Oxygen dissociation curve for chorioallantoic capillary blood of chicken embryo.

JOURNAL

OF

APPLIED

Vol. 40, No. 3, March

PHYSIOLOGY

1976.

Printed

in U.S.A.

Oxygen dissociation curve for chorioallantoic capillary blood of chicken embryo HIROSHI TAZAWA, TSUKASA ONO, AND MASAJI MOCHIZUKI Department of Physiology, Yamaguta University School of Medicine, Yamugata 990-23; and Research Institute of Applied Electricity, Hokkaido University, Sapporo CX?O,Japan

TAZAWA, WIROSHI, TWKASA ZUKI. Oxygen dissociation curve

ONO,

AND

MASAJI

MOCHI-

for chorioallantoic capillary Hood of chicken embryo. J. Appl. Physiol. 40(3): 393398. 1976. -Oxygen dissociation curves for blood in the chorioallantoic capillary of chicken embryos were determined using a microphotometric apparatus made for measuring the reaction velocity of a red blood cell with oxygen and carbon monoxide. The modified Hill’s equations expressing the dissociation curve during development were calculated by two methods. P& at pH of 7.4 were found to be 60.0,54.4,46.2,33.1, and 28.6 mmHg for 10, 12, 14, 16 and 18 days of incubation, respectively. Although the Bohr factor did not show a clear relation to age, the oxygen affinity and the oxygen capacity tended to increase with the lapse of days, and the power of heme-to-heme interaction, to decrease with age. The findings imply that there is a respiratory adaptation of embryos during development. chorioallantoic capillary; Hill’s apparatus; oxygen affinity; P,,

equation;

microphotometric

OXYGEN DISSOCIATION CURVE of blood is an important factor in understanding not only respiratory functions of blood but also adaptation to environmental conditions surrounding an animal. In most mammalian species, it is known that the dissociation curve of a fetus significantly shifts to the left of that of a mother. This relatively high oxygen affinity of hemoglobin is considered favorable to the fetus growing under hypoxic circumstance. Because of the combined effect of increasing demand for oxygen and of diffusion restriction due to an eggshell, the avian embryo must also develop under the condition of oxygen scarcity near term. Unlike the mammalian fetus, however, the gas exchange of avian embryo is completely isolated from the maternal gas exchange system and parameters of gas exchange change markedly during the short period of incubation. Therefore, respiratory adaptation to development appears more distinctly in the avian embryo. The dissociation curve for chicken embryonic blood has so far been measured by Hall (12), Bartels et al. (l), Farooqui and Huehns (9), and Misson and Freeman (18). According to their results, the leftward displacement of the dissociation curve as compared to chick or hen’s blood was also observed in the late period of incubation. In addition, Misson and Freeman (18) showed a drastic change in oxygen &nity of embryonic blood during the last week of incubation and related it to the change in the concenTHE

tration of adenosine triphosphate (ATP). In spite of increasing interest in embryonic respiratory physiology, however, the amount of information concerning the dissociation curve of the embryo still seemsto be insufflcient. The present study was therefore attempted to determine directly the dissociation curve for blood which exists in situ in the chorioallantoic capillary of the White Rock Chicken embryo incubated for the period of lo-18 days. MATERIALS

AND

METHODS

Microphotometric reaction apparatus. A photocolorimetric method employing a microscope was recently developed in our laboratory for the purpose of measuring the reaction velocity of the red blood cell with oxygen and carbon monoxide (19, 25). The precise description of the microphotometric reaction apparatus appears elsewhere (20). The system mainly consisted of a light source, an airtight reaction cuvette and two photomultipliers coupled with interference filters, and a microscope. The reaction with the gas mixture was carried out in the cuvette furnished with an airtight chamber made of thin transparent glass. The cuvette was placed in a constant temperature chamber of 38°C. After blood was placed in it, light was focused on red blood cells monitored through an ocular lens. One red cells was illuminated by narrowing an iris diaphragm installed in the light path of the microscope. Then, the gas mixture was introduced into the chamber of the cuvette to induce the reaction of blood. The color change of the red cell hemoglobin resulting from reaction with the gas mixture was detected with interference filters and photomultipliers at wavelengths of 402 and 418 nm. Since the equilibration of blood with oxygen occurred in a relatively short time, various levels of oxygen saturation (So,) were obtained successively by changing the PO, of gas mixtures used for reaction. The time required for constructing one dissociation curve was within 15 min. Gas mixtures. Since some reports on the dissociation curve of bird blood implied that full oxygenation of blood could not be attained even at high PO, (3-5), the PO, of gas mixture used for attaining the full oxygenation was made well over 250 mmHg. In chicken embryos, the blood Pcoz gradually increases during development (6, 10, 11, 23). The dissociation curve was therefore determined at 3 values of Pcoz, 10, 25, and 40 mmHg, and 911 values of PO, were prepared for each Pcoz. All the

393

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394

TAZAWA,

determinations of gas tension were made in triplicate with a Scholander gas analyzer. Chorioallantoic membrane. The gas exchange of avian embryo takes place in a well-vascularized chorioallantoic membrane developing just beneath the shell membranes. As has been shown previously (241, only a single layer of the gas exchange capillaries grows on the entire surface of the chorioallantoic membrane and the red blood cells existing in the capillary can be observed with the microscope. A small piece of the chorioallantoic membrane was therefore excised together with the shell membranes and placed on a slide glass of the cuvette after gently removing approximately 1.5 cm2 of the eggshell. Excess fluid and blood exuding from large blood vessels were removed with filter paper and the shell membranes were gently peeled off. Then, only the chorioallantoic membrane was placed on the slide of the reaction cuvette, which was directly placed on the microphotometric apparatus. Time required for preparation of the material was only a couple of minutes following the removal of the eggshell. The egg was kept at 38°C during sampling by circulating warmed water in an egg stand. The eggs used in the present experiment were incubated for 10-18 days. In the experiments using gas mixtures with 40 mmHg Pco,, dissociation curves were measured every day throughout the experimental period. Experiments were carried out every other day from 10 to 18 days with gas mixtures having 10 and 25 mmHg Pco2. The chorioallantoic membrane placed on the slide sometimes shrank or swelled in embryos aged 10 days and this caused some difficulty in the measurement of oxygen equilibration. For this reason, the amount of data from the lo-day-old embryos measured with gas mixtures of 25 mmHg Pco2 was not large enough to compare with others. Eight to 12 eggs were used in 1 day’s experiments.

ONO,

AND

MOCHIZUKI

at 25 mmHg. The resultant So, was 55.8% in this example. Five to six levels of So, were obtained in like manner by changing gas mixtures and the dissociation curve was depicted by plotting the So, against the PO, of gas mixture. The dissociation curves of the 14-day-old embryos are represented in Fig. 2, A-C. The Pco2’s of the curves shown in Fig. 2, A-C, are 10, 25, and 40 mmHg, respectively. In Fig. 3, A-C, are shown the dissociation curves during development, where the average value of 8 to 12 determinations of So, was plotted against the PO,. As shown in the figures, the dissociation curve for capillary blood certainly shifts to the left with the lapse of incubation days and the PO, of the halfsaturation (P,,) at 40 mmHg PcoZ (Fig. 3C), for example, changes from 79 mmHg at 10 days to 26 mmHg at 18 days. Since in embryos, bicarbonate concentration of blood increases during development (6, 8, 11, 23), the plasma pH becomes different at each day of incubation despite keeping the Pco2 of equilibrating gas at the same value. Consequently, it is necessary to reconstruct the dissociation curve by converting the Pco2 value to pH in order to quantify the Bohr effect. Using the data previously obtained in arterial and mixed venous bloods during the course of incubation (231, the log Pco,-pH diagram was constructed (Fig. 4). Taking into account this relationship, for example, blood of lo-day-old embryos equilibrated with 40 mmHg Pco2 (Fig. 3C) becomes more acidic (pH = 7.08) than that of 18-day-old embryos (pH = 7.36) and consequently the P,, of the former embryos at the same pH as the latter (pH = 7.36) must become lower than 79 mmHg. To quantify the dissociation curves, the constants in a modified Hill’s equation were determined as follows. The equation is: log PO, = K,- K,.pH + K,.log So,/(lOO - So,), where too-

RESULTS

An equilibration curve of capillary blood with gas is shown in Fig. 1. Blood equilibrated with air was first deoxygenated with nitrogen gas, then reoxygenated partially with an oxygen gas mixture having 48.5 mmHg PO, and finally equilibrated with gas having 260 mmHg PO,. The Pco, in all gas mixtures used was kept -7

r-r-‘0

20

-130

-0

10

PQ (mm

10 20 30 40 50 60 70 80 90 1oc 110

Po2(mm 1. Equilibration curve of blood in the chorioallantoic of 14-day-old embryo measured with a microphotometric apparatus.

FIG.

lary tion

capilreac-

Hg)

1 60

70

80

90

Hg)

0

10 20 30 40 50 60 70 80 90 PO, CmmHg)

2. Oxygen dissociation curves for capillary blood of 14-dayold embryos. A: 10 mmHg Pco, (N = 8); B: 25 mmHg Pcop (N = 10); and C: 40 mmHg Pco2 (N = 10). FIG.


DISSOCIATION

CURVE

FOR

CAPILLARY

395

BLOOD

log P,, = 5.232 - 0.482 pH (r = -0.944, N = 28)

8Oj

In the result, the dissociation curves shown in Fig. 2, AC, are formulated by the Hill’s expression as follows log PO, = 5.232 - 0.482 pH + 0.310 log So,/(lOO - So,)

7oj

The Hill’s equation and P,, for the dissociation curves shown in Fig. 3, A-C, were estimated in like manner and summarized in Table 1. The other approach for estimating the Hill’s expression was to calculate the regression equation using the

Pcoz ,lO mmHg

o

100

10 20

30 40

50 60

Po2

(mm

B

c

50

90

70 80 i 60;

1

o

arterlallzed

l

mlxed

blood

venous

blood

80 11 70 60 i

50 50 i 40

N

::

Hg)

100

90

z

70 80 90 100 110

30

30

20 40 I

20

104

10

10 1

0

10 20 30 40 PO,

50 60 (mm

70

80 90 100 110

Hg)

0

10 20

30 40 50 60 PO?

(mm

70 80 90 100 110

I,

I

7o

.

,

I

72

I

7.4

log So,/(lOO - Soz) = -4.265 + 2.998 log PO, (r = 0.948, N = 8) log So,/(100 - So& = -5.611 + 3.412 log PO, (r = 0.958, N = 10)

I

,

1

7.8

I1

8.0

8.2

FIG. 4. In vivo diagram of log Pco2 vs. pH, which was constructed using the values of Pcoz and pH of blood withdrawn from the allantoic vein and artery. pH of equilibrated blood, which corresponds to the gas mixture Pcoz, was estimated using this diagram.

10.0

constant K, represents the Bohr factor and K, is the reciprocal of n value which indicates the power of hemeto-heme interaction. K, was estimated from a diagram of log SO~/(lOO - So& vs. log PO, and then K, and K, from a diagram of log PsOvs. pH (13). Another method was TV calculate simultaneously three constants of the equation by least squares, substituting the values of pH, PO,, and So, over the range 20-90%. In Fig. 5 are shown the relationships between log SoJ (100 - So,) and log PO, for the dissociation curves represented in Fig. 2, A-C. The relationships a, b, and c were derived from the dissociation curves of Fig. 2, A-C, respectively. The pH values corresponding to the Pco2 of 10, 25, and 40 mmHg were estimated as 7.89, 7.48, and 7.26. The regression equations for the relationships a, b, and c become respectively as follows

I

PH

Hg)

FIG. 3. Oxygen dissociation curves for capillary blood during development. The number written beside each curve indicates the days of incubation and average value of 8-12 determinations of SO, was plotted against the PO, of gas mixture. A: 10 mmHg; B: 25 mmHg; and C: 40 mmHg,

I 7.6

1

8.0 6.0 4.0 -

1.0 1 0.8 0.6 0.4 -

0.2I 10

20 PO,

40

60

100

(mmHg)

FIG. 5. Relationship between log So$(lOO - So,) and log PO, of the dissociation curves of 14-day-old embryos shown in Fig. 2. Curve a was constructed from the results shown in Fig. 2A and the pH estimated was 7.89. pH values for the curves b and c were, respectively, 7.48 and 7.26. PsO was obtained as the value corresponding to So$(lOO - So,) = 1 and the value of n in the Hill’s expression was the slope of the curve*

and log So,/(lOO - Soz) = -5.566 + 3.227 log Paz (r = 0.915, N = 10) The P,, was also able to be determined as 26.5 t 2.9, 44.2 t 2.6, and 53.1 -t- 4.9 mmHg for relationships a, b, and c. A slope of these curves gives the IZ value of the Hill’s equation and an average value became 3.228. The constant KS, which is the reciprocal of the IL, was 0.310. The constants K, and K, were then estimated by using the values of the PsOand pH determined as above. The relationship between log P,, and pH is represented in Fig. 6 and the regression equation becomes

10 1,

,

70

,

72

,

,

r

74

,

7.6

I

]

7.8

,

,

80

PH

6. Relationship between log P,, and pH of 14-day-old embryos whose dissociation curves are shown in Fig. 2, A-C. Constants& and Kg in Hill’s expression were determined from the regression equation of this relation. FIG.


396 TABLE

curves

TAZAWA,

1. Hill% equations expressing dissuciation for capillary blood of developing embryos

Age,

K,

10

log PO, = 4.250

days

12

14 16 18

&

5.133 5.232 4,872 4.216

-

Kl

0.334 pH + 0.259 log So,f(lOO 0.459 0.297 0.482 0.310 0.453 0.395 0.373 0.464

- So,)

least squares method using a minicomputer (DEC, PDP ll/lO). The values of the gas mixture PO,, the measured So, in the range of 2@90% and the pH converted from the gas mixture Pcoz were substituted into the equation: y = K, - K2mxX+ K3mx2,where y corresponds to the value of log PO,, x1 to pH, and x2 to log So,f(lOO - So,), and the three constants were determined. The results of 12,14, 16, and 18 days of incubation are, respectively, as follows log PO, = 5.175 - 0.464 pH + 0.299 log So,/(lOO - So,) log PO2 = 5.154 - 0.472 pH + 0.317 log So,/(lOO - So2) log Po, = 4.872 - 0.453 pH + 0.408 log S0,/(100 - So2) and log PO, = 4.113 - 0.359 pH + 0.466 log So,/(lOO - So,) The equations are all identical to those estimated by the former method. The dissociation curves at pH of 7.4 calculated from the Hill’s equation shown in Table 1 are depicted in Fig. 7. The number written in the figure implies the days of incubation. The P5& at pH’s of 7.4, arterial blood (pH,) and mixed venous blood (pH,) are summarized in Table 2 together with the values of pH, and pHV determined previously (23). DISCUSSION

In a conventional spectrophotometric method, it is necessary to hemolyze blood to measure the oxygen saturation. The hemolysis sometimes causes the results to shift leftward to some extent as compared to those results measured with a manometric method. In addition, both the methods frequently require tonometry to equilibrate blood with known gas mixtures, which produces cumbersome problems such as time delays and the necessity of using large amounts of blood. In the microphotometric method, on the other hand, the equilibration with gas is performed in a short time and the oxygen saturation can be measured in the red blood cell without hemolyzing it. Additionally, there is a great advantage in determining the So, of blood existing in the gas exchange capillary, i.e., the chorioallantoic membrane of chicken embryo. The detailed explanations of the microphotometric reaction apparatus and its specifications were made elsewhere (20). The dissociation curve determined in an individual embryo shows a fairly large scattering (Fig. 2). Generally the other blood gas parameters such as gas tensions and pH, as well as the 0, capacity, also seem to scatter as shown in the previous reports (22, 23). This tendency may be an intrinsic feature in an animal which grows rapidly for a short period, because the gas exchange

ONO,

AND

MOCHIZUKI

parameters alter markedly as it develops. Furthermore, the respiratory parameters in the embryos tend to depend on the flock of hens and the incubated groups of eggs. The dissociation curves illustrated in Fig. 2C, which especially show a larger scattering, include the preliminary results measured in another group of incubated eggs. Comparing the present results with previous studies, the P,, of the U-day-old embryo shown by Bartels et al. (31.3 mmHg) (1) was a bit larger than ours (28.6 mmHg) and the results measured by Misson and Freeman (18) were relatively higher despite the same tendency of the dissociation curve to drastically shift to the left with the lapse of incubation days. The difference between the two results may depend chiefly on the methods and materials used. In our measurement, the dissociation curves were determined in the red cell existing in situ in the gas exchange capillary, and the pH values were estimated as a first approximation from the Pco2 using the in vivo diagram of log Pco2 vs. pH. Bartels et al. (1) and Misson and Freeman (18) determined the dissociation curve in the sampled blood using tonometry and a mixing technique, respectively. In addition, both authors estimated the PO, for obtaining the dissociation curve at a pH of 7.4 by using the correcting factor for human blood. Recently, Lutz et al. (16, 17) reported, concerning the determination of oxygen saturation in bird blood, that the relatively high oxygen consumption of the nucleated red cell of birds might affect the dissociation curve; that is, the So2’smeasured at various Po2’smight be estimated too low because of the high oxygen consumption of blood. If this is also the case in embryonic blood, the measurement of the So, should ideally be

FIG.

ment,

7. Oxygen which were

dissociation constructed

curves at pH of 7.4 during from Hill’s equations shown

developin Table

TABLE 2. Ps;s (mmHg) at pH’s of 7.4, arterial blood (pHa) and mixed venous blood (pHii) during development Age, days 10

At pH At pH,

7.4

50.1

(7.64) 58.6 (7.43)

At pH, Values or mixed

60.0

shown venous

12

14

16

18

54.5 43.1 (7.62) 54.8 (7.39)

46.2 39.8 (7,541 50.2 (7.33)

33.1 29.7 (7.50) 36.1 (7.32)

28.6 26.8 (7.48) 30.3 (7.33)

in the parentheses blood measured

indicate previouslv

pH in the arterial

blood

(23).


DISSOCIATION

CURVE

FOR

CAPILLARY

397

BLOOD

made at the same time as the equilibration, In our method, the So, measurement was carried out during equilibration by continuously flowing an 0, gas mixture to the capillary. Then the 0, consumption by blood as well as by the chorioallantoic membrane may be disregarded. The time needed for measurement might also be an important factor which has influence on the dissociation curve. Misson and Freeman (18) measured organic phosphate when they determined the dissociation curve, suggesting that the leftward shift of the curve during development was due to a decrease in ATP concentration. In addition, they also stated that ATP decreased significantly with the lapse of time after blood withdrawal; this fact suggests that the measurement must be performed in as short a time as possible after sampling. The present measurement of all the So& required for constructing the dissociation curve of one egg was always completed within about 15 min after sampling the chorioallantoic membrane from the egg. In addition, blood to be studied was never withdrawn from the blood vessel. Comparing the P&s between the hen (or chick) and the embryo, both bird and embryo have a large variation of the P,,. In particular, the result measured by Lutz et al. (16) in the adult bird (35.1 mmHg) shows a very low value as compared to those values determined by others (the results from Bartels et al. (l), Chiodi and Terman (3), and Misson and Freeman (18) range from 48 to 57 mmHg). Although the precise comparison between the hen and the embryo must still await further studies, the information obtained from the embryo reflects purely the result of its own adaptation to development and receives no influence from maternal conditions The Bohr effect in the embryo did not show a good correlation with age (Table 3). On the other hand, the change in P,* due to acidification by 0.1 unit from pH 7.4 apparently decreases with the time of incubation. This is chiefly due te the increasing affmity for oxygen during development. Furthermore, the 0, capacity was previously shown to increase with age (22, 25) and in addition the power of heme-to-heme interaction decreases during development. Because all of these three parameters influence the oxygen delivery of the blood, both the values of the Bohr factor and APJApH cannot indicate the oxygen delivery quantitatively for the developing embryos. As shown in Hill’s equations, the costant K, which is the reciprocal of the TZvalue apparently increases with age; that is, the interaction between hemes becomes low and the amount of oxygen released consequently becomes less in the elder embryos even at the same Bohr factor. The change in Soz from 50% due to acidification by 0.1 unit from pH 7.4 without change in the PO, becomes markedly small with the lapse of time. On the other hand, the increasing oxygen capacity with age promotis the efficiency of the oxygen delivery of the blood. The total effect of these factors on oxygen delivery is reflected in the values referred to as the effective Bohr effect (214). Because both the oxygen

3. Oxygen capacity determined previously (Z&23) and values of Bohr factor, AP&pH, ASo,lApH, and effective Bohr effect TABLE

Age, days 14

0, capacity, Bohr factor

16

~01% (Alog P,,/

12.2 -0.37

ApW ~~,0~~P~,.,-%,, AWAPK4-7.3, Effective Bohr vol%

18

-2.5 4.5 0.6

mmHg % effect,

affinity and oxygen capacity are IOW as compared to most mammalian species, the value of the effective Bohr effect in the embryo is smaller than that of most species of mammals and is as small as that of the goat and sheep whose oxygen capacity is also small (2, 14). The younger embryos have a relatively high value of the effective Bohr effect in comparison with that of embryos near term. This fact seems to be favorable to oxygen delivery in the younger embryos, because their metabolic rate per weight is higher than that of the embryos near term. This favorable condition is mainly supported by the low oxygen affinity and the high interaction between hemes which favor the unloading of oxygen into the tissue. The present measurements resulted in the following relationship between the embryonic weight and the PsOat pH of 7.4 P50 = 62.6 - 1.770 weight

(r = -0.985)

A similar relationship between the metabolic mass and the oxygen afKnity is observed both in the adult birds and in the mammalian species; that is, the smaller animals tend to have a lower oxygen affinity (2, 7, 16, 21) The dissociation curve gradually shifts to the left during incubation. The displacement to the left is consistent with the progress of hypoxia and favors uptake of oxygen by the blood under conditions of hypoxia. Additionally, the increasing oxygen capacity is useful to elevate the efficiency of oxygen delivery in the last period of incubation. Similarly, the high oxygen capacity and relatively high affinity for oxygen were observed in the bird which could tolerate hypoxia and the leftward shift of the dissociation curve was also seen in chronic hypoxic hypoxia (15). In brief, the low oxygen affinity and the high power of heme-to-heme interaction observed in the early period of incubation are favorable to oxygen supply to the younger embryos which must consume relatively large amounts of oxygen. The embryos near term, which must develop under the relative oxygen scarcity due to the restricted diffusing capacity of eggshell, can tolerate the hypoxia because of the increasing oxygen affinity and oxygen capacity. These findings clearly show a considerable adaptation of the embryos to the changing gas exchange conditions during development, and in addition may mimic the respiratory adaptation to the environment of mammalian species as well as birds. Received

for tmblication

5 December

1974.


398

TAZAWA,

ONO,

AND

MOCHIZUKI

REFERENCES 1. BARTELS, H,, G. HILLER, AND W. REINHARDT. Oxygen affinity of chicken blood before and after hatching. Respiration Physiol. 1: 345356, 1966. 2. BARTELS, H., P. HILPERT, K, BARBEY, K. BETKE, K. RIEGEL, E. M. LANG, AND J. METCALFE. Respiratory function of blood of the yak, llama, camel, Dybowski deer, and African elephant. Am. J. Physiol. 205: 331-336, X963. 3. CHIODI, H., AND J. W. TERMAN. Arterial blood gases of the domestic hen. Am. J. Physiol. 208: 798-800, 1965. 4. CLAUSEN, G,, R. SANSON, AND A. STORESUND. The HbO, dissociation curve of the fulmar and the herring gull, Respiration Physiol. 12: 6&70, 1971. 5. DANZER, L. A., AND J. E. COHN. The dissociation curve for goose blood. Respiration. Physiol. 3: 302-306, 1967. 6. DAWES, C., AND K. SIMKISS. The acid-base status of the blood of the developing chick embryo. J. ExptZ. Biol. 50: 7%86, 1969. 7. DHINDSA, D. S., A. S. HOVERSLAND, AND J. METCALFE. Comparative studies of the respiratory functions of mammalian blood. VII. Armadillo Dasypus novemcinctus). Respiration Physiol. 13: 19S208, 1971. 8. ERASMUS, B. W., B. J. HOWELL, AND H. RAHN. Ontogeny of acidbase balance in the bullfrog and chicken. Respiration Physiol. 11: 4G53, 1970171. 9. FAROOQUI, A. M., AND E. R, HUEHNS. Oxygen dissociation studies of red cells from very small human and chicken embryos. Intern. Symp. Struktur Funktion Erythrocyten, 4th, BerZin, August 1970. 10. FREEMAN, B. M., AND B. H. MISSON. pH, p0, and pC0, of blood from the foetus and neonate of Gallus domesticus. Comp. Biothem. Physiol. 33: 763-772, 1970. 11. GIRARD, H. Respiratory acidosis with partial metabolic compensation in chick embryo blood during normal development. Respiration Physiol. 13: 34%351, 1971. 12. HALL, F. G. Hemoglobin function in the developing chick. J. Physiol., London 83: 22%228, 1934. 13. HELEGERS, A. E., G. MESCHIA, H. PRYSTOWSKY, A. S. WOLKOFF, AND Da H. BARRON, A comparison of the oxygen dissociation curves of the bloods of maternal and fetal goats at various pHs. Quart. J. ExptZ. Physiol. 44: 215-221, 1959.

14. HILPERT, P,, R. G. FLEISCHMANN, D. KEMPE, AND H. BARTELS. The Bohr effect related to blood and erythrocyte pH. Am. J. Physiol. 205: 337-340, 1963. 15. LENFANT, C., P. WAYS, C. AUCUTT, AND J. CRUZ. Effect of chronic hypoxic hypoxia in the O,-Hb dissociation curve and respiratory gas transport in man. Respiration PhysioZ. 7: 7-29, 1969. 16, LUTZ, P. L., I, S. LONGMUIR, AND K. SCHMIDT-NIELSEN. Oxygen affinity of bird blood. Respiration Physiol. 20: 325330, 1974, 17. LUTZ, P. L., I, S. LONGMUIR, J. V. TU’ITLE, AND K. SCHMIDTNIELSEN. Dissociation curve of bird blood and effect on red cell oxygen consumption. Respiration PhysioZ. 17: 26%275, 1973. 18. MISSON, B, H., AND B, M, FREEMAN. Organic phosphates and oxygen affinity of chick blood before and after hatching. Respiration Physiol. 14: 34%352, 1972. 19. MOCHIZUKI, M., H. TAZAWA, AND T. ONO. Microphotometry for determining the reaction rate of 0, and CO with red blood cells in the chorioallantoic capillary. In: Oxygen Transport to Tissue, edited by D, F. Bruley and H. I. Bicher. New York: Plenum, 1973, p. 997-1006. 20. ONO, T., AND H. TAZAWA. Microphotometric method for measuring the oxygenation and deoxygenation rates in a single red blood cell. Japan. J. PhysioZ. 25: 9%107, 1975. 21, SCHMIDT-NEILSEN, K., AND J. L. LARIMER. Oxygen dissociation curves of mammalian blood in relation to body size. Am. J. Physiol. 195: 424428, 1958. 22. TAZAWA, H. Measurement of respiratory parameters in blood of chicken embryo. J. AppZ. Physiol. 30: 17-20, 1971. 23. TAZAWA, H., T. MIKAMI, AND C. YOSHIMOTO. Respiratory properties of chicken embryonic blood during development. Respiration Physiol. 13: 16&170, 2971. 24. TAZAWA, H., AND T. ONO. Microscopic observation of the chorioallantoic capillary bed of chicken embryos. Respiration Physiol. 20: 81-89, 1974. 25. TAZAWA, H., T. ONO, AND M. MOCHIZUKI. Reaction velocity of carbon monoxide with blood cells in the chorioallantoic vascular plexus of chicken embryos. Respiration Physiol. 20: 161-170, 1974.


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