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The avian embryo develops a structure for gas exchange with its environment that has some of the same functions as the mammalian placenta. The CAM, with its capillary bed, is the respiratory organ of the chick embryo until the 19th d of incubation, at which time the embryo pips internally in the air cell and starts air breathing(1). This internal pipping is the end of the prenatal period of development and initiates the transition from diffusive gas exchange of the CAM to convective gas exchange of the lungs(1). Some similarities between the chick embryo and mammalian fetus have been reported, for instance 1) the CABF represents about 50% of the combined cardiac output(2);2) the combined cardiac output of the chick embryo is 500 mL·kg-1·min-1, a value which is comparable to mammalian fetuses(3); 3) the distribution of the cardiac output as well as the gas tensions are also comparable to that of the sheep fetus(2, 4).

The level of gas exchange in the CAM depends on the rate of blood flow passing the exchange membrane, as in the placenta. From animal(3) and human fetal(5) studies it is known that there is an increase in umbilical blood flow with increasing gestational age. No data are available on the development of the CABF of the chick embryo. In the present study chick embryos from stage 34 to stage 43 (d 9-16 of a 21-d incubation) were used to measure the changes of the heart rate and the CABF with increasing incubation time.

METHODS

Fertile White Leghorn chick eggs were incubated at 38 °C and 60% humidity. They were rotated hourly to prevent adhesions between the embryo and its membranes(6). CABF measurements from the 9th to 16th d of incubation (total incubation time, 21 d), stage 34-43(7), were performed.

All procedures and measurements were done inside a regular clinical infant incubator, in which the temperature was kept at 38 °C and the humidity at 60%. By means of transillumination the limits of the air cell were identified and cut with an electric saw. Afterward the eggs were placed in a small plexiglass holder provided with a continuous gas flow of a N2/O2 mixture (5 L/min) adjusted to 21% oxygen at 38 °C and 60% humidity. After opening of the air cell, the external membrane was visible. To visualize the vessels running in the CAM, the external membrane was made transparent using a cotton swab made wet with 0.9% saline. Using a dissection stereomicroscope(WILD 3M, total magnification 10×), the external shell membrane was removed with great care. A small incision was made in the CAM using cautery to avoid hemorrhage. The embryo was now visible in the amnionic fluid.Figure 1 shows schematically the embryonic circulation and demonstrates where the flow probe was placed. The overall circulation in the chick embryo is comparable to the human fetus, but there are some differences as described by White(8). Major differences are the vitelline veins, which transport the digested yolk material to the liver, and the umbilical blood, which flows directly to the left hepatic vein without entering the liver substance. The dark red pulsating chorioallantoic artery was localized near the embryonic abdomen and placed in the lumen of a 0.5-mm flow probe (0.5VB39, Transonic Systems Inc., Ithaca NY), avoiding any obstruction of the vessel. The flowmeter subtracts the downstream transit time from the upstream transit time using wide-beam ultrasonic illumination. This difference between integrated transit times is a measure of blood flow rather than velocity(9). The space between the vessel and the probe had to be filled with fluid without air bubbles or tissue to obtain a homogeneous ultrasound field. Because only acoustic contact is necessary, a loose fit of the probe is acceptable. This technique has been validated using mechanical calibrations or microsphere injections to measure blood flow in different vessels from several species(10, 11). The heart rate was derived from the blood flow signal. A hemodynamic digitalized acquisition system (sample interval, 2 ms, and system trigger, 2000 ms) (Hemodynamic Acquisition System, University of Limburg) was used for monitoring and saving, beat to beat, the blood flow and heart rate data. The temperature in the egg was measured using a small temperature probe (Ellab electrical thermometer, type AF6, diameter 0.8 mm) placed in the amnionic fluid, to maintain it at 38 °C, because of the sensitivity of the heart rate to temperature changes(12, 13).

Figure 1
figure 1

Schematic view of the chick embryo circulation redrawn after Romanoff(1) and White(7). Abbreviations: Aah, aortic arch; AV, allantoic vein;AVC, anterior vena cava; DA, dorsal aorta; IVC, inferior vena cava; LA and RA, left and right atrium;LAA and RAA, left and right allantoic artery;LDAR and RDAR, left and right ductus arteriosus;LHV, left hepatic vein; LV and RV, left and right ventricle; MV, mesenteric vein; OMV, omphalomesenteric vein; PA, pulmonary artery; PAh, pulmonary arch; PV, pulmonary vein; VV, vitelline vein.

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A total of 100 chick embryos were studied, 10 at each stage. Only those embryos which had no bleeding or malformations were included. A 10-min recording was made after a period of stabilization of the blood flow and heart rate. Each period of 10 min yielded a blood flow which was the mean value for the period. After recording, embryos were removed from the egg and classified using an index of morphologic maturity(7). The weight of the embryo was determined using a laboratory scale (Sartorius, L2200P), after drying the embryo with a hair dryer.

The opening of the air cell changes the diffusion gradient through the egg shell by exposing the embryo directly to the environment, which could induce changes in the embryonic blood gasses(14). To control for this, chorioallantoic artery blood samples for pH, Po2, and Pco2 were obtained from another group of chick embryos at stage 40-41(n = 15) in which the air cell was opened for, respectively, 10, 20, and 30 min. The embryos were kept in the same environmental conditions as the experimental group. For the samples, the chorioallantoic artery was carefully lifted with forceps, and a curved 30-gauge needle was inserted contrary to the blood stream flow. Blood samples (0.2 mL) were collected into heparinized syringes and analyzed at 38 °C (Radiometer ABL3, Copenhagen).

Statistical analysis. Blood flow, heart rate, and blood gas values were expressed as mean ± SD. The coefficient of variation of the mean blood flow was calculated for each egg. The correlation between blood flow or heart rate and the embryonic weight was calculated using the Spearman rank correlation. Significance was accepted at p < 0.01.

RESULTS

To determine the effects of opening the air cell of the egg, embryonic blood gas levels were measured at, respectively, 10, 20, and 30 min after opening the air cell. As can be seen in Table 1, the mean levels for blood pH, Po2, and Pco2 did not change significantly with increasing the time of opening the air cell. The blood gas levels of our study were within the range previously reported for the chick embryo with an intact air cell (pH 7.374-7.530; Pco2, 4.53-5.76 kPa; Po2, 2.93-4.80 kPa)(15–19).

Table 1 Blood gas levels in the chick embryo 10, 20, and 30 min after opening of the air cell at stage 40 and 41
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Table 2 shows embryonic weight, CABF, and heart rate at different stages. The coefficient of variation of the blood flow ranged from 1.93 to 7.8%. This represented the biologic and the methodologic variations of the blood flow measurements. The blood flow increased 10-fold from 0.35 ± 0.18 mL/min at stage 34 (d 9 of incubation) to 3.13± 1.49 mL/min at stage 43 (d 16 of incubation). The embryonic body weight also showed a 10-fold increase during the same incubation period(correlation between stage and weight was R2 = 0.875). The mean ratio of CABF to embryonic weight did not change during the incubation. The CABF significantly correlated with the embryonic weight(R2 = 0.69 and p < 0.0001)(Fig. 2). The heart rate increased, respectively, from 194.67 ± 37.55 beats/min at stage 34 to 288.78 ± 13.48 beats/min at stage 43. This increase was significantly correlated with the embryonic weight (R2 = 0.38 and p < 0.0001)(Fig. 3).

Table 2 CABF and heart rate of the chick embryo from stages 34 to 43
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Figure 2
figure 2

The CABF (mL/min) measured in the chick embryo from stage 34-43 (d 9-16 of 21-d incubation). The CABF increases with embryonic weight.

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Figure 3
figure 3

The heart rate (bpm, beats/min) measured in the chick embryo from stage 34-43 (d 9-16 of 21-d incubation). The heart rate increases with embryonic weight.

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DISCUSSION

The importance of the present study lies, first, in our description of some methodologic aspects of the preparation and its possible uses for hemodynamic studies during the prenatal period, and second, because we report the changes in the CABF and the heart rate in the chick embryo from d 9 (stage 34) to 16(stage 43).

The chick embryo has been extensively used to answer very different questions at different stages of development. Early in development it has been used for studies of the hemodynamics of the cardiovascular system(20, 21) and the development of the CNS(22). Later in development it has become an attractive model for the study of the physiology of gas diffusion and exchange(23, 24). It has also been used for studies on the mechanisms involved in the etiology of intestinal malformations(25).

The circulation in the chick embryo is comparable to that of the mammalian fetus(1, 8). The left and right chorioallantoic arteries bring the deoxygenated blood into contact with CAM where gas exchange through the egg shell occurs and the chorioallantoic vein returns the oxygenated blood to the embryo. These vessels are equivalent to the umbilical circulation. After opening the air cell and the extraembryonic membranes these vessels are easily accessed. Technologic developments have produced transducers that allow reliable continuous blood flow measurements from small vessels(9), so we thought that the chick embryo would be an attractive model for studying the development of hemodynamic variables such as CABF and heart rate at different incubation times. It would also offer possibilities for studying normal and abnormal mechanisms involved in the control of the developing circulation. The preparations necessary for these acute experiments were simple. The measurement of the blood flow was noninvasive, but all steps necessary to have access to the vessel were rather invasive. First, it is important to maintain the normal range of humidity and temperature, because of its influence on cardiovascular variables(12, 13). Second, the air cell and the external membrane had to be removed. Removing the air cell and the external membrane modifies the diffusion characteristics of the egg(14). Pores of the avian eggshell are the only gas exchange organ between the developing embryo and the external environment. The passage of oxygen and carbon dioxide from and into the egg involves a series of membranes and blood barriers. When the air cell is opened hypocapnia might be induced, because of the higher level of the Pco2 in the air cell compared with the external environment(23). To control for that, blood samples were taken from the chorioallantoic artery after opening of the air cell for, respectively, 10, 20, and 30 min. These time intervals were chosen to match the duration of the recordings. The values obtained for Po2, Pco2, and pH were not significantly different from those given in the literature(15–19). However, it is important to note that both our values and those reported in the literature present great variability. Ideally it would be more accurate to measure blood gases at the beginning and at the end of the recordings to be sure that the embryonic blood gases did not change. This would require repetitive sampling from the chorioallantoic artery, which is possible(17), but the use of the same vessel makes the preparation technically more complicated, and the total volume of sampling, around 0.5 mL, represents 15-20% of the embryonic blood volume. This can disturb the physiologic conditions of the preparation.

Our study showed an increase in the CABF and the heart rate with increasing incubation time. There was a 10-fold increase in blood flow, from 0.35± 0.18 mL/min at stage 34 (d 9 of incubation) to 3.13 ± 1.49 mL/min at stage 43 (d 16 of incubation). The increase in CABF correlated with body weight. This correlation is not surprising, because large increases in body mass will increase the total metabolic rate. This causes an increase in the chorioallantoic blood flow to achieve an adequate gas exchange and meet the new metabolic demands(26). Moreover, it is known that the umbilical blood flow in human pregnancy increases with gestational age(5).

There are a few reports on the CABF measured at different incubation times. Tazawa et al.(12) measured a CABF of 4 mL/min using an electromagnetic flow probe in chick embryos at stage 43. This study examined the feasibility of the measurements, but it did not report the number of observations or the variability of the measurements. Most information available on CABF was obtained using the Fick formula and yielded CABF of around 4.44 mL/min(4, 14, 27, 28). The measurements of the CABF obtained in our preparation using a Transonic flowmeter yield values (3.13 ± 1.49 mL/min) that are comparable to those of previous studies. The advantage of our methods is the possibility of measuring blood flow reliably and continuously for periods of 30-60 min. This offers the possibility of designing experiments to study physiologic or pathophysiologic mechanisms involved in embryonic cardiovascular control. This will be object of a future report.

In contrast to other studies(1, 20), we measured an increase in the heart rate from stage 34 to 43. This observation is in agreement with Tazawa et al.(29). In our study the maximum heart rate at stage 43 was 288 ± 14 beats/min, which is comparable to the level of 278 beats/min at stage 43 reported by Tazawa and Mochizuki(27) and Tazawa and Takenaka(28). The difference in results between studies is not easy to explain, but the absence of a progressive increase in heart rate in some studies could have been due to differences in ambient temperature during the recordings. Thus, the chick embryo presents a steady increase in heart rate until d 17, which is followed by a decrease before hatching and a further increase afterward(1, 29). This characteristic behavior is in contrast with the gradual prenatal and postnatal decrease described in the mammalian fetus(1, 3, 5). A possible explanation for the steady increase in the heart rate in the chick embryo is the large metabolic rate per unit body weight and its increase associated with very rapid development(26, 30), suggesting that the control of the cardiac output in the chick embryo is more dependent on heart rate than on the Starling mechanism. Furthermore, the functional autonomic innervation of the embryonic heart occurs late in the development, anatomic parasympathetic and sympathetic neurons being completely functional at stage 42 (d 17)(31, 32). The increase in heart rate before this is thus associated with a relative lack of vagal control.

In summary, our study describes a technique using the chick embryo for studying CABF and heart rate. This technique did not interfere with gas exchange or exert obvious deleterious effects upon the embryo. The reported CABF profiles may serve as a basis for further cardiovascular research in perinatology.