Introduction

The objective of this experiment is to elucidate the mechanism of regulation of

ventilation in the body by observing the respiratory response to increasing levels of
inspired CO2 on the white leghorn chicken (Gallus Domesticus). The respiratory
parameters being measured are tidal volumes, respiratory rate, minute ventilation, and
airway resistance. In addition dinitrophenol (DNP)- a mitochondrial uncoupler is also
used to study the effects of hyper-metabolism on respiratory response. Thus heart rate and
temperature is also being measured as an indicator of an increase in metabolism.
The chicken was used in this study because its airway resistance is separate from
compliance making it easier to study. Also, unique CO2 chemoreceptors in the lung allow
the chicken to hyperventilate under low CO2 conditions and are similar to a feed forward
control system which allows for a more predictable and easier to control response (AVS).
The mechanics of the avian respiratory system starts with the trachea and then a group of
air sacs the caudal group which consists of the caudal thoracic (2) and abdominal (2) air
sacs and the cranial group, consisting of the cranial thoracic (2), clavicular (1), and
cervical (2) air sacs which in total are 9 air sacs. These air sacs do not facilitate gas
exchange directly but act as bellows to draw air into the bird, which is stored until
expiration (cite). As a result, the air sacs allow a continuous stream of air to flow to the
lungs in a unidirectional system. During inspiration air travels to the caudal portion of the
lung where it is divided into the caudal air sacs and the mediodorsales. From there, air
heading into the mediodorsales travel into the parabronchi, which is the source of gas
exchange, and then into the medioventrales, culminating in the cranial air sac (cite).
Upon expiration contraction of the muscles decrease body volume, forcing the exchanged
air in the cranial portion out through the medioventrales, the parabronchi, and the trachea

while air from the caudal air sac travels up the mediodorsales and parabronchi and ending
in the cranial air sacs, completing one respiratory cycle.
Three types of chemoreceptors in birds undertake regulation of the respiratory
cycle. They are the intrapulmonary chemoreceptors (IPC), arterial chemoreceptors, and
the central chemoreceptors. IPCs, which are located in the lungs function similar to
pulmonary stretch receptors in mammals in that they regulate breath size and arterial
PCO2 levels. IPCs regulate ventilation by decreasing breath size as their activity
becomes more frequent (Sturkies). IPC activity is inversely proportional to PCO2 levels,
as PCO2 levels increase, the firing rate of IPCs decrease (Sturkies). Arterial
chemoreceptors are located in the carotid/ thoracic bodies and are innervated by the
vagus nerve. They are sensitive to changes in pH, CO2, and O2 levels and are stimulated
in birds during hypoxia, showing increased action potential frequency as the level of O2
decreases causing an increase in ventilation (Sturkies). The final type of chemoreceptor,
central chemoreceptors lie in respiratory control centers in the brain stem. Central
chemoreceptors, which respond to hypercapnic hypoxia, increase action potential
frequency, causing the depth and rate of respiration to increase (cite).
Consequently, we can postulate that the effect of increasing CO2 inspiration in the
avian lung causes a concerted effort from all three chemoreceptors to increase ventilatory
efforts in a counter balance fashion in order to bring CO2 levels back to the physiological
standard. IPC activity decreases as CO2 levels increase which allows a deeper breath size,
while arterial and chemoreceptor activity increases, causing respiration in the bird to
become deeper and more frequent. Also in the addition of DNP, which causes protons in
the mitochondria to leak, thus uncoupling electron transport and phosphorylation
reactions makes ATP synthesis less efficient (cite). As a result hyper metabolism occurs

to compensate for the inefficiency, which causes local accumulation of CO2 in the tissue,
strengthening the effect of this chemoreceptor regulation causing a further increase in
respired volume.
Methods
In this study white leghorn chickens (Gallus Domesticus) were anesthetized using
a combination of sodium pentobarbital, diazepam, and ketamine. Initial anesthesia was
administered through the medial petal vein in order to prepare the chicken for surgery
(lab manual). Once an appropriate plane of anesthesia was determined, the chicken was
cannulated in the specific order of the ulnar vein, trachea, carotid artery, and the caudal
thoracic airsac (lab manual). For a more in depth look at the procedure look at the NPB
111L lab manual pages 9-21 for an in depth description on the procedures. The ulnar vein
was cannulated in order to introduce anesthesia during the experiment using a dual
stopcock with a large syringe filled with heparinized saline and a smaller syringe filled
with anesthetic which was injected via PE90 tubing (lab). Tracheal cannulation was done
in order to introduce artificial ventilation from the MKS. Carotid arteries were cannulated
in order to observe blood pressure from a blood pressure transducer. The air sac was
cannulated in order to measure oscillatory pressure.
Three direct measurements were made: core body temperature, pressure between
air in the caudal thoracic air sac and the air outside of the trachea, and gas flow during
respiration. The plethysmographic transducer was used to measure the pressure of the
caudal thoracic air sac and the trachea. The pneumotachometer and the statham pressure
transducer were used to measure the flow of gas as the bird respired.

Results

Temperatur
e
Tidal
Volume
Expiratory
Tidal
Volume
Inspiratory
Respiratory
Rate
Minute
Ventilation
Heart Rate
Mean
Arterial
Pressure
Systolic
Diastolic
Pulse
Pressure

Temperatur
e
Tidal
Volume
Expiratory
Tidal
Volume
Inspiratory
Respiratory
Rate
Minute
Ventilation
Heart Rate

Table 1-Pre-DNP injection values

Units
Control 1% CO2 2% CO2
Begin
C
34.12
33.98
33.98

4% CO2
34.2

Control
End
34

mL

19.5

16.2

18.5

28.4

18.1

mL

18.7

16.7

21.1

25.7

16.8

BPM
(Breaths
)
mL/min

11.6

16.1

8.5

15.5

14.34

310

311

417

449

259

BPM
(Beats)
mmHg

155

136

174

165

172

62

59

54

62

64

mmHg
mmHg
mmHg

103
42
61

106
35
71

106
28
78

107
40
67

101
45
56

4% CO2
34.57

Control
End
34.6

Table 2-Post-DNP injection values

Units
Control 1% CO2 2% CO2
Begin
C
34.12
34.17
34.4
mL

18.9

22

25.6

28

21.6

mL

17

20

21

27.3

18.4

BPM
(Breaths
)
mL/min

22.3

26

28

28.9

27

425

626

758

970

755

BPM
(Beats)

224

250

254

263

208

Mean
mmHg
68
64
69
119
77
Arterial
Pressure
Systolic
mmHg
109
113
116
136
118
Diastolic
mmHg
47
40
46
110
57
Pulse
mmHg
62
73
70
26
61
Pressure
Data was gathered and analyzed using Biopac BSL analysis 4.0. For all of the
tabulated values the I-beam tool was used to highlight sections of the graph of the raw
data. Temperature was calculated using the mean function in Biopac and sampling
approximately 10 seconds of data. Inspiratory tidal volume was calculated from the
tracheal airflow raw data source channel using the negative integration function and
highlighting the tracheal flow from peak to trough. Expiratory tidal volume was
calculated from the same data channel as the inspiratory tidal volume but used the
positive integration function. Respiratory rate was calculated from the airflow raw data
source channel and used the RR function where the window was set from 5-60. Minute
Ventilation was calculated by multiplying expiratory tidal volume and respiratory rate.

Figure A:Temperature Change as %CO2 increases

34.8
34.6
34.4
34.2
Pre DNP
Linear (Pre DNP)
Temperature
(Celsius)
34

Post DNP

Linear (Post DNP)

33.8
33.6
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
% CO2

For the
cardiovascular data all measurements used the blood pressure raw data source channel,
heart rate was measured using the BPM function. Systolic pressure was gathered by using
the max function on the peak portion of the blood pressure cycle and averaged over 10

cycles. Diastolic pressure was measured in the same way but used the min function on the
trough portion of the blood pressure. Mean arterial pressure (MAP) was calculated using
the formula [(2 x diastolic)+systolic]/3. Pulse pressure was calculated by subtracting the
systolic and diastolic pressures.

Figure B:Expiratory Tidal Volume as % CO2 increases

30
25
20
15
Pre volume
DNP
Linear (Pre DNP)
Tidal
(mL)
10

Post DNP

Linear (Post DNP)

5
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
% CO2

Figure
A shows the relationship between core temperature as % CO2 inspired by the chicken
increased. As % CO2 increased so did the core body temperature, which is modeled by
both trend lines showing a positive slope. Between the pre DNP and the Post DNP
injection trials, the average temperature for the post DNP trials had a higher core body

temperature than the pre DNP trials, which is also evident in the trend line for the post
DNP trial being shifted farther up than the pre DNP trials.

Figure B is a representation of the relationships between expiratory tidal volume

and % CO2 inspired. It was observed that as CO2 levels increased, so did expiratory tidal
volumes and is shown by the positive slope of the trend lines for the pre and post DNP
trials. Post DNP injection trials tended to have larger expiratory tidal volumes than pre
DNP trials also evident in the trend line for the post DNP that is shifted further up than
the pre DNP trials.
Figure C shows the effect of CO2 on inspiratory tidal volume. In figure C, the
trend line shows a positive slope both pre and post DNP injection as CO2 levels increase.
This is an indication that inspiration is greater as CO2 levels increase. Between pre DNP
and post DNP trials the slope of increase for post DNP is steeper which suggests that

Figure C:Inspiratory Tidal Volume as % CO2 increases

30
25
20
15
Pre DNP
Linear (Pre DNP)
Tidal
Volume (mL)
10

Post DNP

Linear (Post DNP)

5
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
% CO2

DNP plays a
hand in increasing inspiratory tidal volumes.

Figure D:Respiratory Rate as % CO2 increases

35
30
25
20
Pre
DNP
Linear
(Pre
DNP)
Breaths per minute (BPM) 15

Post DNP

Linear (Post DNP)

10
5
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
% CO2

In figure D
respiratory frequencies are plotted against increasing CO2 levels. Like the previous
figures the pre DNP and post DNP trend lines showed a positive slope, which meant that
respiratory rate also increased as CO2 levels increased. The same upward shift of the
trend line was also observed between the pre and post DNP injection trials in figure D. As
a result post DNP respiratory rate also higher than the pre DNP respiratory rate.

Figure E:Minute Ventilation as % CO2 increases

1200
1000
800
Pre DNP
Linear(mL/min)
(Pre DNP) 600Post DNP
Minute
Ventilation
400

Linear (Post DNP)

200
0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
% CO2

The changes in minute ventilation as CO2 levels increase is observed in figure E.

Again we see that the trend lines both pre and post DNP injection display a positive slope
indicating that minute ventilation tends to increase as higher CO2 levels are inspired. In
addition post DNP injection values are also higher than the pre DNP values, evidence that
DNP causes an increase in minute ventilation.
Discussion
Based on figures B-E a general trend that is established is that as inspired CO2
levels increase, tidal volumes, respiratory rate, and minute ventilation also increase.
These trends support the idea that IPCs are not solely responsible for regulating

ventilatory efforts; all 3 chemoreceptors work in synchrony to increase ventilation when

elevated levels of CO2 are detected in the body. Starting with inspiration, in figure C as
CO2 levels increase, inspiratory tidal volume also increases; this mechanism is the result
of CO2 levels inhibiting IPC action potential discharge, which in turn also inhibits the
restriction of inspiration (cite). In turn arterial and central chemoreceptors respond to the
rise in CO2 levels of the body by sending more action potentials down their afferent
nerves to control centers in the brain, which stimulate and cause deeper inspiration. A
similar study conducted by Robert Banzett and Ray Burger tested the response of avian
IPCs in response to increasing pulmonary arterial blood PCO2 and gas PCO2 levels in the
lung. The study concluded that avian IPCs were located in the lung either at the gas
exchange area (parabronchi) or upstream as increasing PCO2 levels in the blood and
airway of the lungs caused an increase in inspiratory tidal volume as IPC discharge
frequency decreased (Banzett). Thus, IPCs function to terminate inspiration, but
increases in CO2 inactivates IPC discharge and subsequent termination during inspiration
while arterial and central chemoreceptors are activated causing tidal volume to increase
(Banzett); supporting the idea that the chemoreceptors work together in a counter balance
fashion. In response to an increase in inspiratory tidal volume, expiratory tidal volume
should also increase due to the resulting increase in air intake, which is seen in figure B.
In figure D we see an increase in respiratory rate; the result of the activation of
arterial and central chemoreceptors in the chicken as a response to lower O2 levels due to
a higher ratio of inhaled CO2. Minute ventilation (Vm), which is calculated from
respiratory rate and expiratory tidal volume, should also increase as inspired PCO2
increases in a concerted, counter balance system, which is observed in figure E. We can
conclude by examining the relationship between these 4 parameters that ventilatory effort

is guided by the interaction of all 3 sets of chemoreceptors in the bird. This idea is
supported in a similar study done by (M.R Fedde et al). In this study ventilatory
sensitivity in response to the inspiration of low CO2 partial pressures were used to test the
idea that IPCs are not the sole chemoreceptors used in response to counteract a rise in
arterial CO2 levels. As inspiration of CO2 partial pressure increased in the study tidal
volume, minute ventilation and arterial CO2 also increased while respiratory frequency
stayed the same. These results indicate that at low partial pressures of CO2 (where IPC
inhibition of tidal volume is the greatest) an observed increase in Vm and tidal volume
indicates that arterial and central chemoreceptors are responsible for the increase in
ventilatory efforts (M.R Fedde).
Effects of DNP
In figures A-E post injection trials of DNP show higher overall body temperature,
tidal volume, respiratory rate, and minute ventilation as inspired CO2 increases. This is
due to the physiological mechanism of DNP as an uncoupler allowing H+ to leak and
makes energy production inefficient. As a result the cell enters hyper metabolism in order
to bring energy production back to normal. A consequence of hyper metabolism is the
accumulation of CO2 and H+ in the blood and tissues causing an elevation of blood CO2
levels that affects ventilatory effort by increasing the signal frequencies of central and
arterial chemoreceptors leading to a stronger response. A study done by (Gleeson)
demonstrated this effect. In this study, respiratory adjustments of DNP were measured,
specifically tidal volume (TV), oxygen consumption (VO2), Vm and blood CO2 (PaCO2)
levels. An Injection of DNP was associated with an increase in VO2, and Vm from the
control however PaCO2 and TV initially increased then returned to control values after 10
seconds (Gleeson). This study suggests that only the central and arterial chemoreceptors

are involved in response to increased metabolic demands as respiratory adjustments made

by these chemoreceptors restore the PaCO2 ratio which keeps IPC discharge constant, and
as a result tidal volume constant.
Conclusion
Ventilatory effort in the chicken is regulated as a concerted effort between
the 3 chemoreceptors. At lower levels of inspired CO2, IPCs play a major role in
ventilation due to its AP frequency being inversely proportional to CO2 levels. As CO2
inspiration increases, arterial and central chemoreceptor regulation takes over control of
ventilation as IPCs are rapidly inactivated by the increase in CO2. The DNP trials
demonstrated that during metabolic excitement, the shift in ventilatory effort is primarily
due to central and arterial chemoreceptors, while IPCs play little to no role.