Respiration 0
P~~fsfof0g.v (1977) 31, 203-215
Elsevier/North-Holland
Biomedical Press
OXYGEN ANALYSES OF CHICKEN EMBRYO BLOOD’
HIROSHI Department
of’ Ph.ysiology,
TAZAWA and MASAJI MOCHIZUKI Yamagata
University
School q/Medicine,
Yamagata
990-23,
Japan
Abstract. According to data obtained previously on the blood gas tensions and the oxygen dissociation curve of chicken embryos, the arteriovefious oxygen saturation difference in the allantoic circulation has been conjectured fairly large. In order to confirm this conjecture as well as to check the validity of the in uirro dissociation curve, both the blood oxygen capacity and content in allantoic artery and vein were measured. The in uiuo 02 saturation measured here resulted in a similar value to that estimated from the dissociation curve. The O2 content in allantoic vein ranges from about 7 to 11.5vol ?/:,during the 10th IO the 18th days of incubation and that in artery is pronouncedly low in a range of I to 2.5 vol’i,,,suggesting that the blood flow rate through the body tissues is fairly larger than that through the gas exchange capillary plexus. Then, the distribution of blood flow was estimated from the analyzed data based on a model of blood circulation and some assumptions. In connection with this estimation, the diffusing capacity for deoxygenation in the tissues was speculated to be much larger than that for oxygenation in the chorioallantoic capillaries. Allantoic artery and vein Arteriovenous oxygen difference Blood flow
Diffusing capacity Oxygen transport Oxygen dissociation curve
Recently we measured the oxygen dissociation curves for capillary blood of chicken embryos at 10, 12, 14, 16 and 18 days of incubation with a microphotometric reaction apparatus (Tazawa et al., 1976). In addition, we had already measured both the oxygen tension and content of arterialized blood in the ailantoic vein (referred to as Paoz and Cao,, respectively) together with the oxygen capacity using a polarographic method (Tazawa, 1970, 1971). Thus, the validity of the in vitro dissociation curves could be checked by comparing the arterialized blood oxygen saturation (SaoJ obtained by putting the Pa o2 on the dissociation curves, with that calculated Accepfed~~r
p~bf~eatio~
28
Aprif
1977.
’ This study was supported in part by Grants No. 948036 and No. 057038 from the Ministry of Education of Japan. 203
204
H. TAZAWA
AND M. MOCHIZUKI
from Cao, and O2 capacity. The agreement between the values obtained in the two ways approximated 90%. However, the comparison was not sufficient, because the incubation days were not consistent between the two reports. Therefore, in the present study we further measured both Ca o2 and venous blood O2 content in the allantoic artery (Cvo,) together with the O2 capacity at 10, 12, 14, 16 and 18 days of incubation and compared the Sa o2 and Svo2 with the values estimated from the dissociation curves knowing Pao, and Pvo2 which were measured previously (Tazawa, 1973). Agreement was observed between both sets of values, suggesting that the in vitrodissociation curve obtained by microphotometry could be used for estimating the So, from the other blood gas parameters in the allantoic artery and vein. Referring to the dissociation curves and blood gas measurements obtained earlier, we conjectured that the ratio of O2 uptake to the chorioallantoic O2 transport capacity (V&J .Cao,) might be as large as 80% (Tazawa and Mochizuki, 1976). This speculation was confirmed by the present direct determination of Co, both in the allantoic artery and vein. Furthermore, we speculated that the blood flow rate through the body tissues (forequarters and hindquarters) must be about twofold larger than that of the allantoic circulation to furnish the developing embryos with required OZ. In this connection, the tissue resistivity for deoxygenation was speculated to be very small compared with the resistivity for oxygenation in the chorioallantoic capillary plexus.
Materials and methods White Leghorn chicken embryos were incubated for 10 to 18 days. A blood sample of about 250 ~1 was obtained from the allantoic artery or vein with a heparinized glass syringe. The location of blood vessels was confirmed previously through the eggshell by candling the incubated eggs. When eggs incubated for 16 to 18 days were used, the sharp end of the egg was usually opened because it was difftcult to locate vessels through the eggshell due to the dark shadow of grown embryo, but an allantoic artery and/or vein could be located through a small window opened through the eggshell. When sampling the blood, the vessel, was occluded by forceps for inserting a needle and sampling the blood easily. The time of occluding the blood vessels was kept as short as possible in order to avoid undesirable changes in Co, and Po,. Usually, the occluding time was less than 1 min and the sampling was completed within it. The blood sample was divided into two aliquots anaerobically; one part was immediately analyzed for Co, and another, for O2 capacity after exposing it to air. Analyses were made with an electrolytic-cell O2 analyzer (Lex-Oz-Con, Lexington Inst. Co., U.S.A.). The accuracy and reproducibility of the analyzer have been reported to be good in comparison with a conventional Van-Slyke apparatus, although the correlation showed a slightly lower 02 content by L:x-02-Con measurement (Kusumi et al.,1973). We confirmed the good reproducibility by repeating the measurement of O2 capacity in the same sample.
02
ANALYSES IN CHICKEN EMBRYO
205
The So2 in arterialized and venous bloods was obtained by dividing the Cao2 and Cvo2 by the respective 02 capacities. The values were referred to as Sao2, invi”0 and Svo2, in “iDofor arterialized and venous bloods, respectively. Using the PO*, Pco2 and pH values measured previously both in the allantoic artery and vein (Tazawa, 1973), the Sa o2 and Svo2 were estimated by referring to the O2 dissociation curves obtained from the microphotometric method (Tazawa et al., 1976). These So2s were respectively referred to as Sao,, dissoci,and Sve*, dissoci. and compared with the Saoz, invi”0and Svo2, in “iv0 in order to check the validity of the in vitro dissociation curves. The curves for 10, 12, 14, 16 and 18 days of incubation are respectively represented by the modified Hill’s equations as follows: log Po2 = 4.250 - 0.334 pH + 0.259 log So,/( 100 - So2), log Po, = 5.133 - 0.459 pH + 0.297 log So,/( 100 - Soz), log Po, = 5.232-0.482 pH+0.310 log SoJ(lOO-SoJ, log PoZ = 4.x72-0.453 pH + 0.395 log So2/(100- So2), and log PO1 = 4.216-0.373 pH+0.464 log SoJ(lOO-So2). Based on the direct measurements of Cao, and CvoZ, the blood flow rate through the allantoic circulation (QJ was calculated by Fick’s principle using the 02 uptake (%‘o,), which had been determined in the previous study (Tazawa, 1973).
Results The Cao2, CvoZ and O2 capacity measured are tabulated in table 1 together with the embryo weight. Number of determinations for each incubation day was 15 for O2 content and 30 for O2 capacity. Both the 02 capacity and CoZ increase with age and show a linear correlation to the embryo weight. The relationship is expressed by the following regression equations : O2 capacity = &240.weight+7.873 (N = 150, r = 0.901), Cao2 = 0.220. weight + 7.131 (N = 75, r = 0.863) and Cvo2 = 0.079. weight + 0.684 (N = 75, r = 0.727). The Student r-test implies that the relationship is statistically significant at P < 0.001 for the former two equations and P < 0.01 for the. last. Comparing the present results with those determined previously with the polarographic method (Tazawa, 1971) the rates of increase in 02 capacity and Cao, per unit weight of embryo were a bit larger in the present study. However, the values of 02 capacity and Caoz agree well with those of previous study after 14 days of incubation. Therefore, the difference between both the coefficients resulted from the 02 difference in the earlier days of incubation. The precise reasons for the difference in the younger embroys are unknown. The So, calculated as CeJOz capacity (Sao,, in“iuoand Svo2, in“iv0for arterialized and venous bloods, respectively) are shown in table 2, where the average values (N = 15) and standard deviations are tabulated. In table 2 are also shown the So2 values obtained from the dissociation curves by referring to the Po, and pH measured
206
H. TAZAWA AND
M.
MOCHIZUKI
TABLE I The embryo weight, oxygen capacity and content __.
_.___ _ Embryo weight
Age
..-
_
(days) (g) _____.~_ -_ 10 12 14 16 18 ____
O2 capacity (vol %)
Ca0,
8.0&0.6 8.810.7 10.2&0.7 11.910.7 12.s+ 1.0
7.2kO.6 8.2kO.6 9.2kO.7 10.9+0.7 il.S~I.1
-_ _.-.__ ~____...
2.0+0.5 4.2& 1.2 8.9-t 1.5 I4.9rf: 1.6 21.552.3
.-.
.-._-.-._-.-
--
Values are means +SD. Number of determinations 02 capacity.
Oxygen saturation
-
__~ --
CVO, fvol%) -.. -
--
--
0.9kO.5 1.0*0.5 1.1+0.4 2.OkO.5 2.5 +oJ5
_._-.-
--_--.
at individual age was 15 for 0~ content and 30 for
TABLE 2 (%) calculated from the present measurements of oxygen content and capacity (SoI, inn:ioo) and obtained from the dissociation curve (So, discmi.) .-- --._-.
Age (days)
Sk, in
10
_._______
--
fvol %)
12 14 16 18 -_--__
oir>o
87.7k4.1 90.5+3.0 90.1 k 1.8 90.6k2.5 88.6k3.8 ~_-._---~-
svo, dissoci. ~~~~-_-
in vie0 .- ._-
87.2 88.2 87.0 88.0 84.5
I l.Ok6.0 12.0 2 5.8 10.9 * 4.4 16.5+3.7 20. I k4.8 .- __~.~
---.
--
-
dissoci. .-.--. 8.2 14.1 8.2 20.3 29.9 _
-.
Values are means + SD for So,. incil,o(N = 15).
previously (Tazawa, 1973) (Sab,, dissaci. and SV~~,dissoci,). Comparison between Soz, in vim and Soz,dissoci.shows that both the results generally agree except for the Svo, at I8 days of incubation. The inconsistency at 18 days may partially be attributed to the steepness of the curve and the Lex-O&on measurement which showed lower than the Van-Slyke measurement, especially at the low range of OX content (Kusumi et al., 1973). Another possible cause may be differences in growth between the groups of eggs. In order to clarify this speculation for the difference between SVO,, in aim and svo2, dissoci. at 18 days, we further measured the Pvoz together with the Cvo, and O2 capacity in the same blood sample collected from l&day-old embryos. The results of eight eggs were 15.5 + 1.9 mm Hg, 2.2 +0.5 ~01% and 12.650.6 ~01% for Pvo,, Cvo2 and 02 capacity, respectively. The embryos weighed 22.1 + 1.5 g. The Svoz, in vi”0 calculated as CvoJO2 capacity was 17.3 & 3.9 %. On the other hand, the SVO*,dissoci.estimated from the Pvoz measured here (15.5 mm Hg on an average) and the dissociation curve was 18.9 f 4.1%, which was significantly low as compared with Svo2,dissoci.estimated from the previously measured Pvo2 (20.6 mm Hg on an average). As a result, the main reason for inconsistency at 18 days
02
ANALYSES
IN CHICKEN
207
EMBRYO
might result from the value of Pvo,. The present embryos at 18 days weighed more than the embryos whose PvoZs were used for calculation of SvoI, dissoci,; the former weighed 21.5 g and the latter 19.0 g. This indicated that the embryos used in the present experiment had grown more at 18 days in comparison with those of the previous study. Since the PO, has a negative correlation with the embryo weight, the PvoZ of the present embryos fell as low as 16 mm Hg and consequently the Svo2, invi”0showed lower value than the SvoI, dissoci,. .0.5
,O.L 7
.c E
.0.3 g h 0 z 02 = c % .O.l $
0' Age Fig. 1. The blood
,O
1
14
12
10
flow rate through (vo2) during
the allantoic development
16
18
(days) circulation from
(Q.) and the 02 uptake
of incubated
eggs
10 to 18 days of incubation.
In fig. 1 are shown the blood flow rates in the chorioallantoic capillary plexus (@) and the O2 uptake (%‘02)during development. The qo2 was obtained previously (Tazawa, 1973). The increasing rates of Qa and \io, with age almost parallel one another.
Discussion The O2 capacity and Cao, determined here (table 1) agreed with those measured previously with the polarographic method (Tazawa, 1970, 1971). Furthermore, the So, values obtained from Co? divided by O2 capacity coincided with those evaluated from the O2 dissociation curve and PoZ, although the So, of arterialized and venous bloods fell on the upper and lower portions of the dissociation curve, respectively. This seems to prove the reliability of analyzed data. The large difference between the Svo?, in uiuoand svoz, dissoci. at 18 days may be due to difference of development between the two groups. Approaching the time of hatching, the embryos must tolerate O2 lack in the air space because of a restricted diffusion of eggshell until the pulmonary respiration begins. The present embryos which weighed more than
H. TAZAWA
208
AND M. MOCHIZLJKI
those used for calculating the Soz, dissoci,seem to encounter more severe O2 scarcity as a result of relatively faster growth. Consequently, the Pvo, of the present embryos might be lower. In fact, the Pv o2 determined later in eight incubated eggs together with the Co, showed lower values. The Cvo, of chicken embryos was surprisingly low as had been estimated indirectly from the O2 dissociation curve and blood gas parameters (Tazawa and Mochizuki, 1976) e.g. about 2 ~01% at 16 days of incubation, This fact is also partially proved by referring to the data reported by other investigators, i.e. Bartels et al. (1966) and Misson and Freeman (1972) showed the O2 dissociation curve and Freeman and Misson (1970) and Girard (1971) measured the Pvo2, The Svo2 estimated roughly from their data ranged from about 15 to 30%. /-\D.A. YQP
I CP
Lungs
ja. cid.
-
Forequarters
cd
ah .Cd
a v.
0.a. Chorioallantois
aa. Fig. 2. A simplified ventricle, allantoic
L.A.;
diagram
left atrium,
Ca
%)acd
of blood circulation
L.V.; left ventricle,
artery, a.“.; allantoic
-
in chicken embryo.
D.A.:
ductus arteriosus.
vein, d.a.; descending aorta.
i.v.c.; inferior
R.A.;
right atrium,
F.: interatrial
R.V.;
right
foramina.
a.a.;
vena cava. and s.v.c.: superior
vena cava.
Figure 2 shows a simplified diagram of blood circulation of chicken embryos. In the mammalian fetus, blood from the inferior vena cava is shunted through the foramen ovale to the left ventricle and distributed to the brain, heart and upper extremities, i.e. forequarters. It then returns through the superior vena cava to the right atrium where it is shunted into the right ventricle, out the pulmonary artery and through the ductus arteriosus to supply the lower extremities and viscera, i.e. hindquarters. Blood circulation shown in fig. 2 is similar to the mammalian fetus, in which the anastomoses between the forequarters and hindquarters were
02
ANALYSES IN CHICKEN EMBRYO
209
neglected. Blood flows from the superior and interior venae cavae proceed to the right ventricle in which they become confluent with the 02-rich blood draining the chorioallantois. The venous returns from these three compartments mix together to some extent in the right atrium so that the O2 saturation in the left ventricle may become higher than in tne descending aorta. In the fetal lambs, the So, in carotid arterial blood was reported slightly higher than in femoral arterial blood (Dawes et al., 1954; Dawes and Mott, 1964). Although there are no data concerning the selective streaming through the right atrium in chicken embryos, we assume that a fraction (X) of confluent flow of Oz-depleted bloods from the forequarters and hindquarters mixes with a fraction (x) of 02-rich blood flow draining the chorioallantois in the right atrium. and they pass through the tricuspid valves. The other fractions (1 -X and 1 -x) of venous returns cross the interatrial foramina to the left atrium. The output from the right ventricle is divided into the pulmonary circulation (& fraction y) and the shunt through the ductus arteriosus (fraction I- y). Then, blood in the pulmonary circulation unloads the 02 to the lungs and mixes with blood shunted through the interatrial foramina. A fraction (z) of the output from the left ventricle proceeds to the descending aorta and distributes to the hindquarters and chorioallantoic capillary plexus, together with the shunted blood through the ductus arteriosus. The 0, taken up through the chorioallantoic membrane (7iro2) is consumed in the lungs, forequarters and hindquarters by u .\jo2, v. iTo and w.vo2, respectively (u, v and w are fractions of 7irozconsumed in the lungs, forequarters and hindquarters, respectively). According to the above assumptions, the following equations are derived. Blood flow rate in the descending aorta (&) is the sum of that in the hindquarters (&) and that in the allantoic circulation (&). Qd=Qh+Qa
(1)
These blood flows and those in the lungs (Q,) and forequarters (Qr) are related to the 02 consumption and 02 content difference by Fick’s principle. Q, = u . %,/c,
- C,“)
Qf = v%2/(cf-cfy)
Gh = w . &/(c,
(2)
(3) -
chv)
(4)
and Q = G/G
-C,)
(5)
where C,, Cr and Cd are respectively blood 0, content in ml.(ml blood)’ in the arteries to the lungs, forequarters and hindquarters (and chorioallantois), and the O2 content in their veins are represented by Cpvr CT”, Chv and C,, respectively. The blood flow rates through the lungs, forequarters and hindquarters are again expressed using the ratios of separation (X, x, y and z) as follows:
Yfx’Qt3+X.(Qf+Q,,)] = Q,
(6)
H. TAZAWA
210 (1 -z).(C(l
-x).Qa+(l
AND M. MOCHIZUKI
-X).(Qr+QlJ]+y.[xQ+X(Qr+Qh)]}
= Q‘
(l-Y)~Cx~Q~+X~(Q~+Qh)]+Z’([(l-x)~Q,+(l-x)~(i)~+Q~)] +Y.[x.Qa+X&+&)]} Y.CX.Qa.Ca+X.(Qt.Cf,+(ih.Chv)]
(1 -z).
= @I (8)
= Q&,
([Cl -x)QC,+(l
(7)
(9)
-X)~(Q&“+Q~Ch”)] +Y.[x.Q,+X.(~f+Qh)].c,,}
= Q&f
(10)
and (1 -Y).[x.Qa.Ca+X~tQr~Cr”+Qh.Ch”)]+Z. ([(I -x).Q;Ca +(l -~)~(~f~~~“+Qh~Ch”)]+y~[x~~a+X~(~~+~~)]~Cp”}
= Q&Cd
(11)
From eqs. (6) to (1 I), the ratios of separation in the right atrium, pulmonary artery and aortic arches are expressed by the blood flow rate and O2 content as follows, x = [% ’ (cf Y =
l/(l
cd)&
-
c,)
+
Q,
-
x
’ /[~p’(Cr-C,)]}
(13)
+Qf.(Cr-Cp)/[Qd’(Cd-Cp)]}
(14)
and z = l/i1 Furthermore, [Qd
’ (cf
from eq. (12) a fraction X must be -
cd)/(cf
/tQf+(ih)
c,)
+
Q, Cd), the relationship of Cd > C, is obtained from eqs. (13) and (14) where y and z express the fraction, i.e. y < 1 and z < 1. Actually, in the fetal lambs
02
211
ANALYSES IN CHICKEN EMBRYO
(Dawes et al., 1954) the So, of blood from the right ventricle was about 90% of that of the descending aorta (i.e. C, = 0.9 C,). If we use this relationship to the chicken embryos, the C, is determined as 0.018 ml. (ml blood))‘. As to the 02 content of the venous blood from the three compartments, we assume that both the O2 contents from the forequarters and hindquarters are identical and 40% less than the O2 content of pulmonary venous blood, i.e., Cf, = Chv = 0.6 C,, (= C,). In fetal lambs (Dawes et al., 1954), Cr, was a bit lower than ChV.However, in chicken embryos, as determined here, the O2 content in arterial blood supplying the tissues is predominantly low (e.g. 2 vol”A in the descending aorta or allantoic artery of 16-day-old embryos). As a result, the venous 02 reserve must be much lower than in other species, then the assumption of Cr, = ChV= 0.6 C,, may be permissible. Then, in order to solve the equations, the values of C, is first approximated and the rates of blood flow are obtained from eqs. of (1) to (5). Next, the range of X is determined by eq. (15) and the ratios of separation (x, y and z) are calculated for X value from eqs. of (12) to (14). If these values satisfy all of the relationships expressed by eqs. of (6) to (1 l), they are right solutions for the equations. The value of X is determined by trial and error until the eqs. of (6) to (11) are all satisfied. In 16-day-old embryos, we have the values of 0.11 and 0.02 ml. (ml blood)- ’ for C, and Cd, and 0.36 ml.min-’ for iTo,, respectively. In case that the So, in bloods of the forequarters and hindquarters were given as above in connection with the values reported in the fetal lambs, the following values were obtained. X = 0.6844,
x = 0.3865,
y = 0.1680,
z = 0.1827,
C, = 0.018,
Cr = 0.032,
Cr, = C,,v = 0.006,
C,, = 0.01,
@, = 2.25,
Qr = 8.31,
Qh = 9.0,
oa = 4.0 and Qd = 13.0.
In fig. 3 are shown the distribution of blood flow (ml. min- ‘) and the percentage saturation with O2 of blood which was calculated by the O2 content divided by O2 capacity of 0.12 ml. (ml blood)-‘. The So, is shown in parentheses. In chicken embryos, the volume of blood flow to the tissues is larger than that through the chorioallantoic membrane, which may compensate for the low 0, content of blood supplying the tissues. The O2 transport capacity expressed by the product of blood flow rate and O2 content (0. Co,) is 0.27 and 0.18 ml. min- ’ for the forequarters and hindquarters, respectively (17.8 and 12.1 ml. min- ’ . (kg weight of embryo)-‘), and the O2 utilization coefficient (\ioJQ . Co,) is about 80 and 70% for the forequarters and hindquarters. On the other hand, hens have a systemic 02 transport capacity of 43 ml .min- ’ (26.9 ml .min-’ .(kg weight)- ‘) and an 02 utilization coefficient of 60 y0 (Piiper et al., 1970). In the fetal lambs, the 02 transport capacity is 4.6 and 5.0 ml. min- ’ . (kg weight of fetus)- ’ for the forequarters and hindquarters, and the 02 utilization coefficient is about 60 and 54% for the former and the latter, respectively (Dawes et al., 1954). The O2 transport capacity in the human subjects is estimated about 20 ml *min- ’ . (kg weight)- ’ and the 02 utilization coefficient about 20 to 30% at rest (Bartels et al., 1955). During
II. TAZAWA AND M. MOCHIZUKI
212
Chorioallantois
4.0
-
(16.7)
Fig. 3. The calculated values of blood flow rate (mI.min _ ‘) in each part of the circulation and the 02 saturation of blood (‘$‘,).The So, is shown in the parentheses T
I-
150 140 130 120 110 ~ loo-
“-_
2
go-
E
Iso
shell
air space ps
altantoic vein
70a”
60-
chorioallantoic/ P”““‘“‘ies ,I I ,’
systemic arteries
I
4 .---___.
systemic veins Pv _-__Fig. 4. The Po, values in eggs incubated for 16 days. The Pi and Pi, are Oz tension of bloods flowing into the forequarters and hindquarters, respectively, and the Pe2 of the output from these tissues (Pi, and Phv)are assumed to have the same value (P”). The mean tissue PO, (I%) is assumed 1 mm Hg less than P,. The solid line indicates the values measured and the dotted line, those assumed or conjectured in the present report.
02
213
ANALYSES IN CHICKEN EMBRYO
exercise, O2 utilization in man increases up to 50 to 60% in association with a significant decrease in’Pvo2 (Cruz et al., 1969). The 02 transport capacity both in the forequarters and hindquarters seems to be equal or a bit larger than in hens and human beings and the O2 utilization coefficient in 16-day-old embryos is much larger than both fetal lambs and human subjects during exercise, emphasizing that in embryos the metabolic rate per unit body weight (24 ml - min- ’ . (kg weight) - ‘) is extremely large in connection with the rapid development. The O2 release in the tissues is proportional to the difference between the mean systemic capillary PO1(i.e. PC‘ and Per, for the forequarters andhindquarters, respectively) and tissue Po, (Pt). The proportional constant may be defined as the diffusing capacity for deoxygenation in the tissues (i.e. Die,= and D$e,X for the forequarters and hindquarters, respectively). The O2 flux through the eggshell- is dependent upon the Po, difference between the environmental air (PO) and air space (Ps) and the diffusing capacity in the eggshell (DJ. Similarly, the O2 quantity taken up by the chorioallantoic capillary blood depends on the difference between the air space PO2 and mean chorioallantoic capillary PoZ (Pee) and the diffusing capacity for oxygenation in the chorioallantoic capillary bed (DoX). That is, v . Vo,
= Dfdeox.(k
- pt)
(16)
(17) and $ro2= D, . (PO-P,) = D,, . (Ps - k).
(18)
Figure 4 shows the Po, values of each part of the eggs incubated for 16 days, where the mean PO, in the tissues (I%) was assumed to be 1 mm Hg less than the systemic venous blood Po, (P”) and the relationship between PO2and SOSwas assumed to be linear in a low range of So2. We used the values of 20.8 mm Hg and 16.5 % for the allantoic arterial PoZ and So2, respectively, from the results of both the previous and the present measurements.. The mean capillary PO2 for chorioallantoic and systemic circulations was estimated by Bohr’s integral procedure. The P, was obtained from the data of Wangensteen and Rahn (1970/71). Using the individual value of Po, shown in fig. 4, the diffusing capacities defined by eqs. of (16) to (18) were calculated. D, = 10.1 x 10-3,
D,, = 4.9 x 10-‘3,
DLox= 29.6 x 10e3 and
Dieox = 29.3 x 10e3 ml-min-’
*mm Hg-‘.
Both the Ddeoxsin the forequarters and hindquarters are much larger than D,,. The resistivity for deoxygenation in the tissues (1 /Ddeox)appears to be very small compared
214
H. TAZAWA AND M. MOCHIZUKI
with that for oxygenation in the chorioallantois in association with the large blood flow rate through the tissues. The experimental evidence that the arterialized bllod PO, in the allantoic vein (P,) was lowered to about the middle level between P, and allantoic arterial Po, (Pd) seems to be attributed to the large resistivity to 02 in the chorioallantoic capillary plexus (l/D,,X). The low value of Po, in the allantoic artery may also be attributed to the high O2 extraction in the tissues. On the other hand.,since the 02 uptake increases as embryos grow (fig. 1) and the diffusion conditions across the shell remain unchanged, the P, decreases with increase in O2 uptake during development and the D, is constant. Therefore, the 02 transport from the environmental air to the tissues may be limited by the resistivity for oxygenation in the chorioallantoic capillary (l/D,,) at the earlier days of incubation, and then with the progress of development of embryos, by the addition of a moderate eggshell resistivity (1 /DS).
References
Bartels, H., R. Beer, E. Fleischer, H. J. Hoflheinz, J. Krail, G. Rodewald, J. Wenner and I. Witt (1955). Bostimmung von Kurzschlussdurchblutung und Diffusionskapazitlt der Lungs bei Gesunden und Lungenkranken. Pfltigers Arch. 261: 99-132. Bartels, H., G. Hiller and W. Reinhardt (1966). Oxygen affinity ofchicken blood before and after hatching. Respir. Physiol.
I: 345-356.
Cruz, J. C., H. Rahn and L. E. Farhi (1969). Mixed venous Po,, Pco2, pH, and cardiac output during exercise in trained subjects. .I. &pi. Physiof. 27: 431-434. Dawes, G. S., J. C. Mott and J. G. Widdicombe (1954). The foetal circulation in the lamb. J. Physiol. (London) 126: 563-587.
Dawes, G. S. and J. C. Mott (1964). Changes in 02 distribution and consumption in foetal lambs with variations in umbilical blood flow. J. Physiol. (London) 170: 524540. Freeman, B. M. and B. H. Misson (1970). pH, pt& and pCO2 of blood from the foetus and neonate of Gailus Domesticus. Camp. Biochem. Phy.&l. 33: 763-772. Girard, H. (1971). Respiratory acidosis with partial metabolic compensation in chick embryo during normal development. Respir. Physiol. 13 : 343-35 1. Kusumi, F., W. C. Butts and W. Ruff (1973). Superior analytical performance by electrolytic cell analysis of blood oxygen content. J. Appl. Physiol. 35: 299-300. Misson, B. H. and B. M. Freeman (1972). Organic phosphates and oxygen affinity of chick blood before and after hatching. Respik. Physioi. I4 : 343-352. Piiper, J., F. Drees and P. Scheid (1970). Gas exchange in the domestic fowl during spontaneous breathing and artiticial ventilation. Respir. Physiol. 9: 234-245. Romanoff, A. L. (1960). The Avian Embryo. New York, The Macmillan Co., 1149 p. Romanoff, A. L. (1967). Biochemistry of the Avian Embryo. New York, John Wiley & Sons, 291 p. Tazawa, H. (1970). Measurement of O2 content in microliter blood samples. J. Appl. Physiof. 29: 414-416. Tazawa, H. (1971). Measurement of respiratory parameters in blood of chicken embryo. J. App!. Physioi. 30: 17-20.
Tazawa, H. (1973). Hypothermal effect ofthe gas exchange in chicken embryo. Respir. Physiol. 17: 21-31.
02 ANALYSES IN CHICKEN EMBRYO
215
Tazawa, H. and M. Mochizuki (1976). Estimation of contact time and diffusing capacity for oxygen in the chorioallantoic vascular plexus. Respir. Physiol. 28: 119-128. Tazawa, H., T. Ono and M. Mochizuki (1976). Oxygen dissociation curve for chorioallantoic capillary blood of chicken embryo. J. Appl. Physiol. 40: 393-398. Wangensteen, 0. D. and H. Rahn (1970/71). Respiratory gas exchange by the avian embryo. Respir. Physiol. 11: 3145.
P~~fsfof0g.v (1977) 31, 203-215
Elsevier/North-Holland
Biomedical Press
OXYGEN ANALYSES OF CHICKEN EMBRYO BLOOD’
HIROSHI Department
of’ Ph.ysiology,
TAZAWA and MASAJI MOCHIZUKI Yamagata
University
School q/Medicine,
Yamagata
990-23,
Japan
Abstract. According to data obtained previously on the blood gas tensions and the oxygen dissociation curve of chicken embryos, the arteriovefious oxygen saturation difference in the allantoic circulation has been conjectured fairly large. In order to confirm this conjecture as well as to check the validity of the in uirro dissociation curve, both the blood oxygen capacity and content in allantoic artery and vein were measured. The in uiuo 02 saturation measured here resulted in a similar value to that estimated from the dissociation curve. The O2 content in allantoic vein ranges from about 7 to 11.5vol ?/:,during the 10th IO the 18th days of incubation and that in artery is pronouncedly low in a range of I to 2.5 vol’i,,,suggesting that the blood flow rate through the body tissues is fairly larger than that through the gas exchange capillary plexus. Then, the distribution of blood flow was estimated from the analyzed data based on a model of blood circulation and some assumptions. In connection with this estimation, the diffusing capacity for deoxygenation in the tissues was speculated to be much larger than that for oxygenation in the chorioallantoic capillaries. Allantoic artery and vein Arteriovenous oxygen difference Blood flow
Diffusing capacity Oxygen transport Oxygen dissociation curve
Recently we measured the oxygen dissociation curves for capillary blood of chicken embryos at 10, 12, 14, 16 and 18 days of incubation with a microphotometric reaction apparatus (Tazawa et al., 1976). In addition, we had already measured both the oxygen tension and content of arterialized blood in the ailantoic vein (referred to as Paoz and Cao,, respectively) together with the oxygen capacity using a polarographic method (Tazawa, 1970, 1971). Thus, the validity of the in vitro dissociation curves could be checked by comparing the arterialized blood oxygen saturation (SaoJ obtained by putting the Pa o2 on the dissociation curves, with that calculated Accepfed~~r
p~bf~eatio~
28
Aprif
1977.
’ This study was supported in part by Grants No. 948036 and No. 057038 from the Ministry of Education of Japan. 203
204
H. TAZAWA
AND M. MOCHIZUKI
from Cao, and O2 capacity. The agreement between the values obtained in the two ways approximated 90%. However, the comparison was not sufficient, because the incubation days were not consistent between the two reports. Therefore, in the present study we further measured both Ca o2 and venous blood O2 content in the allantoic artery (Cvo,) together with the O2 capacity at 10, 12, 14, 16 and 18 days of incubation and compared the Sa o2 and Svo2 with the values estimated from the dissociation curves knowing Pao, and Pvo2 which were measured previously (Tazawa, 1973). Agreement was observed between both sets of values, suggesting that the in vitrodissociation curve obtained by microphotometry could be used for estimating the So, from the other blood gas parameters in the allantoic artery and vein. Referring to the dissociation curves and blood gas measurements obtained earlier, we conjectured that the ratio of O2 uptake to the chorioallantoic O2 transport capacity (V&J .Cao,) might be as large as 80% (Tazawa and Mochizuki, 1976). This speculation was confirmed by the present direct determination of Co, both in the allantoic artery and vein. Furthermore, we speculated that the blood flow rate through the body tissues (forequarters and hindquarters) must be about twofold larger than that of the allantoic circulation to furnish the developing embryos with required OZ. In this connection, the tissue resistivity for deoxygenation was speculated to be very small compared with the resistivity for oxygenation in the chorioallantoic capillary plexus.
Materials and methods White Leghorn chicken embryos were incubated for 10 to 18 days. A blood sample of about 250 ~1 was obtained from the allantoic artery or vein with a heparinized glass syringe. The location of blood vessels was confirmed previously through the eggshell by candling the incubated eggs. When eggs incubated for 16 to 18 days were used, the sharp end of the egg was usually opened because it was difftcult to locate vessels through the eggshell due to the dark shadow of grown embryo, but an allantoic artery and/or vein could be located through a small window opened through the eggshell. When sampling the blood, the vessel, was occluded by forceps for inserting a needle and sampling the blood easily. The time of occluding the blood vessels was kept as short as possible in order to avoid undesirable changes in Co, and Po,. Usually, the occluding time was less than 1 min and the sampling was completed within it. The blood sample was divided into two aliquots anaerobically; one part was immediately analyzed for Co, and another, for O2 capacity after exposing it to air. Analyses were made with an electrolytic-cell O2 analyzer (Lex-Oz-Con, Lexington Inst. Co., U.S.A.). The accuracy and reproducibility of the analyzer have been reported to be good in comparison with a conventional Van-Slyke apparatus, although the correlation showed a slightly lower 02 content by L:x-02-Con measurement (Kusumi et al.,1973). We confirmed the good reproducibility by repeating the measurement of O2 capacity in the same sample.
02
ANALYSES IN CHICKEN EMBRYO
205
The So2 in arterialized and venous bloods was obtained by dividing the Cao2 and Cvo2 by the respective 02 capacities. The values were referred to as Sao2, invi”0 and Svo2, in “iDofor arterialized and venous bloods, respectively. Using the PO*, Pco2 and pH values measured previously both in the allantoic artery and vein (Tazawa, 1973), the Sa o2 and Svo2 were estimated by referring to the O2 dissociation curves obtained from the microphotometric method (Tazawa et al., 1976). These So2s were respectively referred to as Sao,, dissoci,and Sve*, dissoci. and compared with the Saoz, invi”0and Svo2, in “iv0 in order to check the validity of the in vitro dissociation curves. The curves for 10, 12, 14, 16 and 18 days of incubation are respectively represented by the modified Hill’s equations as follows: log Po2 = 4.250 - 0.334 pH + 0.259 log So,/( 100 - So2), log Po, = 5.133 - 0.459 pH + 0.297 log So,/( 100 - Soz), log Po, = 5.232-0.482 pH+0.310 log SoJ(lOO-SoJ, log PoZ = 4.x72-0.453 pH + 0.395 log So2/(100- So2), and log PO1 = 4.216-0.373 pH+0.464 log SoJ(lOO-So2). Based on the direct measurements of Cao, and CvoZ, the blood flow rate through the allantoic circulation (QJ was calculated by Fick’s principle using the 02 uptake (%‘o,), which had been determined in the previous study (Tazawa, 1973).
Results The Cao2, CvoZ and O2 capacity measured are tabulated in table 1 together with the embryo weight. Number of determinations for each incubation day was 15 for O2 content and 30 for O2 capacity. Both the 02 capacity and CoZ increase with age and show a linear correlation to the embryo weight. The relationship is expressed by the following regression equations : O2 capacity = &240.weight+7.873 (N = 150, r = 0.901), Cao2 = 0.220. weight + 7.131 (N = 75, r = 0.863) and Cvo2 = 0.079. weight + 0.684 (N = 75, r = 0.727). The Student r-test implies that the relationship is statistically significant at P < 0.001 for the former two equations and P < 0.01 for the. last. Comparing the present results with those determined previously with the polarographic method (Tazawa, 1971) the rates of increase in 02 capacity and Cao, per unit weight of embryo were a bit larger in the present study. However, the values of 02 capacity and Caoz agree well with those of previous study after 14 days of incubation. Therefore, the difference between both the coefficients resulted from the 02 difference in the earlier days of incubation. The precise reasons for the difference in the younger embroys are unknown. The So, calculated as CeJOz capacity (Sao,, in“iuoand Svo2, in“iv0for arterialized and venous bloods, respectively) are shown in table 2, where the average values (N = 15) and standard deviations are tabulated. In table 2 are also shown the So2 values obtained from the dissociation curves by referring to the Po, and pH measured
206
H. TAZAWA AND
M.
MOCHIZUKI
TABLE I The embryo weight, oxygen capacity and content __.
_.___ _ Embryo weight
Age
..-
_
(days) (g) _____.~_ -_ 10 12 14 16 18 ____
O2 capacity (vol %)
Ca0,
8.0&0.6 8.810.7 10.2&0.7 11.910.7 12.s+ 1.0
7.2kO.6 8.2kO.6 9.2kO.7 10.9+0.7 il.S~I.1
-_ _.-.__ ~____...
2.0+0.5 4.2& 1.2 8.9-t 1.5 I4.9rf: 1.6 21.552.3
.-.
.-._-.-._-.-
--
Values are means +SD. Number of determinations 02 capacity.
Oxygen saturation
-
__~ --
CVO, fvol%) -.. -
--
--
0.9kO.5 1.0*0.5 1.1+0.4 2.OkO.5 2.5 +oJ5
_._-.-
--_--.
at individual age was 15 for 0~ content and 30 for
TABLE 2 (%) calculated from the present measurements of oxygen content and capacity (SoI, inn:ioo) and obtained from the dissociation curve (So, discmi.) .-- --._-.
Age (days)
Sk, in
10
_._______
--
fvol %)
12 14 16 18 -_--__
oir>o
87.7k4.1 90.5+3.0 90.1 k 1.8 90.6k2.5 88.6k3.8 ~_-._---~-
svo, dissoci. ~~~~-_-
in vie0 .- ._-
87.2 88.2 87.0 88.0 84.5
I l.Ok6.0 12.0 2 5.8 10.9 * 4.4 16.5+3.7 20. I k4.8 .- __~.~
---.
--
-
dissoci. .-.--. 8.2 14.1 8.2 20.3 29.9 _
-.
Values are means + SD for So,. incil,o(N = 15).
previously (Tazawa, 1973) (Sab,, dissaci. and SV~~,dissoci,). Comparison between Soz, in vim and Soz,dissoci.shows that both the results generally agree except for the Svo, at I8 days of incubation. The inconsistency at 18 days may partially be attributed to the steepness of the curve and the Lex-O&on measurement which showed lower than the Van-Slyke measurement, especially at the low range of OX content (Kusumi et al., 1973). Another possible cause may be differences in growth between the groups of eggs. In order to clarify this speculation for the difference between SVO,, in aim and svo2, dissoci. at 18 days, we further measured the Pvoz together with the Cvo, and O2 capacity in the same blood sample collected from l&day-old embryos. The results of eight eggs were 15.5 + 1.9 mm Hg, 2.2 +0.5 ~01% and 12.650.6 ~01% for Pvo,, Cvo2 and 02 capacity, respectively. The embryos weighed 22.1 + 1.5 g. The Svoz, in vi”0 calculated as CvoJO2 capacity was 17.3 & 3.9 %. On the other hand, the SVO*,dissoci.estimated from the Pvoz measured here (15.5 mm Hg on an average) and the dissociation curve was 18.9 f 4.1%, which was significantly low as compared with Svo2,dissoci.estimated from the previously measured Pvo2 (20.6 mm Hg on an average). As a result, the main reason for inconsistency at 18 days
02
ANALYSES
IN CHICKEN
207
EMBRYO
might result from the value of Pvo,. The present embryos at 18 days weighed more than the embryos whose PvoZs were used for calculation of SvoI, dissoci,; the former weighed 21.5 g and the latter 19.0 g. This indicated that the embryos used in the present experiment had grown more at 18 days in comparison with those of the previous study. Since the PO, has a negative correlation with the embryo weight, the PvoZ of the present embryos fell as low as 16 mm Hg and consequently the Svo2, invi”0showed lower value than the SvoI, dissoci,. .0.5
,O.L 7
.c E
.0.3 g h 0 z 02 = c % .O.l $
0' Age Fig. 1. The blood
,O
1
14
12
10
flow rate through (vo2) during
the allantoic development
16
18
(days) circulation from
(Q.) and the 02 uptake
of incubated
eggs
10 to 18 days of incubation.
In fig. 1 are shown the blood flow rates in the chorioallantoic capillary plexus (@) and the O2 uptake (%‘02)during development. The qo2 was obtained previously (Tazawa, 1973). The increasing rates of Qa and \io, with age almost parallel one another.
Discussion The O2 capacity and Cao, determined here (table 1) agreed with those measured previously with the polarographic method (Tazawa, 1970, 1971). Furthermore, the So, values obtained from Co? divided by O2 capacity coincided with those evaluated from the O2 dissociation curve and PoZ, although the So, of arterialized and venous bloods fell on the upper and lower portions of the dissociation curve, respectively. This seems to prove the reliability of analyzed data. The large difference between the Svo?, in uiuoand svoz, dissoci. at 18 days may be due to difference of development between the two groups. Approaching the time of hatching, the embryos must tolerate O2 lack in the air space because of a restricted diffusion of eggshell until the pulmonary respiration begins. The present embryos which weighed more than
H. TAZAWA
208
AND M. MOCHIZLJKI
those used for calculating the Soz, dissoci,seem to encounter more severe O2 scarcity as a result of relatively faster growth. Consequently, the Pvo, of the present embryos might be lower. In fact, the Pv o2 determined later in eight incubated eggs together with the Co, showed lower values. The Cvo, of chicken embryos was surprisingly low as had been estimated indirectly from the O2 dissociation curve and blood gas parameters (Tazawa and Mochizuki, 1976) e.g. about 2 ~01% at 16 days of incubation, This fact is also partially proved by referring to the data reported by other investigators, i.e. Bartels et al. (1966) and Misson and Freeman (1972) showed the O2 dissociation curve and Freeman and Misson (1970) and Girard (1971) measured the Pvo2, The Svo2 estimated roughly from their data ranged from about 15 to 30%. /-\D.A. YQP
I CP
Lungs
ja. cid.
-
Forequarters
cd
ah .Cd
a v.
0.a. Chorioallantois
aa. Fig. 2. A simplified ventricle, allantoic
L.A.;
diagram
left atrium,
Ca
%)acd
of blood circulation
L.V.; left ventricle,
artery, a.“.; allantoic
-
in chicken embryo.
D.A.:
ductus arteriosus.
vein, d.a.; descending aorta.
i.v.c.; inferior
R.A.;
right atrium,
F.: interatrial
R.V.;
right
foramina.
a.a.;
vena cava. and s.v.c.: superior
vena cava.
Figure 2 shows a simplified diagram of blood circulation of chicken embryos. In the mammalian fetus, blood from the inferior vena cava is shunted through the foramen ovale to the left ventricle and distributed to the brain, heart and upper extremities, i.e. forequarters. It then returns through the superior vena cava to the right atrium where it is shunted into the right ventricle, out the pulmonary artery and through the ductus arteriosus to supply the lower extremities and viscera, i.e. hindquarters. Blood circulation shown in fig. 2 is similar to the mammalian fetus, in which the anastomoses between the forequarters and hindquarters were
02
ANALYSES IN CHICKEN EMBRYO
209
neglected. Blood flows from the superior and interior venae cavae proceed to the right ventricle in which they become confluent with the 02-rich blood draining the chorioallantois. The venous returns from these three compartments mix together to some extent in the right atrium so that the O2 saturation in the left ventricle may become higher than in tne descending aorta. In the fetal lambs, the So, in carotid arterial blood was reported slightly higher than in femoral arterial blood (Dawes et al., 1954; Dawes and Mott, 1964). Although there are no data concerning the selective streaming through the right atrium in chicken embryos, we assume that a fraction (X) of confluent flow of Oz-depleted bloods from the forequarters and hindquarters mixes with a fraction (x) of 02-rich blood flow draining the chorioallantois in the right atrium. and they pass through the tricuspid valves. The other fractions (1 -X and 1 -x) of venous returns cross the interatrial foramina to the left atrium. The output from the right ventricle is divided into the pulmonary circulation (& fraction y) and the shunt through the ductus arteriosus (fraction I- y). Then, blood in the pulmonary circulation unloads the 02 to the lungs and mixes with blood shunted through the interatrial foramina. A fraction (z) of the output from the left ventricle proceeds to the descending aorta and distributes to the hindquarters and chorioallantoic capillary plexus, together with the shunted blood through the ductus arteriosus. The 0, taken up through the chorioallantoic membrane (7iro2) is consumed in the lungs, forequarters and hindquarters by u .\jo2, v. iTo and w.vo2, respectively (u, v and w are fractions of 7irozconsumed in the lungs, forequarters and hindquarters, respectively). According to the above assumptions, the following equations are derived. Blood flow rate in the descending aorta (&) is the sum of that in the hindquarters (&) and that in the allantoic circulation (&). Qd=Qh+Qa
(1)
These blood flows and those in the lungs (Q,) and forequarters (Qr) are related to the 02 consumption and 02 content difference by Fick’s principle. Q, = u . %,/c,
- C,“)
Qf = v%2/(cf-cfy)
Gh = w . &/(c,
(2)
(3) -
chv)
(4)
and Q = G/G
-C,)
(5)
where C,, Cr and Cd are respectively blood 0, content in ml.(ml blood)’ in the arteries to the lungs, forequarters and hindquarters (and chorioallantois), and the O2 content in their veins are represented by Cpvr CT”, Chv and C,, respectively. The blood flow rates through the lungs, forequarters and hindquarters are again expressed using the ratios of separation (X, x, y and z) as follows:
Yfx’Qt3+X.(Qf+Q,,)] = Q,
(6)
H. TAZAWA
210 (1 -z).(C(l
-x).Qa+(l
AND M. MOCHIZUKI
-X).(Qr+QlJ]+y.[xQ+X(Qr+Qh)]}
= Q‘
(l-Y)~Cx~Q~+X~(Q~+Qh)]+Z’([(l-x)~Q,+(l-x)~(i)~+Q~)] +Y.[x.Qa+X&+&)]} Y.CX.Qa.Ca+X.(Qt.Cf,+(ih.Chv)]
(1 -z).
= @I (8)
= Q&,
([Cl -x)QC,+(l
(7)
(9)
-X)~(Q&“+Q~Ch”)] +Y.[x.Q,+X.(~f+Qh)].c,,}
= Q&f
(10)
and (1 -Y).[x.Qa.Ca+X~tQr~Cr”+Qh.Ch”)]+Z. ([(I -x).Q;Ca +(l -~)~(~f~~~“+Qh~Ch”)]+y~[x~~a+X~(~~+~~)]~Cp”}
= Q&Cd
(11)
From eqs. (6) to (1 I), the ratios of separation in the right atrium, pulmonary artery and aortic arches are expressed by the blood flow rate and O2 content as follows, x = [% ’ (cf Y =
l/(l
cd)&
-
c,)
+
Q,
-
x
’ /[~p’(Cr-C,)]}
(13)
+Qf.(Cr-Cp)/[Qd’(Cd-Cp)]}
(14)
and z = l/i1 Furthermore, [Qd
’ (cf
from eq. (12) a fraction X must be -
cd)/(cf
/tQf+(ih)
c,)
+
Q, Cd), the relationship of Cd > C, is obtained from eqs. (13) and (14) where y and z express the fraction, i.e. y < 1 and z < 1. Actually, in the fetal lambs
02
211
ANALYSES IN CHICKEN EMBRYO
(Dawes et al., 1954) the So, of blood from the right ventricle was about 90% of that of the descending aorta (i.e. C, = 0.9 C,). If we use this relationship to the chicken embryos, the C, is determined as 0.018 ml. (ml blood))‘. As to the 02 content of the venous blood from the three compartments, we assume that both the O2 contents from the forequarters and hindquarters are identical and 40% less than the O2 content of pulmonary venous blood, i.e., Cf, = Chv = 0.6 C,, (= C,). In fetal lambs (Dawes et al., 1954), Cr, was a bit lower than ChV.However, in chicken embryos, as determined here, the O2 content in arterial blood supplying the tissues is predominantly low (e.g. 2 vol”A in the descending aorta or allantoic artery of 16-day-old embryos). As a result, the venous 02 reserve must be much lower than in other species, then the assumption of Cr, = ChV= 0.6 C,, may be permissible. Then, in order to solve the equations, the values of C, is first approximated and the rates of blood flow are obtained from eqs. of (1) to (5). Next, the range of X is determined by eq. (15) and the ratios of separation (x, y and z) are calculated for X value from eqs. of (12) to (14). If these values satisfy all of the relationships expressed by eqs. of (6) to (1 l), they are right solutions for the equations. The value of X is determined by trial and error until the eqs. of (6) to (11) are all satisfied. In 16-day-old embryos, we have the values of 0.11 and 0.02 ml. (ml blood)- ’ for C, and Cd, and 0.36 ml.min-’ for iTo,, respectively. In case that the So, in bloods of the forequarters and hindquarters were given as above in connection with the values reported in the fetal lambs, the following values were obtained. X = 0.6844,
x = 0.3865,
y = 0.1680,
z = 0.1827,
C, = 0.018,
Cr = 0.032,
Cr, = C,,v = 0.006,
C,, = 0.01,
@, = 2.25,
Qr = 8.31,
Qh = 9.0,
oa = 4.0 and Qd = 13.0.
In fig. 3 are shown the distribution of blood flow (ml. min- ‘) and the percentage saturation with O2 of blood which was calculated by the O2 content divided by O2 capacity of 0.12 ml. (ml blood)-‘. The So, is shown in parentheses. In chicken embryos, the volume of blood flow to the tissues is larger than that through the chorioallantoic membrane, which may compensate for the low 0, content of blood supplying the tissues. The O2 transport capacity expressed by the product of blood flow rate and O2 content (0. Co,) is 0.27 and 0.18 ml. min- ’ for the forequarters and hindquarters, respectively (17.8 and 12.1 ml. min- ’ . (kg weight of embryo)-‘), and the O2 utilization coefficient (\ioJQ . Co,) is about 80 and 70% for the forequarters and hindquarters. On the other hand, hens have a systemic 02 transport capacity of 43 ml .min- ’ (26.9 ml .min-’ .(kg weight)- ‘) and an 02 utilization coefficient of 60 y0 (Piiper et al., 1970). In the fetal lambs, the 02 transport capacity is 4.6 and 5.0 ml. min- ’ . (kg weight of fetus)- ’ for the forequarters and hindquarters, and the 02 utilization coefficient is about 60 and 54% for the former and the latter, respectively (Dawes et al., 1954). The O2 transport capacity in the human subjects is estimated about 20 ml *min- ’ . (kg weight)- ’ and the 02 utilization coefficient about 20 to 30% at rest (Bartels et al., 1955). During
II. TAZAWA AND M. MOCHIZUKI
212
Chorioallantois
4.0
-
(16.7)
Fig. 3. The calculated values of blood flow rate (mI.min _ ‘) in each part of the circulation and the 02 saturation of blood (‘$‘,).The So, is shown in the parentheses T
I-
150 140 130 120 110 ~ loo-
“-_
2
go-
E
Iso
shell
air space ps
altantoic vein
70a”
60-
chorioallantoic/ P”““‘“‘ies ,I I ,’
systemic arteries
I
4 .---___.
systemic veins Pv _-__Fig. 4. The Po, values in eggs incubated for 16 days. The Pi and Pi, are Oz tension of bloods flowing into the forequarters and hindquarters, respectively, and the Pe2 of the output from these tissues (Pi, and Phv)are assumed to have the same value (P”). The mean tissue PO, (I%) is assumed 1 mm Hg less than P,. The solid line indicates the values measured and the dotted line, those assumed or conjectured in the present report.
02
213
ANALYSES IN CHICKEN EMBRYO
exercise, O2 utilization in man increases up to 50 to 60% in association with a significant decrease in’Pvo2 (Cruz et al., 1969). The 02 transport capacity both in the forequarters and hindquarters seems to be equal or a bit larger than in hens and human beings and the O2 utilization coefficient in 16-day-old embryos is much larger than both fetal lambs and human subjects during exercise, emphasizing that in embryos the metabolic rate per unit body weight (24 ml - min- ’ . (kg weight) - ‘) is extremely large in connection with the rapid development. The O2 release in the tissues is proportional to the difference between the mean systemic capillary PO1(i.e. PC‘ and Per, for the forequarters andhindquarters, respectively) and tissue Po, (Pt). The proportional constant may be defined as the diffusing capacity for deoxygenation in the tissues (i.e. Die,= and D$e,X for the forequarters and hindquarters, respectively). The O2 flux through the eggshell- is dependent upon the Po, difference between the environmental air (PO) and air space (Ps) and the diffusing capacity in the eggshell (DJ. Similarly, the O2 quantity taken up by the chorioallantoic capillary blood depends on the difference between the air space PO2 and mean chorioallantoic capillary PoZ (Pee) and the diffusing capacity for oxygenation in the chorioallantoic capillary bed (DoX). That is, v . Vo,
= Dfdeox.(k
- pt)
(16)
(17) and $ro2= D, . (PO-P,) = D,, . (Ps - k).
(18)
Figure 4 shows the Po, values of each part of the eggs incubated for 16 days, where the mean PO, in the tissues (I%) was assumed to be 1 mm Hg less than the systemic venous blood Po, (P”) and the relationship between PO2and SOSwas assumed to be linear in a low range of So2. We used the values of 20.8 mm Hg and 16.5 % for the allantoic arterial PoZ and So2, respectively, from the results of both the previous and the present measurements.. The mean capillary PO2 for chorioallantoic and systemic circulations was estimated by Bohr’s integral procedure. The P, was obtained from the data of Wangensteen and Rahn (1970/71). Using the individual value of Po, shown in fig. 4, the diffusing capacities defined by eqs. of (16) to (18) were calculated. D, = 10.1 x 10-3,
D,, = 4.9 x 10-‘3,
DLox= 29.6 x 10e3 and
Dieox = 29.3 x 10e3 ml-min-’
*mm Hg-‘.
Both the Ddeoxsin the forequarters and hindquarters are much larger than D,,. The resistivity for deoxygenation in the tissues (1 /Ddeox)appears to be very small compared
214
H. TAZAWA AND M. MOCHIZUKI
with that for oxygenation in the chorioallantois in association with the large blood flow rate through the tissues. The experimental evidence that the arterialized bllod PO, in the allantoic vein (P,) was lowered to about the middle level between P, and allantoic arterial Po, (Pd) seems to be attributed to the large resistivity to 02 in the chorioallantoic capillary plexus (l/D,,X). The low value of Po, in the allantoic artery may also be attributed to the high O2 extraction in the tissues. On the other hand.,since the 02 uptake increases as embryos grow (fig. 1) and the diffusion conditions across the shell remain unchanged, the P, decreases with increase in O2 uptake during development and the D, is constant. Therefore, the 02 transport from the environmental air to the tissues may be limited by the resistivity for oxygenation in the chorioallantoic capillary (l/D,,) at the earlier days of incubation, and then with the progress of development of embryos, by the addition of a moderate eggshell resistivity (1 /DS).
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Cruz, J. C., H. Rahn and L. E. Farhi (1969). Mixed venous Po,, Pco2, pH, and cardiac output during exercise in trained subjects. .I. &pi. Physiof. 27: 431-434. Dawes, G. S., J. C. Mott and J. G. Widdicombe (1954). The foetal circulation in the lamb. J. Physiol. (London) 126: 563-587.
Dawes, G. S. and J. C. Mott (1964). Changes in 02 distribution and consumption in foetal lambs with variations in umbilical blood flow. J. Physiol. (London) 170: 524540. Freeman, B. M. and B. H. Misson (1970). pH, pt& and pCO2 of blood from the foetus and neonate of Gailus Domesticus. Camp. Biochem. Phy.&l. 33: 763-772. Girard, H. (1971). Respiratory acidosis with partial metabolic compensation in chick embryo during normal development. Respir. Physiol. 13 : 343-35 1. Kusumi, F., W. C. Butts and W. Ruff (1973). Superior analytical performance by electrolytic cell analysis of blood oxygen content. J. Appl. Physiol. 35: 299-300. Misson, B. H. and B. M. Freeman (1972). Organic phosphates and oxygen affinity of chick blood before and after hatching. Respik. Physioi. I4 : 343-352. Piiper, J., F. Drees and P. Scheid (1970). Gas exchange in the domestic fowl during spontaneous breathing and artiticial ventilation. Respir. Physiol. 9: 234-245. Romanoff, A. L. (1960). The Avian Embryo. New York, The Macmillan Co., 1149 p. Romanoff, A. L. (1967). Biochemistry of the Avian Embryo. New York, John Wiley & Sons, 291 p. Tazawa, H. (1970). Measurement of O2 content in microliter blood samples. J. Appl. Physiof. 29: 414-416. Tazawa, H. (1971). Measurement of respiratory parameters in blood of chicken embryo. J. App!. Physioi. 30: 17-20.
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Tazawa, H. and M. Mochizuki (1976). Estimation of contact time and diffusing capacity for oxygen in the chorioallantoic vascular plexus. Respir. Physiol. 28: 119-128. Tazawa, H., T. Ono and M. Mochizuki (1976). Oxygen dissociation curve for chorioallantoic capillary blood of chicken embryo. J. Appl. Physiol. 40: 393-398. Wangensteen, 0. D. and H. Rahn (1970/71). Respiratory gas exchange by the avian embryo. Respir. Physiol. 11: 3145.
