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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