Capillary transit times and kinetics of oxygenation in the primary respiratory organ of early chick embryo.

In early avian embryos respiratory gas exchange between environment and blood takes place via the blood vessels of the yolk sac. To investigate the principles governing oxygen transfer we determined the RBC velocity, the length of the a-v channels, and the RBC transit time in the vitelline capillary network of 4-day-old chick embryos. Measurements were carried out using a digital video analyzing system for on-line velocity measurements (modified dual video window technique) and morphometric determinations. Velocity measurements were performed on fluorescent (FITC)-labeled erythrocytes. All measured parameters exhibited a large scatter. The mean RBC velocity was 193 +/- 117 (+/- SD) microns/sec and the mean length of the a-v channels was 434 +/- 291 microns. The transit time ranged between 0.1 and 17 sec (median value 2.5 sec). Using previously determined oxygen hemoglobin dissociation curves and blood oxygen partial pressure (Meuer and Baumann, 1988, Respir. Physiol 71, 331-342; Meuer et al., 1989, J. Dev. Physiol. 11, 354-359) the kinetics of the oxygen uptake by the blood was calculated. This gave an estimated transit time required for 99% oxygen saturation of 1.5 sec. The data show that in more than a third of all a-v channels the transit time is shorter than required for complete blood oxygenation. This is consistent with recent determinations of the blood oxygen saturation of mixed arterialized blood (89% at Day 4; Meuer and Baumann, 1988).

Tissue PO2 and growth rate in early chick embryos.

We determined dry mass, frequency distribution of tissue PO2 and microvascular density of head and trunk of early chick embryos between day 4 and day 6 of incubation. During this period the percentage of the dry mass of the head on total dry mass increased from 31% to 48% indicating that the head grows faster than the trunk. Tissue PO2 values ranged between zero and arterial PO2. About half of the numbers were less than 5 Torr. Mean tissue PO2 was significantly higher in the head than in the trunk. This was paralleled by a significantly higher microvascular density in the head. The high frequency of low tissue PO2 values found at each day at all measuring sites suggests that the embryonic tissue of both head and trunk extracts as much oxygen from the capillary blood as possible. Consequently, growth rate strongly depends on oxygen availability. Since the arterial PO2 is the same in the head and in the trunk (Meuer and Baumann, 1988), the diversity of tissue PO2 and growth rate found in this study is probably caused by differences in the structure of the microvascular bed resulting in variations of tissue blood perfusion, capillary transit time and diffusion distance.

Erythrocyte velocity and total blood flow in the extraembryonic circulation of early chick embryos determined by digital video technique.

RBC velocity was determined in the major blood vessels of the extraembryonic circulation of early chick embryos between Day 4 and Day 6 of development using fluorescent-labeled erythrocytes. Measurements were performed by applying a digital frame-by-frame video technique. The expenditure of operator interaction was minimized by computer support. Velocity measurements of more than 15,000 labeled blood cells were evaluated for mean RBC velocity and volume flow of 354 venous blood vessel segments. Linear regression for the power function of the calculated volume flow vs the vessel diameter yielded an exponent of 2.77 at Day 4, increasing to 2.96 by Day 6. Applying Murray's model of energetic cost, these data indicate that in the course of development the newly formed extraembryonic vascular system is optimized in terms of minimizing cardiac work. The total extraembryonic blood flow as calculated from the sum of the volume flows of the main veins was 656 +/- 218 and 1169 +/- 409 nl/sec at Day 4 and Day 6, respectively. Using previously determined values of blood oxygen concentration, embryonic oxygen uptakes of 9.6 nl/sec (Day 4) and 40.2 nl/sec (Day 6) were calculated.

Changes in blood PCO2 and acid-base status in chick embryo between day 4 and 6 of incubation.

PCO2 was measured in extraembryonic blood vessels of early chick embryos (first third of incubation period) using PCO2 microelectrodes with liquid ion exchanger. Between day 4 and day 6 of incubation mean PCO2 in the vitelline veins increased from 4.2 to 7.0 torr and in the vitelline arteries from 6.9 to 10.6 torr. From the CO2 titration curves obtained in ovo by measurements in hypercapnic environment buffer values of the blood were evaluated (28.8 mmol/l at day 4 and 21.4 mmol/l at day 6). Calculated standard bicarbonate concentration was 29.4 mmol/l at day 4 and decreased to 24.4 mmol/l by day 6 indicating metabolic acidification. These results are compatible to the data previously determined for older embryos.

Measurement of pH in blood vessels and interstitium of 4 and 6 days-old chick embryos.

The pH in intra- and extraembryonic blood vessels and in the embryonic interstitium has been measured in 4 and 6 days-old chick embryos using pH microelectrodes. The mean pH of the lateral vitelline vein was 8.0 at day 4 and 7.89 at day 6 and that of the vitelline artery 7.8 and 7.66, respectively. The pH of the blood in the embryonic heart was the same at day 4 and 6 (7.68 and 7.67). The lowest blood pH was recorded in the jugular vein (7.42 at day 6). The pH values measured in the interstitium show less variability. However a clear cranio-caudal gradient was apparent at day 4 where pH fell from 7.68 to 7.47. At day 6 the pH values of the interstitium ranged between 7.61 and 7.52 showing no preferential distribution. Using previously determined values for blood PO2 and the oxygen haemoglobin equilibrium curves, we calculated the oxygen saturation of the blood at the measured pH. In the vitelline vein the O2 saturation is 89% at day 4 and more than 95% at day 6, whereas that of the vitelline artery is 37% and 25%, respectively. When ambient PCO2 is raised to about 8 torr the pH falls to 7.81 (7.68) in the vitelline vein at day 4 (6) and the oxygen saturation decreases to 64% and 79%, respectively. Thus the high pH values found in the vitelline vein seem to be necessary for adequate oxygen uptake of the embryo.

Oxygen pressure in intra- and extraembryonic blood vessels of early chick embryo.

Oxygen partial pressure (PO2) was measured in vitelline and intraembryonic vessels of 4- and 6-day-old chick embryos in ovo using recessed-tip oxygen microelectrodes. At day 4 mean PO2 values were 6 kPa in the vitelline arteries and 8.5 kPa in the oxygenated blood of the lateral vitelline veins. These values increased significantly by day 6 to 6.5 kPa in the vitelline arteries and 10.8 kPa in the lateral vitelline veins. The PO2 in intraembryonic arteries was found to be close to the PO2 in the vitelline arteries. In intraembryonic veins PO2 values ranged from 2.1 to 4.6 kPa displaying a gradient from cranial to caudal. Our measurements show that the major part of the diffusion resistance between environment and gas exchange vessels is formed by the albumen layer separating the yolk sac from the inner shell membrane, and that the oxygen transport is limited by diffusion. The low PO2 values in the intraembryonic venous vessels suggest that the gas transport system works at the limit of its capacity.

Oxygen supply of early chick embryo in normoxia and hypoxia.

The oxygen partial pressure (PO2) was measured in the large vessels of the yolk sac of 4- and 6-day old chick embryos using recessed-tip oxygen microelectrodes. The eggs were either incubated in air or in a hypoxic gas mixture (13.5% O2/86.5% N2). For the measurements, a part of the eggshell and the shell membranes were removed. Between 4 and 6 days of development, the mean PO2 in the anterior and posterior vitelline veins, which is approximately equal to the PO2 of the blood leaving the gas exchange vessels, was about 12 kPa (92 torr) in the normoxic group and about 9.3 kPa (70 torr) in the hypoxic group, suggesting that oxygen transport is diffusion-limited. The mean PO2 in the vitelline arteries, which are supplied by the dorsal aorta, was found to be in the range of 6-6.5 kPa (45-49 torr) in both normoxia and hypoxia. Taking into account that the eggshell and the shell membranes form an additional diffusion resistance, we have evaluated the PO2 and oxygen saturation of the blood of intact normoxic and hypoxic embryos.