concern ourselves with the multicomponent diffusion coefficient since our control values were also on air. However, kinetic gas theory predicts that the diffusion coefficient for any gas is inversely proportional to the absolute pressure and one would therefore predict that the control Pco2 should double. This process, of course, should take time since CO, must initially be stored in the tissues (fig. 2B). If we assume that after a four hour exposure a new steady-state equilibrium has been achieved, then we can compare the values observed for the 13, 15 and 16 day old embryos (table 2). Assuming a doubling of the control Pcol value after 4 hours exposure, we note that these 3 groups of embryos attained, respectively, 88, 73 and 87 y0 of the predicted value. However, if the rate of metabolism at these observed Pco, tensions is reduced by a similar amount as that observed during the Pco2 rise in the SF, mixture (table 3), then we must allow for a 16 y0 reduction in Mco2. This would reduce the predicted Pcol increase from 100 % to 84 % which can be compared to the average rise of 83 % for the 3 groups of embryos. Similar considerations can be applied to the eggs which were exposed to 0.5 atm. If we assume an increase in the metabolic rate, one would predict that the control Pcol should fall to 58 % of the initial value instead of to 50 %. Instead we observed only a fall to 70 and 69 %, respectively, for the 14 and 16-day-old embryos (table 2). The longest exposure period for these eggs was 2 hours and possibly not long enough to wash out all the CO,. In summary, if allowances are made for the changes in metabolism, the experimental 2 atm would indicate that CO, gas exchange of the chicken embryo obeys rather well the inverse pressure law for the CO, diffusion coefficient. The 0.5 atm experiments indicate a slight deviation from ideality. It is of interest to note that these experiments confirm indirectly the observations carried out with water vapor conductance in eggs exposed to changes in ambient pressures (Paganelli et al., 1975). In this case the water vapor difference across the eggshell, dPn20, was kept constant and the flux measured which changed inversely with the absolute pressure.
THEEFFECTSOFA
He AND SF, ATMOSPHERE
In these experiments the atmospheric pressure remained constant and 21 % 0, in He or SF, was exchanged for air. The binary diffusion coefficient for CO, in air is 0.166 and for CO, in He 3.8 times larger, namely 0.634 (Chapman-Enskog equation, Reid and Sherwood, 1966). In other words, one would predict a Pco2 reduction to about i, while our observations indicate a reduction of about f. The difficulty that one encounters is the fact that the diffusion coefficient for CO, or any other gas is only known or calculable for binary mixtures. As Chang eial. (1975) point out, in a multicomponent gas system, the transfer of one component is not only
B. DEW, BRASMJS AND H. RAHN
62
a function of its own concentration but also a function of the concentration of other components and Fick’s law of diffusion no longer applies. As a consequence a component of a given ternary gas mixture may ‘diffuse’ even though the pressure gradient is zero, may not ‘diffuse’ even though there exists a pressure difference or a component may ‘diffuse against’ a pressure difference. Chang et al. (1975) have shown in their theoretical model how for a constant pressure difference for 0,, CO, and inert gas the CO, and 0, fluxes are altered when N, is exchanged for He or SF,. In our case we assume that the metabolic gas flux is constant or known and since we know the partial pressure differences between the inside of the eggshell and the environment, one can calculate what we have called the ‘effective’ diffusion coefficient for CO,, DE. It allows us furthermore to compare such a value with the binary diffusion coefficient and to appreciate the differences between binary and quaternary diffusion. It should be pointed out that the binary diffusion coefficient, for example, for CO, in N, should be different from that in air. However this difference is negligible, a fact which has also been demonstrated empirically and is due to the similarity of the molecular weight of 0, and N,.
THE EFFECTIVE DIFFUSION COEFFICIENT, DE IN A
21 % O,, He
OR
SF,
MIXTURE
Eq. (3) can be rearranged and describes the steady state in our control where Dco2 in air is equal to 0.166 cm’ * set- ‘.
(4)
Dco* =
&j [
I’ co2 .,2
The equation is also assumed to represent the steady state after 2-4 hours exposure to a new gas composition when we replace the known DCo2 in air by the unknown Dnco2, the effective diffusion coefficient in the new gas composition: (4a) The bracketed term in eqs. (4) and (4a) remains constant and if the change in metabolic rate for the new gas composition is corrected one may then solve for the effective diffusion coefftcient, Dnco2, by comparing the control and experimental P&o,. Dividing eq. (4a) by eq. (4): (5)
DECO2= DCOl . f * cp~o,/p“%o,l
where Deco, = the effective diffusion coefficient for a new quaternary gas mixture (cm’ * set- ‘) ; DCo2 = the binary diffusion coefficient in N, or air at 1 atmosphere (0.166 cm2 * set-‘); Pko2 = the control air-cell CO, tensions (torr); P’kol = the
co,
63
TRANSPORT IN EGGS
air-cell CO, tension during exposure to the abnormal gas com~sition (torr); (f) = fraction of the control metabolic rate which is equal to 0.84 during SF, exposure and 1.15 during He exposure (see table 3). In table 4 we compare the control CO, tension in air at 1 atm with the CO, tensions after 2-4 hours of exposure to the new gas composition as well as the effective D~co~ calculated from eq. (5). In the next column are shown the binary diffusion coefficients calculated from Chapman-Enskog equation for 37 “C (Reid and Sherwood, 1966). It will be noted that there is a negligible difference between the diffusion coefficients in SF, (binary) and SF,-0, (quaternary). On the other hand, the coefficients in He are nearly twice that in He-O,. This difference in behavior was predicted by Chang et al. (1975). Whether or not the difference in the coefficients observed and predicted for 40 % 0, in N, at 0.5 atm is significant is difficult to ascertain since the coefficients at 2.0 atm air are very close to the predicted value. TABLE4 Comparison of the binary and quaternary diffusion coefficients for CO,, D and DE, respectively, calculated from the changes in the air-cell CO, tensions (P,& Experimental gas composition
Air-cell Pco, Control
Effective
De/D
DECO,
Binary D co1
0.166
0.166
1.00
Experimental
Air or N, SF&,
35.7
65.4
0.076
0.071
1.O7
He-O,
32.0
ld.4
0.361
0.635
0.57
33.3 32.4
22.9 21.8
0.281
0.332
0.85
34.0 25.2
59.2 44.1 I
0.080
0.083
0.96
0.5 atm 40%0,,6O%N, 2.0 atm air
EFFECTS OF
He AND INCRBASBD PRESSURE UPON EMBRYONIC DEVELOPMENT
Our observations suggest that many of the failures in development of chick embryos and hatching success of eggs exposed to He-O, atm (Ferguson et al., 1973; Weiss, 1975; Weiss and Grirnard, 1972) or to pressures of 2.5 to 10 atm of air or He (Akers and Thompson, 1969) must be attributable to changes in the diffusion constants of the metabolic gases producing abnormal CO, tensions which could not be properly compensated. In addition, the water vapor diffusion coefficient would be equally affected (Paganelli et al., 1975) by these abnormal gas environments. Thus an increase in DHIO would increase the water loss and dehydrate the eggs, unless the incubator
3. DEW. ERASMUS AND H. RAHN
64
humidity was appropriately increased, Such dehydrations were observed by Ferguson Weiss (1975), and Weiss and Grimard (1971). Compression will reduce the water vapour diffusion coeffkient (Paganelli et al., 1975) which would prevent the at~i~ent of an optimal air-cell volume (about 15 % of the egg volume) which is essential for the initial inflation of the lung and the rebreathing maneuvers prior to pipping of the shell and the final hatching act. et al. (1973),
References Akers, T. K. and R. E. Thompson (1969). Reaction of the chick embryo development to various hyperbaric gas mixtures. Aerospace Med. 40: 1361-l 364. Cain, S. M. (1970). Increased oxygen uptake with passive hyperventialtion of dogs. .7.Appl. Physiol. 28 : 4-7. Chang, H. K., R. C. Tai and L. E. Farhi (1975). Some implications of ternary diffusion in the lung. Respir. Physiol. 23: 109-120. Dejours, P. (1974). Recent concepts in the physiology of gas exchange. Proc. Int. UK Physiol. Sci. 10: 25-26.
Erasmus, B. and H. Rahn (1973). Effects of inert gases upon the 0, and CO, gradient across the eggshell of the incubating hen’s egg. Physiologist 16: 307. Ferguson, T. M., J. Valera, D. H. Miller and C. E. Sewell, Jr. (1973). Effect ofa helium-oxygenatmosphere on the developing chick embryo and subsequent growth of chicks. Aerospace Med. 44: 27-32. Karetzky, M. S. and S. M. Cain (1970). Effect of carbon dioxide on oxygen uptake during hy~~entilation in normal man. J. Appl. Physiol. 28: 8-12. Kutchai, H. and J. B. Steen (1971). Permeability of the shell and shell membranes of the hen’s egg during development. Respir. Physiol. 11: 265-2’78. Paganelli, C. V., A. Ar, H. Rahn and 0. D. Wangensteen (1975). Diffusion in the gas phase: the effects of ambient pressure and gas composition. Respir. Physiol. 25: 247-258. Piiper, J., P. Dejours, P. Haab and H. Rahn (1971). Concepts and basic quantities in gas exchange physiology. Respir. Physiol. 13 : 292-304. Rahn, H., and B. Erasmus (1973). Role of diffusive conductance in metabolic gas transport across the incubating avian egg shell: effects of barometric pressure. Fed. Proc. 32: 342. Rahn, H., C. V. Paganelli and A. Ar (1974). The avian egg: air-cell tension, metabolism and incubation time. Respir. Physiol. 22: 297-309. Reid, R. C. and T. K. Sherwood (1966). Properties of Gases and Liquids. New York, McGraw-Hill, Chapt. 11. Romijn, C. and J. Roos (1938). The air space of the hen’s egg and its changes during tbe period of incubation. J. Physiol. (London) 94: 365-379. Scholander, P. (1949). Volumetric respirometer for aquatic animals. Reu. Sci. Inst. 20: 885-887. Wangensteen, 0. D., D. Wilson and H. Rahn (1970/71). Diffusion of gases across the shell’of the hen’s egg. Respir. Physiol. 11: 16-30. Wang~~n, 0. D. and H. Rahn (1970~71). Respiratory gas exchange by the avian embryo. Respir. Physiol. 11: 31-45. Wangensteen, 0. D. (1972). Gas exchange of a bird’s embryo. Respir. Physiol. 14: 64-74. Weiss, H. S. and M. Grimard (1971). Embryogenesis in 100 % 0, at reduced pressure. Space Life Sci. 3: 118-124. Weiss, H. S. and M. Grnnard (1972). Inert gas effects on embryonic develppment. J. A@. Physidl. 33: 375-380.
Weiss, H. S. (1975). Improved hatch ratein helium-oxygen by reducing shell diffusion area. Proc. Sot. Exp. Biol. Med. 148: 937-941.
North-Holland Publishing Companv, Amsterdam
EFFECTS OF AMBIENT PRESSURES, He AND SF, ON 0, TRANSPORT IN THE AVIAN EGG’
AND CO,
B. DEW. ERASMUS and H. RAHN Department of Physiology, School of Medicine, State University of New Yorkat Buffalo, Buffalo, N. Y. 14214, U.S.A.
Abstract. The CO, and 0, tensions were determined in the air cells of l-4-16 day old chicken eggs before and after transfer to a 21 % 0, in He or SF, atmosphere. In the former gas mixture the air-cell Pco, (which reflects the arterialized blood Pco,) fell rapidly from 32 torr in air to 17 torr in He-O, attaining a new steady state in 24 h. In the SF,+, mixture Pco, rose from 36 torr in air to 66 torr in a similar period. A similar Pm, increase was also observed when eggs were compressed to 2 atm of air while exposure to 0.5 atm with a 40 % 0,-N, mixture decreased Pco2. Since gas transport across the eggshell is by gas phase diffusion, these findings can be explained by the changes in the diffusion coefficient of CO, which increase in the presence of He and decrease in SF,. Furthermore, the diffusion coefficient is also inversely related to the absolute pressure. Quantitative prediction of the changes of Po,, in the He and SF, mixture cannot be made, since the binary diffusion coefftcients are not necessarily applicable in a ternary gas mixture. However, effective diffusion coefficients in these multicomponent mixtures can be derived on the basis of the observed Pco, changes.
Ambient pressure Chicken eggs, air-cell Diffusion coefftcients
He, SF, gas mixture Metabolic gas exchange O,, CO, tension
The gas exchange of the avian egg during development differs from the usual transport systems in air-breathing animals because there is no convective flow between the environment and the capillaries of the chorioallantois. The metabolic exchange is carried out by diffusion of gases across the air-filled pores of the eggshell and the air-tilled spaces between the fibers of the outer and inner membrane (Wangensteen et al., 1970/71; Wangensteen and Rahn, 1970/71; Kutchai and Steen, 1971). During the normal course of development water vapor is lost to the environment which Accepted for publication 9 February 1976. ’ This study was supported in part by NIH Grant 5 PO1 HL 14414.
53
B. DEW. ERASMUS AND H. RAHN
54
produces an evergrowing air cell at the blunt end of the egg between the outer and inner membrane. This air cell composition can be conveniently sampled and reflects the CO, tension of the arterialized chorio-allantois blood returning to the embryo (Wangenst~n, 1972). Gas phase diffusion transport across the eggshell can be described as follows: the metabolic CO, elimination, &lco2, is proportional to the product of the diffusion coefficient, DcoI, and Pko2 of the air cell. The latter value represents the CO, difference across the shell if the CO, of the atmosphere is essentially zero. Thus: (1)
Qco2 = k Dcoz ..PAco2
where k = Ap/LRT and Ap represents the total pore area, L the length of the diffusion path or shell thickness, and RT the gas constant and temperature (Paganelli et al., 1975). Since the magnitude of the diffusion coefficient of a gas is not only inversely proportional to the absolute pressure but also modified by physical properties of the other gas (see Chapman-Enskog equation, Reid and Sherwood, t966), one would predict that for a given metabolic rate and a constant pore geometry of the shell, PAN-,*should be inversely related to any induced changes of the diffusion coefficient. To test this hypothesis the air cell Pcoz and Paz were measured in 14- to 16-day-old embryos incubated in air and compared with values after exposure to 0.5 and 2.0 atm or to 21 % 0, in He and SF,. Prelimina~ results were presented previously (Erasmus and Rahn, 1973; Rahn and Erasmus, 1973).
METHODS AND MATERIALS
White Leghorn chicken eggs were incubated at 37 “C and 60 % humidity in a Jamesway incubator for 13 to 16 days, at which time they were placed on a circular rack in the air-tight chamber sketched in fig. 1. Air temperature was maintained at 37.5 “C by a heat tape. To insure adequate gas circulation, the flow rate was monitored to maintain the incubator 0, above 20 % and the CO, below 0.3 %.
&f Gas k
Fig. 1. Schematic representation of the incubation chamber showing how the eggs were incubated with the various gas mixtures and how they were removed for sampling the air-cell gases.
co,
55
TRANSPORT IN EGGS
After an initial control period the air was replaced by 20.3 % 0, in sulfur hexafluoride (SF,) or by 20.7 % 0, in helium for periods up to four hours. Eggs were withdrawn from the bottom of the chamber into the acidulated water bath. The air cell space could, therefore, be sampled by syringe as previously described (Rahn et al., 1974) without exposure to air, a procedure particularly important in the heliumincubated eggs. Oxygen and carbon dioxide concentration were analyzed by Scholander 3 cc analyzer. To study the effect of a change in atmospheric pressure on diffusive gas exchange, the entire apparatus was moved into a large altitude or high pressure chamber. After a control period at sea level, pressure was changed to 0.5 or 2 ATA and gas space samples withdrawn at pressure at intervals up to four hours. Eggs were ventilated with air at 2 atm; however, at 0.5 atm 40% 0, in N, was used to prevent hypoxia. Since our theoretical analysis depended upon the assumption of a constant metabolic flux (eq. l), the oxygen consumption of individual eggs in the helium-oxygen and SF,-oxygen mixtures was measured using a modified Scholander technique (Scholander, 1949) in separate experiments.
Rt!!dts EFFECTSOF HE AND
SF, ON AIR-CELL COMPOSITION
Results of replacing the incubator air with the He or SF, mixture are shown in table 1 and illustrated in fig. 2A. After a 4-hour exposure in the He mixture the air cell Pm, was reduced to 16 torr compared with 32 torr in air. The 0, differences between ambient gas and air cell gas were similarly reduced. During the exposure to the SF, mixture, CO, tension increased above control values and the oxygen-gradient TABLE 1 Effects of SF, and He atmospheres on the 0, and CO, air-cell tensions (torr) in 16-day-old chicken eggs compared with their control values in air (mean values + SEM) Control
Age
20.3 % 0, in SF, 15 min
16days
CO, 0, n
35.7kO.8 107.8kl.l 43
45.1 k1.9 109.5 f 3.2 11
30 min 48.5 k2.6 93.9k2.1 13
1 hour
2 hours
4 hours
59.9k2.3 95.5 +2.8 4
65.4k2.2 72.9k2.4 12
64.3k3.6 76.7k3.8 21
15.OkO.9 129.3 f 1.9 14
17.3 kO.6 131.5k1.2 12
16.4+ 1.2 128.9& 1.6 12
20.7 % 0, in He 16days
CO, 0, n
32.0 +0.8 109.0+1.0 42
19.8k2.6 119.4k2.9 8
16.2+ 1.4 124.7k2.1 5
56
B. DEW.
EIUSMUS
140 -
1
AND
H. RAHN
I
1
ho2urs
3
Air 120 -
lOO-
80 -
torr
604020 -
0
0
1
4
Fig. 2A. Changes in air-cell 0, and CO, tensions after exposure to He-O, and SF&, Data from table 1, 16-day-old embryo.
torr
1
I
1
gas mixtures.
1
1 ATA
l-
Ml-
-
,__-_-““‘*’
0’ 8% / r’
0 2
I’
40 ,:
co
2
0
ATA
‘h ATA
Fig. 2B. Air-cell CO, tensions after exposure to 2 atm air and 0.5 atm 40% 0, in N,. Data from table 2. 16day-old embryo.
co,
TRANSPORT
IN EGGS
57
across the shell was greater. These trends were predicted from eq. (1) since the Dcoz and Do2 in He is larger and in SF, smaller than the respective diffusion coefficients in air. The halftime of the change in gas composition during exposure to He also was faster than during exposure to SF,, reflecting the large differences in densities of these two gas mixtures.
EFFECTS OF CHANGES IN AMBIENT PRFSSURB ON AIR-CELL
COMPOSITION
Table 2 and fig. 2B show the changes in gas tension after exposure to 2 atm in air and 0.5 atm of 40 y0 0, in N,, reflecting the change of the diffusion coeffkient of gases which are inversely proportional to the absolute pressure. Air-cell CO, tension increased from 34 to 59 torr at 2 atm in 16 day eggs and fell from 32 to 22 torr at 0.5 atm.
Effects of doubling
TABLE 2 or halving the ambient pressure on the 0,
and CO, air-cell tensions
(tort) (mean
values +_SEM)
Age
Control
2 ATA - air 1 hour
13 days
CO, 0, n
15 days
CO, 0, n
16 days
CO, 0, n
2 hours
25.2k2.1
44.1 k2.9
123.2* 1.4
260.0 k4.4
9
10
32.2f2.1
48.5k2.9
113.2k2.1
248.5 k5.9
7 34.Ok1.8 109.5fl.B 6
6 51.3*7.1
CO, 0, n
6
7
16 days
CO, 0, n
33.3kl.l
23.9kl.3
22.9 kO.9
85.Ok4.3
86.3 k4.6
32.4* 1.5 109.0*0.9 27
5 17.9* 1.2 115.9k2.7 12
6
242.8k4.3
112.8k1.6 I
4l.Ok4.4 258.6k4.4
59.2 f 3.2
242.8 f 8.9
1/2ATA-4O%G, 14 days
4 hours
6 21.8kl.O 93.8 k2.8 14
58
B. DEW.ERASMUS
EFFECT OF
AND H.RAHN
He AND SF, ON EMBRYONICOXYGEN
CONSUMPTION
One of our objectives was to calculate the change in diffusion coefficient on the basis of the observed Pco, changes (eq. 1) and to compare these values with published or theoretically derived diffusion coefficients. In order to do so it becomes imperative to know any changes in the metabolic rate that occurred when the gas mixture or the ambient pressure was altered. To test the effects of He and SF, mixtures on the metabolic rate the oxygen consumption of individual eggs was measured after 30-60 minutes incubation in the He-O, or SF,-0, mixtures. The results of these
TABLE3 0, consumption
of individual
eggs incubated
in O,-SF,
and O,-He
mixtures
20.3 % 0, in SF, Age
ire, (ml/min
Percentage
STPD)
change
(days) Air
O,-SF,
14 15
0.100 0.259
0.101 0.219
+1 -18
16
0.146
0.130
-12
16
0.181
0.173
16
0.247
0.199
0.247
-25
16
0.265
0.217
0.265
-22
17
0.168
0.131 Average
Air (post)
-5
-28 percentage
change
- 15.7
20.7 “/, 0, in He \iol (ml/min Air
srPD) O,-He
Air (post)
16
0.181
0.257
16
0.249
0.273
+9
16 16
0.305
0.332
0.374
0.370
+8 -1
16
0.243
0.315
16
0.253
0.292
16
0.254
0.343
16
0.235
0.308
16
0.350
0.350
0.310
17 17 17
0.280 0.260
0.320 0.310
0.280
+12
0.160
0.210
0.320 0.190
+16 +24
Average
+30
0.251
+23 +13
0.253
percentage
+26 +24 0
change
+ 15.3
for 30 min
co,
TRANSPORT
IN EGGS
59
experiments are shown in table 3. Incubation in SF&, resulted in a reduction in the &IO2averaging 15.7 % in 7 eggs. In two of these cases, the eggs were returned to air and I& restored to control levels. Conversely, incubation in He-O, mixture resulted in an average 15 % increase in the oxygen consumption of 12 eggs.
Discussion COMPARISON
OF CONVECTIVE
AND DIFFUSION TRANSPORT
To replenish the 0, deficit and to eliminate the metabolic CO, in a lung, fresh air must be transported from the environment to the exchange surface at regular intervals. This transport is achieved by convection. Not only does this require the expenditure of energy, but the ventilation is coordinated with the metabolic demands in such a manner that the CO, and 0, differences between the alveolar surface and the environment stay relatively constant. In the avian embryo similar 0, and CO, differences exist between the air spaces of the fibrous membranes inside the eggshell and the ambient environment. However, there is no convection and the 0, and CO, fluxes across the eggshell depend entirely upon the thermal motion of molecules which by random collision produce a net flow from areas of high concentrations to areas with low concentration. Since the pore geometry of the shell is fixed it becomes mandatory that the 0, and CO, differences across the diffusion barrier increase as metabolic demands increase during embryonic development. These have been described by Romijn and Roos (1938) and Wangensteen and Rahn (1970/71). We can express these two transport mechanisms by conventional equations. Considering CO, elimination, for example, we have for convective and diffusive transport:
(2)
(convection) h;lcol = [VA] * fico2 . P&o,
(3)
(diffusion)
h;rco2 =
1
*’ *LDco’* &oz [
. p&o,
where hjlGOl= CO, flux (ml.min-‘) (STPD); *A = alveolar ventilation (ml*min- ‘); PACO* = alveolar CO, or air cell CO, (torr); /Icol = l/864 = l/RT (ml*ml-‘*torr-‘); Ap = total pore area of shell (cm”); L = length of pore or shell thickness (cm); D co1 = diffusion coefficient of CO, in air (cm2 * min- ‘). The dimensions for the term in brackets in the equation are the same, ml*min- ‘, and Bco, is the transport coefficient for both transport systems and remains constant when different inert gases are introduced or the ambient pressure is changed (Piiper et al., 1971; Dejours, 1974). Thus in a typical convection system \i~ increases as h;lcol and keeps PACT,constant while it must increase in a diffusion transport system with a fvted pore geometry,
60
B. DEW. ERASMUS AND H. RAHN
Ap/L. Furthermore, a convection system carries all metabolic and inert gases with equal dispatch. For a given metabolic rate, alveolar ventilation and PO2 of the inspired gas, the ~~~~~remains constant when one exchanges other inert gases for N, or changes the ambient pressure or both. In this sense convection is a nondiscriminating or egalitarian transport system. On the other hand, diffusion transport discriminates between O,, CO, and other gases on the basis of their molecular weight and other physical properties. Furthermore, for a given pore geometry and metabolic gas flux, the partial pressure differences across the diffusion barrier will depend not only upon the ambient pressure but also the particular inert gas environment.
CHANGES IN co,
PRESSURES
While the changes in CO, tension resulting from changes in the gas composition or ambient pressure were in the direction expected, it is of interest to inquire how close they came to the prediction. There are two problems which confront us, namely the metabolic rate does not remain the same in the He and SF, mixture and must be corrected for. Secondly, the established or calculated binary diffusion coefficients for CO, in He and SF, are no longer applicable since we are dealing with multicomponent diffusion of 4 gases, CO, and water vapor in the presence of 21 % 0, in He or SF, (Chang et al., 1975). Since binary diffusion coefficients in these gases cannot be used to predict the ~~~~~changes, then it should be possible to calculate the correct multicomponent diffusion coefficient for CO, from the observed changes in PAcol.
EFFECT OF Pco2
CHANGES ON METABOLIC RATE
Upon exposure to He-O, we observed an average 15 % increase in oxygen consumption presumably due to the reduced CO, tensions and H+ ion concentration. Similar changes have been reported in man (Karetzky and Cain, 1970) and anesthetized dog (Cain, 1970). When dogs were hyperventilated and their Pcol reduced to one-half of normal, Cain observed an increase of about 14 % in their oxygen uptake. This value is similar to our increase of 15 % (table 3) when the CO, tension in He fell by 50 % (table 1). The mechanism for the increased metabolic rate is unknown but presumably triggered by the reduced H+ concentration. An increase in arterial Pco, in dogs gave less consistent results in decreasing oxygen uptake (Karetzky and Cain, 1970). In our observation exposure to SF, decreased oxygen uptake by 16 % (table 3) which presumably was attributable to the increase of Pco2 and H + concentration (table 1).
co,
TRANSPORTINEGGS
61
EFFECTSOFCHANGINGTHEAMBIENTPRESSURE
During exposure of eggs to 2 atm of air we need not concern ourselves with the multicomponent diffusion coefficient since our control values were also on air. However, kinetic gas theory predicts that the diffusion coefficient for any gas is inversely proportional to the absolute pressure and one would therefore predict that the control Pco2 should double. This process, of course, should take time since CO, must initially be stored in the tissues (fig. 2B). If we assume that after a four hour exposure a new steady-state equilibrium has been achieved, then we can compare the values observed for the 13, 15 and 16 day old embryos (table 2). Assuming a doubling of the control Pcol value after 4 hours exposure, we note that these 3 groups of embryos attained, respectively, 88, 73 and 87 y0 of the predicted value. However, if the rate of metabolism at these observed Pco, tensions is reduced by a similar amount as that observed during the Pco2 rise in the SF, mixture (table 3), then we must allow for a 16 y0 reduction in Mco2. This would reduce the predicted Pcol increase from 100 % to 84 % which can be compared to the average rise of 83 % for the 3 groups of embryos. Similar considerations can be applied to the eggs which were exposed to 0.5 atm. If we assume an increase in the metabolic rate, one would predict that the control Pcol should fall to 58 % of the initial value instead of to 50 %. Instead we observed only a fall to 70 and 69 %, respectively, for the 14 and 16-day-old embryos (table 2). The longest exposure period for these eggs was 2 hours and possibly not long enough to wash out all the CO,. In summary, if allowances are made for the changes in metabolism, the experimental 2 atm would indicate that CO, gas exchange of the chicken embryo obeys rather well the inverse pressure law for the CO, diffusion coefficient. The 0.5 atm experiments indicate a slight deviation from ideality. It is of interest to note that these experiments confirm indirectly the observations carried out with water vapor conductance in eggs exposed to changes in ambient pressures (Paganelli et al., 1975). In this case the water vapor difference across the eggshell, dPn20, was kept constant and the flux measured which changed inversely with the absolute pressure.
THEEFFECTSOFA
He AND SF, ATMOSPHERE
In these experiments the atmospheric pressure remained constant and 21 % 0, in He or SF, was exchanged for air. The binary diffusion coefficient for CO, in air is 0.166 and for CO, in He 3.8 times larger, namely 0.634 (Chapman-Enskog equation, Reid and Sherwood, 1966). In other words, one would predict a Pco2 reduction to about i, while our observations indicate a reduction of about f. The difficulty that one encounters is the fact that the diffusion coefficient for CO, or any other gas is only known or calculable for binary mixtures. As Chang eial. (1975) point out, in a multicomponent gas system, the transfer of one component is not only
B. DEW, BRASMJS AND H. RAHN
62
a function of its own concentration but also a function of the concentration of other components and Fick’s law of diffusion no longer applies. As a consequence a component of a given ternary gas mixture may ‘diffuse’ even though the pressure gradient is zero, may not ‘diffuse’ even though there exists a pressure difference or a component may ‘diffuse against’ a pressure difference. Chang et al. (1975) have shown in their theoretical model how for a constant pressure difference for 0,, CO, and inert gas the CO, and 0, fluxes are altered when N, is exchanged for He or SF,. In our case we assume that the metabolic gas flux is constant or known and since we know the partial pressure differences between the inside of the eggshell and the environment, one can calculate what we have called the ‘effective’ diffusion coefficient for CO,, DE. It allows us furthermore to compare such a value with the binary diffusion coefficient and to appreciate the differences between binary and quaternary diffusion. It should be pointed out that the binary diffusion coefficient, for example, for CO, in N, should be different from that in air. However this difference is negligible, a fact which has also been demonstrated empirically and is due to the similarity of the molecular weight of 0, and N,.
THE EFFECTIVE DIFFUSION COEFFICIENT, DE IN A
21 % O,, He
OR
SF,
MIXTURE
Eq. (3) can be rearranged and describes the steady state in our control where Dco2 in air is equal to 0.166 cm’ * set- ‘.
(4)
Dco* =
&j [
I’ co2 .,2
The equation is also assumed to represent the steady state after 2-4 hours exposure to a new gas composition when we replace the known DCo2 in air by the unknown Dnco2, the effective diffusion coefficient in the new gas composition: (4a) The bracketed term in eqs. (4) and (4a) remains constant and if the change in metabolic rate for the new gas composition is corrected one may then solve for the effective diffusion coefftcient, Dnco2, by comparing the control and experimental P&o,. Dividing eq. (4a) by eq. (4): (5)
DECO2= DCOl . f * cp~o,/p“%o,l
where Deco, = the effective diffusion coefficient for a new quaternary gas mixture (cm’ * set- ‘) ; DCo2 = the binary diffusion coefficient in N, or air at 1 atmosphere (0.166 cm2 * set-‘); Pko2 = the control air-cell CO, tensions (torr); P’kol = the
co,
63
TRANSPORT IN EGGS
air-cell CO, tension during exposure to the abnormal gas com~sition (torr); (f) = fraction of the control metabolic rate which is equal to 0.84 during SF, exposure and 1.15 during He exposure (see table 3). In table 4 we compare the control CO, tension in air at 1 atm with the CO, tensions after 2-4 hours of exposure to the new gas composition as well as the effective D~co~ calculated from eq. (5). In the next column are shown the binary diffusion coefficients calculated from Chapman-Enskog equation for 37 “C (Reid and Sherwood, 1966). It will be noted that there is a negligible difference between the diffusion coefficients in SF, (binary) and SF,-0, (quaternary). On the other hand, the coefficients in He are nearly twice that in He-O,. This difference in behavior was predicted by Chang et al. (1975). Whether or not the difference in the coefficients observed and predicted for 40 % 0, in N, at 0.5 atm is significant is difficult to ascertain since the coefficients at 2.0 atm air are very close to the predicted value. TABLE4 Comparison of the binary and quaternary diffusion coefficients for CO,, D and DE, respectively, calculated from the changes in the air-cell CO, tensions (P,& Experimental gas composition
Air-cell Pco, Control
Effective
De/D
DECO,
Binary D co1
0.166
0.166
1.00
Experimental
Air or N, SF&,
35.7
65.4
0.076
0.071
1.O7
He-O,
32.0
ld.4
0.361
0.635
0.57
33.3 32.4
22.9 21.8
0.281
0.332
0.85
34.0 25.2
59.2 44.1 I
0.080
0.083
0.96
0.5 atm 40%0,,6O%N, 2.0 atm air
EFFECTS OF
He AND INCRBASBD PRESSURE UPON EMBRYONIC DEVELOPMENT
Our observations suggest that many of the failures in development of chick embryos and hatching success of eggs exposed to He-O, atm (Ferguson et al., 1973; Weiss, 1975; Weiss and Grirnard, 1972) or to pressures of 2.5 to 10 atm of air or He (Akers and Thompson, 1969) must be attributable to changes in the diffusion constants of the metabolic gases producing abnormal CO, tensions which could not be properly compensated. In addition, the water vapor diffusion coefficient would be equally affected (Paganelli et al., 1975) by these abnormal gas environments. Thus an increase in DHIO would increase the water loss and dehydrate the eggs, unless the incubator
3. DEW. ERASMUS AND H. RAHN
64
humidity was appropriately increased, Such dehydrations were observed by Ferguson Weiss (1975), and Weiss and Grimard (1971). Compression will reduce the water vapour diffusion coeffkient (Paganelli et al., 1975) which would prevent the at~i~ent of an optimal air-cell volume (about 15 % of the egg volume) which is essential for the initial inflation of the lung and the rebreathing maneuvers prior to pipping of the shell and the final hatching act. et al. (1973),
References Akers, T. K. and R. E. Thompson (1969). Reaction of the chick embryo development to various hyperbaric gas mixtures. Aerospace Med. 40: 1361-l 364. Cain, S. M. (1970). Increased oxygen uptake with passive hyperventialtion of dogs. .7.Appl. Physiol. 28 : 4-7. Chang, H. K., R. C. Tai and L. E. Farhi (1975). Some implications of ternary diffusion in the lung. Respir. Physiol. 23: 109-120. Dejours, P. (1974). Recent concepts in the physiology of gas exchange. Proc. Int. UK Physiol. Sci. 10: 25-26.
Erasmus, B. and H. Rahn (1973). Effects of inert gases upon the 0, and CO, gradient across the eggshell of the incubating hen’s egg. Physiologist 16: 307. Ferguson, T. M., J. Valera, D. H. Miller and C. E. Sewell, Jr. (1973). Effect ofa helium-oxygenatmosphere on the developing chick embryo and subsequent growth of chicks. Aerospace Med. 44: 27-32. Karetzky, M. S. and S. M. Cain (1970). Effect of carbon dioxide on oxygen uptake during hy~~entilation in normal man. J. Appl. Physiol. 28: 8-12. Kutchai, H. and J. B. Steen (1971). Permeability of the shell and shell membranes of the hen’s egg during development. Respir. Physiol. 11: 265-2’78. Paganelli, C. V., A. Ar, H. Rahn and 0. D. Wangensteen (1975). Diffusion in the gas phase: the effects of ambient pressure and gas composition. Respir. Physiol. 25: 247-258. Piiper, J., P. Dejours, P. Haab and H. Rahn (1971). Concepts and basic quantities in gas exchange physiology. Respir. Physiol. 13 : 292-304. Rahn, H., and B. Erasmus (1973). Role of diffusive conductance in metabolic gas transport across the incubating avian egg shell: effects of barometric pressure. Fed. Proc. 32: 342. Rahn, H., C. V. Paganelli and A. Ar (1974). The avian egg: air-cell tension, metabolism and incubation time. Respir. Physiol. 22: 297-309. Reid, R. C. and T. K. Sherwood (1966). Properties of Gases and Liquids. New York, McGraw-Hill, Chapt. 11. Romijn, C. and J. Roos (1938). The air space of the hen’s egg and its changes during tbe period of incubation. J. Physiol. (London) 94: 365-379. Scholander, P. (1949). Volumetric respirometer for aquatic animals. Reu. Sci. Inst. 20: 885-887. Wangensteen, 0. D., D. Wilson and H. Rahn (1970/71). Diffusion of gases across the shell’of the hen’s egg. Respir. Physiol. 11: 16-30. Wang~~n, 0. D. and H. Rahn (1970~71). Respiratory gas exchange by the avian embryo. Respir. Physiol. 11: 31-45. Wangensteen, 0. D. (1972). Gas exchange of a bird’s embryo. Respir. Physiol. 14: 64-74. Weiss, H. S. and M. Grimard (1971). Embryogenesis in 100 % 0, at reduced pressure. Space Life Sci. 3: 118-124. Weiss, H. S. and M. Grnnard (1972). Inert gas effects on embryonic develppment. J. A@. Physidl. 33: 375-380.
Weiss, H. S. (1975). Improved hatch ratein helium-oxygen by reducing shell diffusion area. Proc. Sot. Exp. Biol. Med. 148: 937-941.