232
THE BLOOD PRESSURE OF THE CHICK EMBRYO
DURING DEVELOPMENT
BY A. F. W. HUGHES
Sir Halley Stewart Research Fellow
From Strangeways Research Laboratory, Cambridge
(Received 31 May 1942)
(With 4 Text-figures)
INTRODUCTION
This investigation was made as part of a wider inquiry into the developmental physiology
of chick circulation in relation to the histology of the developing vascular system.
One observation on the blood pressure of the chick embryo has already been made
by Hill & Azuma (1927) who measured the pressure in the arteries of the chick blastoderm after 2 days' incubation and found it to be 2\ cm. of water.
Their method, like that used in the present work, is a sphygmomanometric one. To
the blastoderm freed from its surroundings they applied an external air pressure, transmitted through an elastic transparent membrane thereby compressing the blastoderm
against a glass plate; the least external pressure necessary to collapse the arteries was then
measured.
Hill & Azuma's method as it stands cannot be readily applied to later stages of incubation because of the difficulty of freeing the larger area vasculosa of older embryos from
yolk and white without injury.
The method described below has been developed for measurements on the arteries
of the chorio-allantois and does not involve interference with the yolk sac. It can only
be used, however, when the chorio-allantois has reached a certain size which prohibits
measurements earlier than the sixth day of incubation.
METHODS
Thirty-three successful measurements have been made oh embryos of 6-19 days' incubation, the cborio-allantoic arteries ranging in diameter from 0-25 to 0-75 mm. The final
curve of arterial pressure during development (Fig. 4) includes the value for the 2-day
chick obtained by Hill & Azuma.
The artery in which the blood pressure is to be measured is compressed between a
glass probe, inserted through an incision in the vascular membrane, and a capsule,
2 cm. in diameter, to which air is admitted under pressure and of which the floor is
formed by a membrane of thin rubber with a hole in it (Fig. 1). This hole is smaller
in diameter than the glass probe. The hole and probe are placed concentrically when
measurements are made, and the vertical distance between them is adjusted with great
care. If they are too close, the artery is compressed without any air pressure being applied
The blood pressure of the chick embryo during development
233
o the chamber, but if they are too far apart, bubbles of air escape.* The artery is compressed directly by the air in the chamber.
If a continuous rubber membrane was employed, any curvature imparted to it would
introduce an error into the measurements, for the pressure on the two sides of the membrane would not be the same, and it would be almost impossible to use the device with
Rubber membrane
with hole
Air pressure
and manometer
Light source
\
Artery in which
blood pressure is
being measured
\
Glass rod inserted into chorio-allantoic
cavity through incision in membrane
Diagram of apparatus
Fig. 1. Apparatus for measurement of arterial pressure in chorio-allantoic artery of chick.
no curvature of the membrane. The errors thus introduced would be serious as the
pressures to be measured are small in comparison with those usually measured by
sphygmomanometric methods. The earliest trials of this apparatus were made with
continuous membranes and although the thinnest rubber sheeting obtainable was used,
no uniform readings could be obtained.
The probe and the brass tube attached to the capsule are both carried by special,
micro-manipulator heads, which give a coarse movement over a wide range in all three
• In practice, the distance between hole and probe is kept as large a3 is possible without bubbles of air
escaping. The error in the result due to the pressure inside the air bubble is less than 0-5 cm. of water.
A. F. W. HUGHES
234
dimensions. In the upper end of the glass probe a 6 V. 'flash-lamp' bulb is mounted
The probe thus serves as a glass rod illuminator, and the artery is clearly seen under a
low-power microscope through the hole in the rubber membrane, and the cover-slip
forming the roof of the pressure capsule. The inside of this cover-slip needs moistening
with glycerine to prevent condensation.
The vessel containing the saline, in which egg and device are immersed, has a capacity
of about 250 c.c. and is heated by a compact form of water-bath, controlled to give the
necessary temperature of 38—390 C.
20
25
15
30
35
Min. after immersion
Fig. a. Arterial pressure observations during the progress of an individual experiment.
10
The procedure is as follows. A hole is made in the air space of the egg which is then
immersed in the warm saline, and with extreme care the shell is gradually picked off,
using an outward motion. When most of the shell is removed, the shell membrane is
peeled off, with even greater care. The last third of shell and shell membrane can usually
be floated away from their contents. The whole chorio-allantoic membrane is then exposed, covering nearly all the yolk sac and albumen. In stages up to 12 days or so, it is
advisable to free the chorio-allantois from the yolk sac by careful tearing at the edge of
the former where the two embryonic membranes are attached to each other. When the
operation is successful, a preparation can be made of the embryo in its amnion with yolk
sac and chorio-allantois intact on each side.
Next, an accessible chorio-allantoic artery is selected, and a small incision made in
the membrane near it, through which the probe is inserted. The capsule is cautiously
lowered, and the artery made to lie suitably across the device, by gently pulling on the
The blood pressure of the chick embryo during development
235
Membrane. The probe and membrane are brought near together to compress the artery,
and air pressure is admitted to the capsule. Probe and membrane are now separated until
air bubbles, are on the point of escaping from the hole in the membrane, and the air
pressure is varied until the artery can be compressed by this agency alone. The pressure
is then adjusted until a point midway between systolic and diastolic pressure is found,
so that the blood pulsates across the hole from the side nearest the heart with each heart
beat, being squeezed out during diastole. Separate measurements for systolic and diastolic
pressures are not attempted, as only in this intermediate position is a clear end-point
obtainable. The air pressures applied are measured on a water manometer, and pressure
and time of observation are noted down over a period of 10-30 min. The arterial pressure
o
o
o
o
S,
0
2
4
6
8 10 12 14 16 18 20
Incubation age in days
Fig. 3. Average rate of fall of arterial pressure during an individual experiment
related to the age of the embryo.
gradually falls as the preparation deteriorates (Fig. 2). Up to the tenth day of incubation,
the fall in arterial pressure with time under observation is very gradual. This is due to
the extreme ease with which eggs of this age can be decanted, as described above, with
almost no haemorrhage and with the chorio-allantoic circulation almost unaffected.
In the second half of the incubation period, however, increasingly more damage is done
by this treatment. Since the chorio-allantoic capillaries go into stasis with the slightest
mechanical stimulus and haemorrhage is unavoidable with any manipulation at all, conditions for the measurements of the arterial pressure become less favourable, as the eggs
become older. This is reflected in the curves of Fig. 2 in which the arterial pressure in
embryos of different ages is plotted against time of observation; the curve at 17 days is
much steeper than at earlier stages. Fig. 3 expresses the slope of these curves for twelve
sets of observations, and it is seen that they usually, though not always, become much
steeper in later stages.
The question thus arises as to how our final estimate of the arterial pressure at each
stage is to be made. Up to 10 days of incubation, a simple average of all values obtained
in each set of observations is clearly sufficient, but in later stages this gives merely an
average value of the arterial pressure of embryos dying from anoxemia. It seems more
A. F. W. HUGHES
reasonable to plot at each stage the curve of decrease in arterial pressure with time
observation where the constituent points can obviously be represented by a straight lin
and to read off from this line the estimated arterial pressure at the beginning of observation. In Fig. 4 the circles express these estimated arterial pressures, and to them most
weight has been given in drawing the final curve of arterial pressure against incubation
time. The solid points in Fig. 4 represent the averaged values, and where estimated and
averaged values are given for the same set of observations, the two points are joined by
a vertical line. These lines express the divergence between averaged and estimated arterial
pressures.
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I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21 1 2 3 4 5"
Days of incubation
Days after hatching
Fig. 4. Arterial pressure of chick embryo and chick respectively in respect of age. • = average values for
a given determination. O = value calculated from extrapolation of individual observation (see Fig. 2).
The arterial pressure in newly hatched chicks was measured by a similar method,
except that a continuous rubber membrane and a mercury manometer were used. The
chicks were anaesthetized with ether, and the common carotid arteries in the neck were
exposed, an illuminated glass spatula was inserted between the arteries and neck muscles,
and the capsule applied to the upper surface of the arteries. Six sets of observations were
made on chicks in the first week after hatching and others at later stages. The slope of
the curve for stages after hatching in Fig. 4 is taken from these observations.
DISCUSSION OF RESULTS
It is proposed to discuss these results at length in a subsequent paper, together with
other data from the embryonic circulation. Meanwhile the data on arterial pressure in
the chick embryo can be compared with the corresponding results from mammalian
foetuses whidi have been obtained by various authors, notably by Barcroft (1935, 1936).
The blood pressure of the chick embryo during development
237
Arterial pressure for the sheep (Barcroft, 1936) plotted against foetal age gives a curve
of the same general shape as mine for the chick embryo, rising, at first gradually, then
progressively more steeply. The time scales are respectively 150 and 21 days. It is
interesting to compare the sheep and chick embryos when their size and arterial pressure
are about the same. The sheep foetus at 44 days and the chick embryos at 14^ days
both have the same arterial pressure (18 mm. mercury) and weight (10 g., Needham,
1931). The arterial pressure of the chick embryo is rising very rapidly at that stage,
while that of the sheep, embryo is still in the initial slow rise. The chick embryo also is
developmentally much further advanced than the sheep embryo of the same weight.
A further resemblance can be seen between the curves for arterial pressure during
development in chick and sheep embryos. In both the rapid rise in arterial pressure in
the latter half of development is followed by a period of stationary pressure just before
birth. Barcroft gives the arterial pressure of the sheep foetus at 140 days—10 days before
term—at 76 mm. of mercury; in the continuous tracing of arterial pressure during .birth
by Caesarian section given in the 1935 paper, the pressure at first is 63 mm. of mercury.
In the chick embryo the pressure rises very little after 16 days.
Again, at birth, when pulmonary respiration is established, the arterial pressure in
both animals rises to a figure which continues the preceding rapid rise, interrupted towards
the end of embryonic life. In the sheep foetus the rise in arterial pressure at birth is
about 20%, but in the chick embryo the rise appears to be larger and in the data of
Fig. 4 lies between 34 and 100%.
The stationary period in arterial pressure at the end of development in the chick embryo
is a very interesting problem in developmental physiology. The reason for it certainly
does not lie in the heart itself, the weight of which continues to increase at the end
of development as rapidly as before and doubles between 16 and 21 days (Olivo, 1930).
On opening an egg at the end of the third week of incubation, one is struck by the
extreme darkness of the blood in the chorio-allantoic vessels. The blood in the arteries
appears fully reduced and that in the veins far from saturation with oxygen. Unfortunately, no measurements appear to have been made on the oxygen content of the blood
of the chick embryo at this stage, a problem which becomes all the more interesting in
view of the investigations on mammalian foetuses by Barcroft and co-workers. Appearances, however, suggest that the chick embryo at the end of development is in a state
of anoxaemia so far advanced that the action of the heart may be adversely affected.
SUMMARY
1. An apparatus is described for the measurement of the pressure in the chorioallantoic artery of the chick embryo.
2. Data are given of such measurements ranging in time from the second to the
nineteenth days of incubation.
3. Some measurements are also given of the arterial pressure of the chick within the
first five days after incubation.
REFERENCES
BARCROFT, J. (1935). Irish J. Med. Sci. Purser Lecture, September.
BARCROFT, J. (1936). Phytiol. Rev. 16, no. i, January.
HILL, R. & AZUMA, Y. (1927). J. Phytiol. 27, 62.
NKKDHAM, J. (1931). Chemical Embryology, 3. Cambridge.
OLIVO, O. (1930). Proceedings 4th World's Poultry Congress, London.