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HEMATOCRIT, HEMATOCRIT REGULATION AND ITS EFFECT ON OXYGEN
CONSUMPTION IN LATE STAGE CHICKEN
EMBRYOS (Gallus domesticus)
Sheva Khorrami, B.S.
Thesis Prepared for the Degree of
MASTER OF SCIENCE
UNIVERSITY OF NORTH TEXAS
August 2004
APPROVED: Warren W. Burggren, Major Professor and
Dean of the College of Arts and Sciences Kathy Bishop, Committee Member Edward M. Dzialowski, Committee Member Art Goven, Chair of the Department of Biological
Sciences Sandra L. Terrell, Dean of the Robert B.
Toulouse School of Graduate Studies
Khorrami, Sheva, Hematocrit, hematocrit regulation and its effect on
oxygen consumption in the late stage chicken embryo (Gallus domesticus).
Master of Science (Biology), August 2004, 34 pp., 1 table, 9 figures,
references, 41 titles.
Hematocrit and hematocrit regulation have the potential to affect
developing embryos. To examine the ability of chicken embryos at day 15 to
regulate hematocrit, they were subjected to either repeated saline injections
(5% of total blood volume) or repeated blood removal (5% of total blood
volume). Embryos showed an ability to maintain hematocrit (~20%) despite
blood volume increases up to 115% of initial blood volume. Embryos were not
able to maintain hematocrit in the face of dramatic blood volume loss.
Oxygen consumption of embryos could be affected by their level of
hematocrit. To examine this, chicken embryos at day 15, 16, and 17 of
incubation were given a high hematocrit (~50-60%) sample of blood (400 µl)
to artificially increase the hematocrit of the embryos (~10-12%). Despite the
increase in oxygen availability, when monitored over a period of six hours,
embryos showed no difference (0.36 ± 0.01 (ml O2 • min-1• egg-1) in
metabolism from baseline measurements at day 15, 16 and 17.
ACKNOWLEDGEMENTS
I would like to thank all of the people that have made my years at UNT so
enjoyable. Special thanks go to my committee members, Dr. Kathy Bishop and Dr.
Ed Dzialowski for their support and guidance in my undergraduate and graduate
career. I would like to thank Angie for keeping our lab together in times of calm and
chaos, Ed for fielding hundreds of stupid and not-so-stupid questions, and my
labmates for their 24 hour support and for making graduate school entertaining in the
lab and out. I would especially like to thank Warren for being a wonderful mentor and
role model, for knowing when to push and when to support, and for giving me
wonderful opportunities within academia and out of it. Finally, I would like to thank
my parents and all of my siblings for their love and support. They are the rock that I
stand on; whatever I accomplish is owed to them. This research was supported by a
NSF grant to W.W.B.
ii
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS…………………………………………………………. ii
LIST OF TABLES …………………………………………………………………..iv
LIST OF FIGURES……………………………………………………………..…...v
CHAPTER
1. Introduction And Literature Review…………………………………….1
2. Materials And Methods…………………………………………………10
3. Results ……………….………..………………………………….… ….18
4. Discussion……………………………………………..…………...........26
REFERENCES..…………………………………………….…………………….34
iii
LIST OF TABLES
Table Page 4.1 Previous reported values of hematocrit for the days of incubation
used in this study……………………………………………………….. 33
iv
LIST OF FIGURES
Figure Page
1.1 Oxygen pressure gradients from nest to tissue in domestic fowl embryos …......9
2.1 Apparatus used oxygen consumption determination ……………..………….......16
2.2 Timeline of experimental procedure …………...………………..…….……..…..…17
3.1 Frequency distribution of hematocrits at various stages ……...….…..….……… 20
3.2 Effect of graded blood removal on hematocrit in Day 15 embryos ...…...……….21
3.3 Effect of graded saline injection on hematocrit in Day 15 embryo ……...……… 22
3.4 Effect of artificial erythrocythemia on hematocrit in chicken embryo…...………. 23
3.5 Effect of catheterization on oxygen consumption………………….……………. ..24
3.6 Effects of artificial erythrocythemia on oxygen consumption ...…………………. 25
v
CHAPTER 1
INTRODUCTION AND LITERATURE REVIEW
Importance of the Cardiovascular System
William Harvey, in the 17th century, was the first to describe human blood
circulation (Deshpande, 2002). Since then, the primacy of the cardiovascular system
has historically been emphasized because it sustains and connects many other
physiological systems. The importance of the cardiovascular system is heightened in
the context of development. As the first physiological system to come online during
development, it must support the development of other systems while maintaining its
own development. The study of blood and circulation in development holds
importance because any limitation or enhancement of it may limit or enhance other
developing systems as well (Burggren and Keller, 1997).
The Chicken Embryo as a Developmental Model
Birds have long been classical models for cardiovascular development. The
chicken model system has several advantages such as easy access and non-
seasonal availability. Compared to other models, the chicken embryo has a rapid
cardiovascular development and limited locomotion due to the shell (Tazawa and
Hou, 1997). When the egg is laid, nutrient transfer from the mother is complete and
the egg is analogous to a packet of energy that exchanges ammonia, water, oxygen
and carbon dioxide with the environment. The use of resources throughout
development can be determined with ease because of this characteristic.
The development of gas exchange in avian embryos closely parallels
mammalian fetal development. The use of chicken embryos is advantageous
compared to mammalian development because the chicken embryo develops
independently from its mother. This allows exploration of fetal gas exchange without
1
the complications of maternal influence. Also, an avian embryo model system allows
easy control and manipulation of respiratory factors, some of which cannot be so
easily controlled in the mammalian fetus, to determine their effects on gas exchange
(Tazawa, 1972). For example, due to a lack of convective ventilation in unpipped
avian embryos, this model system is effective in the study of respiration, specifically
diffusive gas exchange (Tazawa et al., 1992).
Ontogeny of Avian Respiration
In early development, the mesoderm of the yolk sac is well vascularized. The
vessels from the yolk sac connect with the dorsal aorta and blood begins to circulate
through a structure called the area vasculousa. From Day 3 to 5, the area vasculosa
grows out in a circular fashion from the embryo (Romanoff, 1960). During these early
stages, the area vasculousa and diffusion play a major role in the delivery of oxygen
to metabolizing tissues (Burggren et al., 2000). At Day 6, primary gas exchange
switches to the chorioallantoic membrane (CAM), which continues to grow rapidly
along the inside of the shell. By Day 9, it has grown over three fourths of the shell
and by Day 12 it has completely covered the egg (Romanoff, 1960). The CAM
remains the primary gas exchanger until the embryo internally pips and aeration of
the lungs proceeds. By the time the embryo externally pips, the CAM is completely
shut down and the bird begins to use its lungs for ventilation (Tazawa and Whittow,
2000).
The chicken embryo grows quickly during the first 2 weeks of incubation and
oxygen uptake of the egg increases geometrically. The increase in oxygen uptake
becomes asymptotic, reaching a plateau prior to pulmonary respiration around day
17 and increases again after internal pipping at day 20 (Tazawa and Whittow, 2000).
As the embryo’s amount of metabolically active tissue increases from growth, the
2
amount of oxygen received through the porous shell is increasingly inadequate in
satisfying the amount of oxygen needed. Thus the embryo experiences a
progressive “developmental hypoxia”.
Factors Affecting Blood O2 Transport
Oxygen transport in blood is dependent upon several different factors
including oxygen affinity, blood flow, rate of hemoglobin oxygen loading and
unloading, and blood oxygen carrying capacity (Piiper and Scheid, 1992). In
developing birds, atmospheric oxygen must come in contact with blood in the CAM
vessels. Oxygen must first cross the barrier of the eggshell and the outer
membrane. Oxygen movement, MX (in cm3 STPD sec-1 ) across the shell is almost
exclusively determined by a combination of the ratio of shell pore area, Ap (in cm2),
and thickness, L( in cm), properties of the gas being diffused, Dx (cm2 sec –1) and the
driving force of partial pressure difference, ∆Px (in torr) (Wangensteen et al.,
1970/71, Paganelli, 1980, Metcalf et al., 1981, Rahn et al., 1987). This relationship
can be expressed in this simplified form of Fick’s Equation:
MX= (Ap/L) ( Dx )(ßg)( ∆Px)
Though shell thickness and porosity can vary among avian species and even
individual eggs, the amount of oxygen that can get into the air cell is first limited at
this calcareous barrier. In diffusion, air partial pressure difference between the
environment and the air cell drives transport. While this outer shell barrier is fixed
throughout development, the inner barrier, composed of an inner membrane and the
chorioallantoic endothelium, changes with development (Tazawa, 1980). Diffusion
and convection are both in operation in this area as oxygen must diffuse across the
barrier but is then carried away by convection. In contrast to diffusive transport,
gases are transported in convective processes by hydraulic pressures (Paganelli,
3
1980). This inner barrier is where red blood cells in the capillaries come into contact
with the gases of the air cell.
The amount of oxygen that is in the blood is determined by air cell gas values,
metabolic rate and diffusion across the inner membrane (Tazawa, 1980). The
diffusing capacity of the inner diffusion barrier (DO2) has been expressed by Tazawa
and Whittow (2000) as:
DO2 = VC* Fc ox * Hct
= Qa * tc * Fc ox* Hct
where VC is the capillary volume of the gas exchanger (in µl), Qa is blood flow
through it (in ml · min-1), Fc ox is the mean corpuscular oxygenation velocity during
contact time (in sec –1 · torr-1), tc is the contact time of erythrocytes with O2 when they
pass through the CAM capillaries (in sec), and Hct is the hematocrit. The eggshell
and embryonic oxygen demand determine the oxygen level in the air cell and the air
cell determines the oxygen level in the blood.
Once oxygen comes in contact with the circulating blood, analysis of oxygen
transport is complicated by perfusion. Perfusive conductance is affected by the flow
of blood (Q) and the slope of the dissociation curve of the gas (ßb). Piiper and
Scheid (1992) also reported that the various conductive limitations of oxygen and
carbon dioxide create a gradient from predominantly perfusion-limited to a range of
perfusion and diffusion limitations. Animals may make physiological adjustments in
perfusion during respiration to deal with diffusion limitations. For example, as a gas
diffuses down a partial pressure gradient across a barrier, they may reduce flow so
that contact time increases between embryonic blood and the diffusive barrier,
leading to better oxygenation of the blood. In contrast, if gas diffuse down a steep
partial pressure gradient of into the blood it is possible that an animal may increase
4
blood flow by increasing blood pressure or heart rate so that the oxygenated blood
will reach metabolizing tissues more quickly. For each situation, physiological
adjustments must be made to find the right combination of perfusion and diffusion
along this gradient. There is often a seamless change from one combination to
another as physiological conditions change.
To what extent is respiration perfusion-limited or diffusion-limited in the
chicken embryo? Consider the general pathway of oxygen in respiration, best
described by the “cascade model” (Weibel, 1984). As oxygen is transported from the
air or water through convection to the blood there is a fall in PO2. Then, through
diffusion, oxygen diffuses into cells to the oxygen sink, the mitochondrion of
metabolically active tissue, causing another drop in PO2. The oxygen cascade in
chicken embryos involves a drop in PO2 through the shell, CAM, arteries and veins,
and finally the tissues (Fig 1.1). Metcalfe (1969/70) suggests that since diffusion is a
large part of O2 transport and diffusion is driven by a Po2 gradient, any factor that
increases Po2 at any point in the O2 transport chain will serve to improve tissue
oxygenation. One way to increase Po2 is artificial erythrocythemia.
Artificial Erythrocythemia and Oxygen Consumption
In embryonic avian gas exchange, much attention has been given to diffusion-
limited mechanisms (Wangensteen and Rahn, 1970/71; Wangensteen et al.,
1970/71; Erasmus and Rahn, 1976; Ar et al., 1980, 1991; Tazawa et al., 1981; Rahn
and Paganelli, 1982; Wagner-Amos and Seymour, 2002) but perfusion limited
mechanisms are in place as well. Within embryonic gas exchange, it is possible that
there is a mixture of these two types of limitations. Oxygen consumption is
determined by oxygen transport, which depends upon a combination of oxygen
content of the blood and cardiac output. Oxygen transport can then be manipulated
5
by changing either arterial oxygen content of the blood or cardiac output. One could
reason that if the amount of oxygen carried in the blood is increased then the amount
of oxygen that reaches the tissues is increased as well. In humans, Gledhill et al.
(1999) found that an increase in hemoglobin when blood volume is kept constant
leads to an increase in metabolic rate. This could be due to the increase in the
oxygen capacity of the blood. Experiments with artificial manipulation of hematocrit
are informative in this regard. In recent years, endurance athletes have begun the
illegal practice of induced erythrocythemia or “blood doping”. This involves the
removal of an amount of their own blood, which is stored. The body replaces what
was lost over a period of weeks, and the stored blood is then re-infused causing a
dramatic rise in hematocrit and presumably blood oxygen transport. The result is an
increase in aerobic power in the athletes (Eichner et al., 1996).
Elevated hematocrits can occur during human development, as well.
Exposure of the fetus to hypoxia leads to an elevated hematocrit in the fetus
(Edwards et al., 1968). For example, in diabetic mothers high maternal and fetal
glucose levels promote excess nutrient storage, while fetal oxygen levels are
depleted. Periods of fetal hypoxia are accompanied by surges in adrenal
chatecholamine levels among other changes such as increased hematocrit. High
hematocrit values in the neonate can lead to vascular sludging, poor circulation, and
postnatal increased levels of bilirubin (Linderkamp, 1996). A second example of high
hematocrit in human development can be found in the fetuses of pregnant mothers
that smoke cigarettes, which exposes the fetus to low levels of carbon monoxide.
Bureau et al. (1983) found a significant positive correlation between the number of
cigarettes smoked per day by pregnant mothers and the levels of hemoglobin and
hematocrit in the fetus. A final example is pregnancy at high altitudes. Khalid et al.
6
(1997) reported that fetal hematocrit and hemoglobin were significantly higher at high
altitude while birth weights were lower. The authors attribute these results to
placental hypoxia experienced at high altitudes.
Erythrocythemia also occurs in chicken embryos exposed to high altitude.
Embryos from low altitude chickens moved and acclimated to high altitude respond
with an increase in hematocrit and a decrease in metabolic rate (Wangensteen et al.
1974). Tazawa et al. (1992) tested metabolic responses in chicken embryos at
various ambient oxygen levels. Late stage embryos in hyperoxic environments
increased their metabolic rates and embryos in 10% O2 environment reduced their
metabolic rates. This suggests that as long as there is not a significant diffusion
limitation (i.e. the amount of oxygen needed is less than the amount that can diffuse
across the shell), an increase in the availability of oxygen leads to an increase in
tissue oxygen consumption. That hematocrit increases in the face of hypoxia during
development leads to the question of whether there is a direct effect of high
hematocrit on oxygen usage in developing animals.
Objectives and Hypothesis
The hypothesis of this thesis is that an increase in hematocrit through artificial
erythrocythemia during late (D15-17) incubation chicken embryos will directly
produce an increase in oxygen consumption by increasing blood oxygen capacity of
the blood by making more oxygen available for consumption. To test this hypothesis,
the ability of Day 15 embryos to regulate hematocrit was examined through graded
saline injections or blood loss. Also, the oxygen consumption of chicken embryos
with artificially high hematocrits was measured for an extended period of time after
treatment to examine the rate of oxygen consumption.
7
P O2 (
mm
Hg)
0
20
40
60
80
100
120
140
160
Nest Shell TissuesShell Membranes Chorioallantois
Outer Inner Vein Artery
Figure 1.1 Oxygen pressure gradients from nest to tissue in domestic fowl embryos (after Wangensteen and Rahn, 1970/71).
8
CHAPTER 2
MATERIAL AND METHODS
Source and Incubation of Eggs
Fertilized White Leghorn (Gallus domesticus) eggs were shipped from Texas
A&M University (College Station, Texas) to the University of North Texas (Denton,
TX), where they were incubated in commercial incubators at a temperature of 38°C
and a relative humidity of 60%. Eggs were turned automatically every 4 hours.
CAM Venous Catheterization
A vein in the chorioallantoic membrane (CAM) was catheterized in each
embryo for the withdrawal and injection of blood or saline solution, adapted from the
method of Tazawa et al (1980). The egg was candled to find the largest CAM vein in
the blunt end of the egg. A piece of eggshell (~ 4mm in diameter) above the selected
vein was removed and the egg was quickly half buried in a sand bath set at 39°C to
maintain egg temperature during surgery. The inner membrane was carefully
removed to reveal the underlying vein, which was isolated and the direction of blood
flow in it determined. The IV catheter consisted of a 30 gauge needle, bent at 90°
approximately 2mm from the tip, glued into 100mm of PE 10 tubing glued into
100mm of PE 50 tubing (Figure 2.1). A small piece of modeling clay was placed on
the shell to the side of the hole to hold the needle after insertion. The catheter was
secured to a Hamilton syringe and filled with approximately 30µl heparinized saline.
The vein was then lifted with forceps to occlude blood flow and the needle was
inserted into the direction of flow. The vein was gently placed back down and a small
amount of super glue gel (3M) was placed on the point of insertion to stop
hemorrhaging and help secure the needle in the vessel. The end of the needle was
then secured in the modeling clay. The catheter was checked for blood flow to insure
9
successful insertion of the needle. After the glue had dried on the point of insertion,
the hole was covered with a small piece of clay and again checked for blood flow in
the catheter.
Hematocrit Determination
Hematocrit, the ratio of the volume of red blood cells to total blood sample
volume, was determined on a 50µL volume of blood drawn with a Hamilton syringe
through the implanted IV catheter. The blood was drawn into a capillary tube, which
was then sealed and centrifuged for five minutes in a mircocentrifuge (ACCU-STAT
MP Readacrit) before determining hematocrit.
Hematocrit Manipulation
1) Hematocrit Reduction
Eggs were catheterized as described previously and maintained in an
incubator maintained at 38°C during the procedure. Two methods- saline injection
and blood withdrawal- were used to attempt to alter hematocrit acutely in Day 15
embryos. In an attempt to induce reduced hematocrit by hypervolemia, every 30
minutes embryos were given 150 µL of heparinized saline using a Hamilton syringe.
Approximately 10 minutes after each injection, 25 µL of blood was drawn through the
implanted catheter for hematocrit determination, resulting in an acute net blood
volume increase of 125 µL.
To reduce hematocrit through blood removal, 125 µL of blood was removed
through the implanted catheter using a Hamilton syringe every 30 minutes. This
blood was then used for hematocrit determination. Romanoff (1967) reported a total
blood volume of 2,250 µL for Day 15 embryos. Therefore, an increase or decrease of
125 µL acutely results in a 5% change of volume.
10
2) Artificial Erythrocythemia
Artificial erythrocythemia is a form of "blood doping" that increases hematocrit
by adding red cells to the circulating blood of an embryo. The blood to be injected
was combined from 2-3 donor chicken embryos at Day 15. A preliminary experiment
showed that re-injection of the combined blood is well tolerated in chicken embryos,
with no obvious agglutination or similar reactions. Approximately 1.5 mL of blood
was collected taken through chorioallantoic vein puncture from donor embryos. This
donor blood was centrifuged to separate red blood cells from plasma. Approximately
700 µL of plasma was removed from the pooled sample and the red blood cells were
re-suspended in a vortex mixer for 20 seconds in the remaining plasma. This
procedure yielded a high hematocrit sample (approximately 50-65% hematocrit).
After re-suspending the sample, it was visually observed for a color change in the
blood induced by aeration during re-suspension to ensure the blood could still be re-
oxygenated. Then 400 µL of the high hematocrit blood sample was injected through
the surgically implanted IV catheter into the recipient egg. After multiple samplings of
blood through the procedure and the injection of high hematocrit blood, each embryo
had a total blood volume acutely elevated by 300 µL, which is an increase of
approximately 12-14% for embryos of these stages of development.
Oxygen Consumption Measurements
Oxygen consumption was measured by placing an egg in a watertight, airtight
respirometer (volume = 296mL). The temperature of the respirometer was
maintained at 38°C by submerging it in a water bath (Fisher ISOTEMP 1028P).
Each respirometer containing an egg was submerged in the water bath for a
minimum of 30 minutes for thermal equilibration before measurements were taken.
Warmed (38°C) air flowed through a port into the bottom of the chamber and out a
11
port at the top at a preset rate of 70 mL /min, ensuring continual replenishment of the
gas in the respirometer. Gas leaving the chamber then passed through Drierite (to
remove water) and soda lime (to remove carbon dioxide) before reaching the multi-
channel oxygen sampler (Model# FC-1B Sable Systems Inc.) (Figure 2.1 B). Three
respirometers were run at the same time with repeated measurements taken on
each egg. The O2 differential between in flowing and out flowing gas was ~ 0.4-0.6%.
VO2 was calculated by Sable System Data Analysis software. All oxygen
consumption measurements reported are mass specific (corrected for egg mass).
Experimental Protocol for Determining Oxygen Consumption
An egg was retrieved from the incubator and placed in a ventilated metabolic
chamber for 30 minutes and a baseline (pre-catheterization) oxygen consumption
determination then made (Figure 2.2). The egg was then removed and an IV
catheter was surgically implanted as described previously. The embryo was allowed
to recover in an incubator for one hour, before being returned to the metabolic
chamber for the remainder of the procedure. After recovery, the egg remained in the
chamber for another 30 minutes before another oxygen consumption measurements
was taken and hematocrit determined. The egg was then “blood doped” as,
described above and hematocrit was determined again to ensure hematocrit had
increased in response to the treatment. After blood doping, oxygen consumption was
determined every 30 minutes over a course of six hours to look for any changes in
oxygen consumption. Then a final hematocrit determination was made.
Statistical Analysis
All oxygen consumption data and hematocrits for each stage were tested for
normality and equality of variances. Hematocrit data for blood volume change was
non-parametric, resulting in the use of a Kruskal–Wallis One Way Analysis of
12
Variance (ANOVA) on Ranks to determine statistical significance. Significance
between different groups was tested for using Dunn’s Method.
A One Way ANOVA was utilized to determine significance between normal
hematocrits at each stage, followed by a Holm-Sidak pairwise multiple comparison
test. Hematocrits determined during the experimental procedure described above
were tested for significance with either a paired t-test or a Mann- Whitney ranked
Sums Test, depending on normality.
Oxygen consumption data was analyzed using a Kruskal-Wallis ANOVA on
Ranks to determine statistical differences between each treatment group, followed
by a Two Way Repeated Measures ANOVA to determine the effects of treatment
and stage.
SigmaStat Version 3.0 (SPSS, Inc.) was used to conduct all statistical
analyses. All statistical decisions were made using a 0.05 level of significance. All
averages are represented as mean ± S.E.M.
13
A
PE 10 Tubing 30G needle with bent tip
PE 50 Tubing
To syringe containing heparinized saline
Flow In
296 mL clear, airtight, metabolic chamber
Catheter Flow out to gas analyzer B
Egg
Figure 2.1 Apparatus used oxygen consumption determination. A) Diagram of catheter used for blood/ saline injections. B) Diagram of respirometer.
14
Injection of red cells
½ hour ½ hour 1 hour 6 hours1 hour
Recovery
½ hour
CatheterizationOxygen Consumption Oxygen
Consumption Oxygen Consumption (repeated every 30 minutes)
Figure 2.2 Timeline of experimental procedure. Thick arrows indicate withdrawal of blood sample for hematocrit
determination.
15
CHAPTER 3
RESULTS
Hematocrit and Developmental Stage
Mean values and variation of control hematocrit values are shown in Figure 3.1.
Hematocrit was significantly affected by developmental stage (One Way ANOVA,
p<0.01). Hematocrit did not increase significantly from Day 15 to Day 16 or Day 16 to
Day 17, but did increase significantly from Day 15 to Day 17 (One Way ANOVA, P=
0.023). Embryos at Day 15 of incubation (n=33) had an average hematocrit of 25±0.8%
(with a range of 15-35%). Average hematocrit in Day 16 embryos was 26±1.27%
(n=14), with several individuals showing hematocrits in the range of 30-32%. Day 17
embryos (n=15) had an average hematocrit of 29 ± 1.06%.
Blood Volume Change and Hematocrit
1) Effect of graded blood removal.
The protocol for blood removal caused an acute blood loss of 5% of the initial
volume every 30 minutes in Day 15 chicken embryos (Figure 3.2 A & B).
Accompanying this blood loss was a significant and progressive decrease in hematocrit
(Kruskal Wallis p<0.001; n=10) which was significant after a blood volume loss of
>40%(Dunn’s Method, p<0. 05).
2) Effect of graded saline addition.
A second group of embryos (n=5), were given 150 µL saline injection equivalent
to a 5% increase of blood volume every 30 minutes (Figure 3.2 A & B). Despite
repeated injections of saline up to 115% of initial blood volume over a 6-12 hour period,
16
there was no significant change in hematocrit from baseline measurements (Kruskal
Wallis, P>0.1).
Artificial Erythrocythemia
Embryos at all developmental stages exhibited a significant increase in
hematocrit of 10-15% immediately following injection of blood with an artificially high
(30-45%) hematocrit (Kruskal–Wallis One Way ANOVA on Ranks, p< 0.001). This
artificially induced erythrocythemia persisted for six hours following injection (Figure
3.4). In Day 15 and 17 embryos, there was no significant change in hematocrit during
the six hour post-injection period (One-Way Repeated Measures ANOVA). In Day 16
embryos, however, there was a significant but small decrease in hematocrit back
towards control values (One-Way Repeated Measures ANOVA, p=0.032). Hematocrit
measurement did not differ significantly between stages. (One way ANOVA, p= 0.01 )
VO2 following Artificial Erythrocythemia
Embryos in Day 15 and 17 of incubation (n= 13, 12) showed no significant
difference in VO2 after catheterization of the chorioallantoic vein (Paired t-test p= 0.109,
0.855,). In embryos at Day 16, however, VO2 decreased significantly (Paired t-test, p=
0.011 Figure 3.5).
In all developmental stages, artificial erythrocythemia did not cause a significant
difference in VO2 at any point over a period of six hours (Kruskal-Wallis, p≥ 0.985, 0.328,
0.946) nor any statistically significant interaction between stage and treatment. (Two
Way ANOVA on Ranks, Figure 3.6B).
17
Day 17
Hematocrit15-17%
18-20%21-23%
24-26%27-29%
30-32%33-35%
0
5
10
15
20
25
30
Day 16
15-17%18-20%
21-23%24-26%
27-29%30-32%
33-35%0
10
20
30
40
Day 15
15-17%18-20%
21-23%24-26%
27-29%30-32%
33-35%0
5
10
15
20
25
30
% o
f Em
bryo
s
n=33 n=14 n=1525 %
26 ±1.27%
HematocritHematocrit
27%
Figure 3.1 Frequency distribution of hematocrits at various stages. Dashed line represents median
hematocrit for each stage. Embryos at Day 15 and 16 and embryos at Day 16 and 17 are not significantly different. Day 17 embryos have an average hematocrit significantly higher than animals at Day 15 (Holm-Sidak Method, P=.006).
18
% of Initial Blood Volume Removed
-100-80-60-40-200
Hem
atoc
rit (%
)
5
10
15
20
25
30
35
A
% o f In itia l B lood V o lum e R em oved
-100-80-60-40-200
Hea
mto
crit
(%)
5
10
15
20
25
30
35 B
Figure 3.2 Effect of graded blood removal on hematocrit in Day 15 embryos (n=10). A) Individual responses of 10 embryos showing considerable variation in the ability to maintain hematocrit. B) Mean responses +/- 1 S.E.M. Hematocrit did not fall significantly from baseline until 40% of the blood volume had been removed (Kruskal Wallis p<0.001). Shaded area indicates control values. Open symbols represent a mean value significantly different from baseline.
19
% of In itia l B lood Volum e Added
0 20 40 60 80 100 120 1
Hem
atoc
rit (%
)
405
10
15
20
25
30
35
A
% of Blood Volume Added
0 20 40 60 80 100 120 140
Hem
atoc
rit (%
)
5
10
15
20
25
30
35
B
Figure 3.3 Effect of graded saline injection on hematocrit in Day 15 embryos (n=5). A) Individual responses of 5 embryos. B) Mean responses +/- 1 S.E.M. Hematocrit did not change significantly from baseline measurements although blood volume was increased by up to 115% (Kruskal Wallis p=.998). Shaded box indicates control values.
20
Time (hours)
Hem
atoc
rit (%
)
20
25
30
35
40
45
Day 15Day 16Day 17
AfterInjection
*
**
65321 40 7
***
Figure 3.4 Effect of artificial erythrocythemia on hematocrit in chicken embryos. The measurement at 0 minutes is taken just before 400 µL injection of high hematocrit blood (See methods). Forty minutes later another hematocrit reading is taken. Measurements at 40 minutes and six hours were all significantly higher than the control measurements before injection (One-Way Repeated Measures ANOVA, p< 0.001). Six hours later the hematocrit remained elevated in embryos at Day 15 and 17. The embryos in Day 16 of incubation showed a significant decrease from initial post-injection six hours after injection. (One-Way Repeated Measures ANOVA, p=0.032). Asterisk (*) represents a significant difference from control measurements. Shaded area indicates control values. A box surrounds data points that are not significantly different from each other.
21
Pre- Catherization Post-Catherization
VO
2 (ml O
2 2 •
min
-1• g
egg
-1)
0 .0
0.1
0.2
0.3
0.4
0.5
*
Day 17
Day 15Day 16
Figure 3.5 Effect of catheterization on oxygen consumption. Day 16 (n=12) embryos showed a small but significant decrease in metabolic rate after catheterization; embryos at Day 15 (n=13) and Day 17 (n=12) did not (Paired t-test, p= .011). Asterisk (*) represents significance.
22
H
emat
ocrit
(%)
2 0
2 5
3 0
3 5
4 0
4 5 A
Tim e (m in post-treatm ent)
0 m in 40 80 120 160 200 240 280 320 360 400
V O2 (m
l O2 •
min
-1• g
egg
-1)
0 .20
0.25
0.30
0.35
0.40
0.45
0.50
Day 15Day 16Day 17
Figure 3.6 Effects of artificial erythrocythemia on oxygen consumption. A) Average hematocrits during VO2 measurements (see Fig. 3.4). B) VO2 within each stage, none of the measurements taken differed significantly from the baseline measurement (0 minutes).
23
CHAPTER 4
DISCUSSION
Hematocrit and Developmental Stage
The hematocrit values of control chicken embryos at Day 15-17 reported in this
study (approximately 25-29%) are within the range of those reported by others (Table
4.1). Hematocrit increases progressively with development in chicken embryos (see
Romanoff, 1967). That hematocrit increased significantly from day 15 to 17, but not
from day 15 to day 16, though, suggests that hematocrit may not increase linearly with
day of incubation.
Hematocrit and Blood Volume Regulation
Regulation of hematocrit is an important component in maintaining blood oxygen
homeostasis. Hematocrit regulation requires a balance between fluid flux from blood to
tissues as well as red blood sequestration and release. Mature animals have several
methods for manipulating these factors. Many animals are able to sequester red blood
cells and then release them when oxygen transport to the tissues is required. An
extreme example is the Weddell seal, which sequesters red blood cells (20.1L) in its
spleen during long dives. When the seal is diving and needing to maintain oxygen
transport to active tissues, sequestered red blood cells are released increasing
hematocrit and tissue oxygenation (Hunford et al., 1996). This red blood cell release
could be triggered experimentally by epinephrine injection. This sudden increase in
hematocrit is likely to lead to a large increase in blood volume. It is not known how the
Weddell seal deals with the hemodynamic implications, though presumably there will be
some filtration of plasma to reduce circulating blood volume. In Salmonid fishes,
24
hematocrit is quickly elevated in response to maximal exercise, likely due to
catecholamine-mediated splenic contractions that release stored erythrocytes
(Yamamot et al.1980, Milligan and Wood, 1986, Yamamoto, 1987, Jensen, 1987).
Chickens that fasted for 48 hours released sequestered red blood cells under
alpha or beta-adrenergic control (Hughes et al., 1984). However, the possibility that red
blood cells can be sequestered for hematocrit regulation has been only poorly studied in
birds, and the ontogeny of any such regulation has not been explored. Day 15 chicken
embryos subjected to graded blood removal in the present study showed no ability to
maintain hematocrit, which fell progressively with each blood withdrawal (Fig.3.1).
Apparently Day 15 chicken embryos are unable to release sequestered red blood cells,
at least in sufficient numbers to offset red blood cell loss. This response may not
develop until after hatching.
Adjustments in plasma volume also play a role in hematocrit regulation. Snakes,
for example, are able to deal with a change in blood volume by changing capillary
permeability. When subjected to graded hemorrhaging (a decrease of 4% every thirty
minutes until a cumulative deficit of 32% is reached), snakes are able to maintain or
quickly restore blood volume (Smits and Lillywhite, 1985). Increasing capillary
permeability would allow extra plasma volume to be filtered into extravascular spaces.
Similarly, if volume were to decrease dramatically, an increase in capillary permeability
would allow for quick movement of fluid from interstital spaces into circulation. This
would allow the animal to maintain blood volume and therefore blood pressure in the
event of significant volume fluctuation. In amphibians subjected to graded hemorrhaging
(a decrease of 1% of body mass every thirty minutes), Hillman and Withers (1988)
25
found that maximal blood flow rates were sustained until a blood loss of up to 5% of
body mass. These animals were found to compensate for blood volume loss with lymph.
For chicken embryos given repeated injections of saline, hematocrit did not
change significantly despite dramatic increases in blood volume. It is likely that either
capillary permeability or blood pressure (or both) increases, causing injected saline to
be filtered out of the circulation as quickly as it is injected. If this is true, it could mean
that blood volume did not actually increase. Whether this is an active reflex or a passive
response requires further investigation. Crossley and Altimiras (2000) found baroreflex
function in chicken embryos progressively matures beginning around Day 18 of
incubation, so the beginnings of physiological control mechanisms for blood volume
could be in place in Day 15-17 embryos. It would be interesting to determine the blood
pressure and a direct measure of blood volume through the course of graded blood
removal or repeated saline injections in late incubation chicken embryos.
Artificial Erythrocythemia and Oxygen Consumption
Increases in environmental oxygen, hemoglobin concentration, or the number of
red blood cells available can increase blood oxygen transport to the tissues. Adjusting
hematocrit is potentially a way of manipulating systemic oxygen transport, because
vertebrates generally show a linear relationship between oxygen capacity in blood and
hematocrit (Tazawa, 1971, Hillman et al. 1985). Hillman et al. (1985) induced graded
erythrocythemia (31-57%) in toads and frogs with an isovolemic loading of packed red
blood cells. These amphibians exhibited oxygen consumption proportional to the
maximal systemic oxygen transport capacity.
26
Day 15, 16, and 17 chicken embryos in this study showed no increase in oxygen
consumption despite a 10-15% artificially induced increase in hematocrit and
concomitant similar level of increase in oxygen capacity. That the increase in hematocrit
and oxygen capacity did not perturb the embryos shows a metabolic tolerance for
hematocrit fluctuation. This may be an important attribute of late embryonic physiology,
given the considerable range in hematocrit evident in at least Day 15 embryos (Fig 3.1).
It is also possible that the acute increase in blood plasma volume from artificial
erythrocythemia caused plasma filtration moving extra fluid to extravascular spaces. It
was hypothesized in this study that an increase in oxygen capacity in chicken embryos
would increase VO2, especially in late incubation when developmental hypoxia is
maximal. In rejecting this hypothesis, several alternatives arise.
Although the increase in blood volume from artificial erythrocythemia is not
certain, it is likely that blood viscosity increased, as blood viscosity has an exponential
relationship with hematocrit in the avian circulation (Berne and Levy, 1992). Also, blood
flow is inversely related to blood viscosity. The consequence of increased viscosity and
associated decreased flow is that diffusive capacity is decreased (Piiper and Scheid,
1992). That oxygen consumption did not change with elevated hematocrit could
demonstrate that respiration in chicken embryos at these stages is primarily
diffusion\limited as opposed to perfusion-limited. On the other hand, perhaps these
results could mean that despite the increase in oxygen capacity, the decrease in
diffusive capacity from a possible decreased flow due to increased viscosity prevents an
increase in metabolic rate. If normal hematocrit in embryos at these stages were at
27
optimal hematocrit (Hillman et al., 1985), then an increase in volume or hematocrit
would work antagonistically toward oxygen transport.
Returning to the suggestion of Metcalfe et al. (1969/1970) that any increase in
the PO2 gradient anywhere along oxygen transport would improve tissue oxygenation,
the data in this study would argue the contrary. To view oxygen transport as only mainly
diffusion-limited overlooks the many perfusion-related factors. Other aspects of
respiration could be affected as well. The interplay of perfusion and diffusion in
respiration is complex. Even when there is a gross inhomogeneity in the
diffusion:perfusion ratio, embryos are still able to maintain metabolic rate (Wagner-
Amos and Seymour, 2002). The delicate and fickle balance between the two is a subtle
tool in respiration for developing chickens.
Future Directions
Day 15 chicken embryos were able to protect hematocrit from large injections of
volume over several hours. Missing from this study was a measurement of blood
volume during experimental volume change. To examine this, a tracer dilution method
similar to Smits et al. (1985) could be utilized. Though day 15 chicken embryos were not
able to maintain hematocrit during blood removal, this does not remove the possibility of
the development of a mechanism like splenic red blood cell sequestration. The
exploration of such a mechanism could prove interesting. The chatecholamine-mediated
release of stored erythrocytes could correspond to other chicken embryo hormonal
cardiovascular regulations. Also, the spleen could be weighed to determine a change in
size before and after volume change. The mechanisms for regulating hematocrit
28
remain unexplored in the developing chicken. What are the mechanisms they employ in
regulating their hematocrit and how do they develop?
Despite a sustained increase in hematocrit, embryos did not or could not utilize
the increase in oxygen capacity of blood from artificial erythrocythemia. To better
understand these results, simultaneous blood pressure, viscosity, heart rate, and
oxygen affinity measurements during erythrocythemia would help to elucidate the
degree to which these certain factors are involved during respiration in response to a
high hematocrit. An understanding of the degree of involvement of these factors would
tease apart the effects of diffusion and perfusion. Also, it would be helpful to see a wider
range of stages. An interesting avenue to explore would be to examine, in vitro, different
concentrations of avian blood exposed to various levels of oxygen. This experiment
could determine the extent to which the oxygen consumption of the blood interferes with
oxygenation of the blood.
29
Day 15 Day 16 Day 17 Romanoff, 1967 26.49 28.5 28.53 Tazawa et al., 1971 29.6 33 34.7 Tazawa, 1972 28.8 34.9 36.8 Tazawa, 1976 28.8 32.2 34.6 Tazawa, 1980 27.3 29.7 30.9 Dzialowski et al., 2002 26.3 28.5 31.7 Current Study 25.3 26.8 29.2 Table 4.1 Previous reported values of hematocrit for the days of incubation used in this study.
30
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