Electron Transport Chain Defect and Inefficient Respiration May Underlie
Pulmonary Hypertension Syndrome (Ascites)-Associated
Mitochondrial Dysfunction in Broilers
D. Cawthon, K. Beers, and W. G. Bottje1
Department of Poultry Science, University of Arkansas, Fayetteville, Arkansas 72701
RCR as well as the adenosine diphosphate (ADP) to O
ratio (an index of oxidative phosphorylation) between
control and PHS mitochondria were accentuated by sequential additions of ADP to isolated mitochondria. In a
second experiment, similar improvements in functional
indices following sequential additions of ADP and responses to respiratory chain inhibitors were observed in
liver mitochondria isolated from Single Comb White Leghorn (SCWL) males (resistant to PHS) similar to that observed in control broiler mitochondria in Experiment 1.
The combined results indicate the presence of a site-specific defect at either Complex II, ubiquinone, or both in
liver mitochondria obtained from broilers with PHS that
may be responsible for the oxidative stress and mitochondrial dysfunction observed in this costly metabolic
disease.
ABSTRACT By using a series of chemical inhibitors of
mitochondrial respiration, a site-specific defect in the electron transport chain was identified in mitochondria obtained from broilers with pulmonary hypertension syndrome (PHS; ascites). Located at the succinate:ubiquinone
oxido-reductase (Complex II:CoQ) interface, this defect
would allow electrons to leak from the respiratory chain
and consume oxygen by forming reactive oxygen species
at a greater rate than in control mitochondria. Lower
levels of the primary antioxidants, α- and β-tocopherol,
and glutathione (GSH) in PHS mitochondria confirmed
the presence of oxidative stress. Respiration studies of
PHS liver mitochondria also revealed disease-associated
decreases in the respiratory control ratio (RCR, an index
of electron transport chain coupling). Differences in the
(Key words: mitochondrial dysfunction, Complex II, broilers, glutathione, tocopherol)
2001 Poultry Science 80:474–484
stress (Enkvetchakul et al., 1993) and defects in liver
(Cawthon et al., 1999) and lung (Iqbal et al., 2001) mitochondrial oxygen consumption. As mitochondria are a
major contributor to oxidative stress (Chance et al., 1979)
and the primary oxygen consumer, defects in the electron
transport chain may be involved in the development of
the mitochondrial dysfunction now associated with PHS.
A diagram of the electron transport chain of mitochondria is provided in Figure 1. The electron transport chain
uses electron flow to create a proton gradient (proton
motive force) that is used to drive adenosine triphosphate
(ATP) synthesis. Oxygen serves as the terminal electron
acceptor in the chain of oxidation-reduction reactions that
occur in the inner mitochondrial membrane (Lehninger et
al., 1992). Proton movement through the f1f0 ATP synthase
effectively “couples” the energy-releasing reactions of ox-
INTRODUCTION
Pulmonary hypertension syndrome (PHS, ascites) is a
metabolic disease in broilers that may cost the poultry
industry worldwide over $1 billion annually (Maxwell et
al. 1996). Most recently the etiology of ascites has been
broadly classified into three categories: (1) pulmonary
hypertension, (2) miscellaneous cardiac pathologies, and
(3) cellular damage caused by reactive oxygen species
(Currie, 1999). Common to all three categories is impaired
oxygen utilization. Most of today’s fast growing broilers
are unable to deliver sufficient oxygen to satisfy the metabolic demands of their rapid growth rates. A marginal
cardio-pulmonary capacity has been elegantly demonstrated (Wideman and Kirby, 1995a,b; Wideman et al.,
1997). Key to oxygen supply and demand are mitochondria that account for 85 to 90% of cellular oxygen consumption (Shigenaga et al., 1994). Recent data indicate
that broilers with PHS suffer from increased oxidative
Abbreviation Key: ACR = acceptor control ratio; ADP = adenosine
diphosphate, ATP = adenosine triphosphate; EGTA = ethylene glycolbis (β-aminoethylether)-N,N,N′,N′ tetraacetic acid; GSH = glutathione;
HEPES = N-[2-Hydroxyethyl-piperizine]-N′-[2-ethanesulfonic acid];
PHS = pulmonary hypertension syndrome; ROS = reactive oxygen species; RCR = respiratory control ratio; RV/TV = right ventricular weight
ratio, SCWL = Single Comb White Leghorn; TTFA = 4,4,4-Trifluoro-1[2-thienyl]-1,3-butanedione.
Received for publication May 18, 2000.
Accepted for publication November 20, 2000.
1
To whom correspondence should be addressed: wbottje@comp.
uark.edu.
474
LIVER MITOCHONDRIAL DYSFUNCTION IN ASCITES
FIGURE 1. Diagrammatic representation of the electron transport
chain adapted from Lehninger et al. (1992). The electron transport chain
consists of four protein complexes (Complex I, II, III, and IV). Electrons
(e−) that enter the electron transport chain from energy substrates such
as malate (Complex I) and succinate (Complex II) are passed down the
electron transport chain (solid arrows) to the terminal electron acceptor,
oxygen that is reduced to water. Coenzyme Q (CoQ, ubiquinone) accepts
electrons from Complex I and II and moves the electrons to Complex III.
Associated with the movement of electrons along the electron transport
chain is the movement of protons (H+, dashed arrows) from the mitochondrial matrix into the intramembranous space, setting up a proton
motive force. The movement of protons through the adenosine triphosphate (ATP) synthase (ATPase) provides the energy to support ATP
synthesis. ADP = adenosine diphosphate.
idation to the energy-storing reaction of phosphorylation;
therefore, the addition of adenosine diphosphate (ADP)
to isolated mitochondria increases electron flow and oxygen consumption until the excess ADP has been phosphorylated to ATP.
Function of isolated mitochondria is assessed by polarographic measurement of oxygen consumption under
various conditions (Estabrook, 1967). Isolated mitochondria will exhibit an initial slow rate of oxygen consumption that is designated State 2 (initial) respiration. In the
presence of energy substrate (e.g., succinate), the addition
of ADP stimulates electron transport chain activity and
initiates a rapid consumption of oxygen during State 3
(active) respiration. When all ADP has been converted to
ATP during State 3 respiration, the demand for energy
(high ADP levels) declines causing oxygen consumption
to decrease and the mitochondria enter State 4 (resting)
respiration. The ADP:O ratio is the amount of ADP per
nanoatom (Estabrook, 1967) of monomeric oxygen consumed during State 3 respiration and is an index of oxidative phosphorylation. A decrease in the ADP:O ratio may
represent functional damage to mitochondrial oxidative
phosphorylation (Nakahara et al., 1998), possibly due to
the generation of reactive oxygen species (ROS) following
univalent reduction of oxygen. The respiratory control
2
Randall Road Hatchery, Tyson Foods, Inc., Springdale, AR 72762.
Cal Maine Foods, Inc., Lincoln, AR 72744.
3
475
ratio (RCR) represents the degree of coupling or efficiency
of electron transport chain activity and is calculated as
State 3 divided by State 4 respiration rate. “Proton leak,”
involving proton flow back into the matrix other than
through the f1f2 ATP synthase, increases State 4 (resting)
respiration, which in turn causes a decrease in the RCR
due to inefficient oxygen utilization (Brand et al., 1994).
Brand et al. (1994) have argued that in the complete absence of proton leak, State 4 respiration would be zero.
A final index of mitochondrial function is the acceptor
control ratio (ACR), which is calculated as State 3 divided
by State 2 respiration rate.
Mitochondria are a major site of oxygen consumption
as well as a major source of endogenous oxidative stress.
It has been estimated that rather than being completely
reduced to water, between 1 and 4% of oxygen consumed
by mitochondria is incompletely reduced by univalent
reduction to form superoxide as a result of leakage of
electrons from the electron transport chain (Chance et al.,
1979). The generation of mitochondrial ROS has now been
linked to a variety of metabolic diseases such as cystic
fibrosis (Fiegel and Shapiro, 1979) and diabetes (Kristal
et al., 1997) as well as aging (Hagen et al., 1997; Herrero
and Barja, 1998; Lass et al., 1998). Various chemical inhibitors have been used to identify site-specific defects in the
respiratory chain where electrons leak and increase ROS
production (e.g., Boveris et al., 1976; Chance et al., 1979;
Turrens and Boveris, 1980, Kristal et al., 1997). Thus, a
major objective of this study was to determine if sitespecific defects in the electron transport chain of broilers
with PHS could account for the impaired mitochondrial
oxygen utilization observed in PHS (Cawthon et al., 1999;
Iqbal et al., 1999). Biochemical assays of antioxidants
would be performed to confirm the presence of oxidative
stress in PHS mitochondria. A second experiment was
conducted using liver mitochondria obtained from Single
Comb White Leghorn (SCWL) males to determine if responses observed in a line of poultry highly resistant to
developing PHS were similar to those in control broilers
in the first experiment.
MATERIALS AND METHODS
Animals and Diet
Male broiler chicks in Experiment 1 (Cobb 500) were
obtained from a local hatchery.2 The chicks were raised
in an environmental chamber on wood shaving litter.
Temperatures in the chamber were 32 and 30 C for Weeks
1 and 2, lowered to 15 C during Week 3, and maintained
between 10 and 15 C for the rest of the study to induce
a high incidence of PHS (Wideman et al., 1995). Birds
were provided a starter diet (23% protein, 3,300 kcal ME/
kg) and water ad libitum. A second experiment was conducted with SCWL males that are highly resistant to developing PHS. The SCWL chicks obtained from a local
hatchery,3 were placed in cages for 6 wk with free access
to feed (23% protein, 3,300 kcal ME/kg) and water. After
6 wk, the birds were moved to litter floor pens with
476
CAWTHON ET AL.
temperature maintained between 20 to 25 C. The birds
were maintained on the same diet to eliminate feed as a
variable between experiments.
Sampling Procedure
Between 3 and 7 wk, broilers in Experiment 1 were
selected that appeared clinically healthy or specifically
exhibited overt symptoms of PHS, i.e., cyanosis of the
comb and wattle or ascites fluid accumulation in the abdomen. The selection of a PHS or control bird was alternated
on successive days during the experiment. The birds were
killed with an overdose of sodium pentobarbital by i.v.
injection into the leg vein, and weights of the right ventricle (RV) and total ventricle (TV) were determined to calculate the right ventricular weight ratio (RV/TV). The RV/
TV is a sensitive indicator of prior exposure of the heart
to increased pulmonary arterial pressures (Burton et al.,
1968). Broilers with an RV/TV < 0.27 without fluid in the
abdomen were designated as controls, whereas those with
an RV/TV ≥ 0.30 with fluid accumulation were designated as PHS. In Experiment 2, SCWL males were selected
between 47 and 62 d of age. The RV/TV for SCWL were
all <0.20. Mean age of broilers and SCWL in these studies
were 37 ± 3 d and 53 ± 3 d, respectively.
Preparation of Mitochondria
Hepatic mitochondria were obtained by differential
centrifugation as outlined by Olafsdottir and Reed (1988)
with modifications (Cawthon et al., 1999). Approximately
20 g of liver tissue was suspended in 50 mL of isolation
media (pH 7.4) containing 220 mM d-mannitol, 70 mM
sucrose, 2 mM N-[2-Hydroxyethylpiperizine]-N′-[2-ethanesulfonic acid] (HEPES), 0.5 mg/ml BSA (fatty acid
free), and 1 mM ethylene glycol-bis (β-aminoethylether)N,N,N′,N′ tetraacetic acid (EGTA). The tissue was then
homogenized with a tissue grinder4 and diluted to 250
mL in isolation media. Aliquots were transferred into
polycarbonate centrifuge tubes and centrifuged twice for
10 min at 600 × g. The pellets containing nuclei and cell
debris were discarded, and the supernatant was centrifuged for 15 min (7,750 × g). The mitochondrial pellets
were resuspended in an isolation buffer (pH 7.0) containing 220 mM d-mannitol, 70 mM sucrose, 2 mM
HEPES, and 0.5 mg/mL BSA (fatty acid free) and were
washed twice. Mitochondria were resuspended in incubation media (210 mM d-mannitol, 70 mM sucrose, 2 mM
HEPES, and 10 mM succinate) and placed on ice.
Mitochondrial Function
Mitochondrial function was determined according to
Estabrook (1967). Oxygen consumption of mitochondria
4
Thomas Tissue Grinders; Thomas Scientific, Swedesboro, NJ
08085-0099.
5
Yellow Springs Instrument Co. Inc., Yellow Springs, OH 45387.
(expressed in nmol/min/mg protein) was measured polarographically with a Clark-type oxygen electrode in duplicate 3-mL thermostatically controlled chambers
equipped with magnetic stirring.5 All functional measurements were made in duplicate and averaged. State 2 respiration of mitochondria was monitored followed by State
3 and State 4 respiration rates with excess and inadequate
levels of ADP, respectively. Aliquots (0.5 mL) of the mitochondrial incubation were removed and added to the
reaction vessel containing 1 mL of RCR reaction buffer
(220 mM d-mannitol, 70 mM sucrose, 2 mM HEPES, 3
mM KH2PO4; 5 µL of 1.5 mM rotenone, 50 µL of 1 M
succinate, pH 7.0). State 3 (active) respiration was induced
by the addition of 25 µL of 10 mM ADP (155 µM ADP,
final concentration), and the RCR (an index of electron
transport chain coupling) was calculated by dividing
State 3 by State 4 (resting) respiration. The ACR, which
is similar to the RCR, was calculated by dividing State 3
by State 2 respiration. The efficiency of ATP synthesis
coupled to cell respiration, the ADP:O ratio, was determined by dividing the quantity of ADP added by the
amount of oxygen consumed during State 3 respiration.
Identification of Site-Specific Defects
in the Electron Transport Chain
To determine if site-specific defects exist in the respiratory chain of PHS liver mitochondria, the amount of oxygen consumed during State 3 respiration in the presence
or absence of various electron transport chain inhibitors
was monitored as previously described (Kristal et al.,
1997). A site-specific defect can be observed as continued
oxygen consumption following inhibition of respiration
when electrons leak from the respiratory chain and react
with oxygen. This continuing oxygen consumption is due
to univalent reduction of oxygen to superoxide and is an
indirect measure of ROS formation.
After functional measurements were obtained as described above, mitochondria were again treated with ADP
(310 µM, final concentration). The amounts of ADP (155
µM and 310 µM) added to mitochondria were determined
in pilot studies to be sufficient to obtain functional measurements and for sufficient time to insure inhibition during State 3 respiration. Shortly after the second State 3
respiration was initiated, respiration was inhibited by
chemicals that block specific sites on the electron transport chain that included: malonate and 4,4,4-Trifluoro-1[2-thienyl]-1,3-butanedione (TTFA ) (Complex II, succinate dehydrogenase), antimycin-A (Complex III, cytochrome b566), myxothiazol (Complex III, Q cycle), and
potassium cyanide (KCN) (Complex IV, cytochrome oxidase). A diagram showing sites of chemical inhibition is
provided in Figure 2. The final concentrations for each
inhibitor were 1 µM for TTFA, antimycin-A, myxothiazol,
and KCN, and 5 µM for malonate. Oxygen consumption
that remained following chemical inhibition was determined by comparing the slopes of State 3 oxygen consumption between the first and second additions of ADP.
Oxygen consumption observed following electron trans-
LIVER MITOCHONDRIAL DYSFUNCTION IN ASCITES
FIGURE 2. Diagrammatic representation of the electron transport
chain showing sites of chemical inhibition used in this study. Electron
movement (e−) is blocked at Complex II by A) malonate and 4,4,4Trifluoro-1-[2-thienyl]-1,3-butanedione (TTFA), at Complex III by B)
myxothiazol (Q cycle) and C) antimycin-A (cytochrome b566), and at
Complex IV (cytochrome oxidase) by D) potassium cyanide (KCN).
Also shown is the transfer of electrons through coenzyme Q (CoQ,
ubiquinone) and cytochrome C (Cyt C). If a site-specific defect exists
in electron transport at any of these sites of chemical inhibition, electrons
will leak (dotted arrows) from the respiratory chain and consume oxygen by univalent reduction that results in the formation of superoxide
(O2䊉−) that, in turn, can be converted to other reactive oxygen species (ROS).
port chain inhibition is expressed as percentage activity
remaining. A significant difference in the percentage activity remaining is indicative of univalent reduction of
oxygen following electron leak and a site-specific defect
in electron transport chain activity (Kristal et al., 1997)
between PHS and control mitochondria. No defect is present if there is no oxygen consumption following respiratory chain inhibition.
Biochemical Analysis
Glutathione. Reduced glutathione (GSH), glutamate,
and γ-glutamyl cysteine were determined using methods
described by Fariss and Reed (1987). Mitochondria and
liver tissue were treated with 10% perchloric acid to precipitate proteins followed by reaction of iodoacetic acid
to form S-carboxy-methyl derivatives and derivatization
of amino groups in the supernatant with 1-fluoro-2,4dinitrobenzene. Derivatized compounds were separated
by ion-exchange column chromatography and identified
by retention times of authentic standards. Concentrations
of each compound were calculated from peak integrated
areas. All chemicals were obtained from Sigma Chemical
Company. Protein concentration in the mitochondrial incubate was determined using a kit (P5656).6
6
Sigma Chemical Co., St. Louis, MO 63178-9916.
Waters and Associates, Milford, MA 01757.
Hoffman LaRoche, Nutley, NJ 07110-1199.
7
8
477
Tocopherols. Determination of α-, γ-, and δ-tocopherol
was carried out by a modified method of Warren and
Reed (1991). The mitochondria were first treated with icecold ethanol containing ascorbic acid (1 g/L) to precipitate proteins. After extracting the sample homogenate
twice with 2 mL hexane, the combined organic layer was
evaporated under nitrogen. The tocopherols were redissolved in methanol:acetonitrile (1:3) and centrifuged for
5 min × 12,000 g; 20 µL of the supernatant was used for
reversed-phase HPLC using Waters system7 with a C18
Nova-Pak8 column (3.9 × 150 cm). The tocopherols were
separated using isocratic conditions with a mobile phase
consisting of 25% methanol and 75% acetonitrile at a flow
rate of 1 mL/min and were monitored by fluorescence
detector set at excitation/emission wavelengths of 298/
328 nm, respectively. Tocopherols were identified and
quantified by comparison to the retention times and peak
areas of authentic standards. Extraction efficiency of tocopherol was based on the theoretical response of the
internal standard.
Statistical Analysis
Data were analyzed by one-way ANOVA and by Student’s t-test. The statistical analysis was accomplished
using the general linear models procedure of SAS威 software (SAS Institute, 1996). A probability level of P ≤ 0.05
was considered statistically significant.
RESULTS
Physiological and biochemical analyses of mitochondria from control and PHS broilers (Experiment 1) and
SCWL males (Experiment 2) are presented in Table 1.
Lower body weights and elevations in the RV/TV and
hematocrit were observed in broilers with PHS that are
typical indicators of this metabolic disease. There was
no difference in the mitochondrial protein concentration
between groups. The PHS liver mitochondria had lower
levels of α- and γ-tocopherols, GSH, and glutamate, but
not δ-tocopherol or γ-glutamyl cysteine, than did controls.
The GSH levels were also lower in hepatic tissue obtained
from broilers with PHS than in controls (data not shown).
SCWL birds in Experiment 2 had much lower body
weights, but exhibited similar hematocrit, and RV/TV as
control broilers in Experiment 1. Despite an elevation in
mitochondrial protein, GSH levels in SCWL liver mitochondria were comparable to those in control broilers.
Tocopherol, glutamate, and γ-glutamyl cysteine were not
determined in SCWL mitochondria in Experiment 2.
In the presence of malonate and TTFA (Complex II
inhibitors), oxygen consumption (percentage activity remaining) was higher in PHS than in control hepatic mitochondria (Figure 3A). There were no differences in residual oxygen consumption with the other respiratory chain
inhibitors antimycin-A (AA), myxothiazol, or KCN. There
were also no differences in percentage activity remaining
between groups when mitochondria were treated with
478
CAWTHON ET AL.
TABLE 1. Physiological and mitochondrial biochemical analyses of broilers in Experiment 1 (controls and
those with pulmonary hypertension syndrome, PHS) and in Single Comb
White Leghorn (SCWL) males in Experiment 21
Experiment 1
2
Variable
Physiological analyses
BW (g)
RV/TV
Hematocrit (%)
Mitochondrial biochemical analysis
Protein (mg/g tissue)
GSH (nmol/mg protein)
Tocopherol (pmol/mg protein)
αγδglutamate (nmol/mg protein)
γ-glutamyl cysteine
(nmol/mg protein)
Control
(n = 8)
PHS
(n = 8)
Experiment 2,
SCWL
(n = 6)
2,349 ± 309
0.19 ± 0.01
33 ± 1
1,684 ± 191a
0.39 ± 0.13a
54 ± 3a
705 ± 24
0.18 ± 0.01
32 ± 2
2.11 ± 0.22
4.43 ± 0.26
2.32 ± 0.10
3.63 ± 0.20a
3.11 ± 0.20
4.11 ± 0.25
53.1
12.7
0.1
2.67
1.47
±
±
±
±
±
8.3
2.8
0.1
0.34
0.31
24.3
3.2
0.2
1.64
1.05
±
±
±
±
±
7.7a
1.9a
0.1
0.25a
0.26
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Mean PHS values are different (P < 0.05) than controls.
Each value represents the mean ± SE (n = 5 to 8).
2
RV/TV = right ventricular to total ventricular weight ratio; GSH = glutathione.
a
1
FIGURE 3. Percentage activity remaining in oxygen consumption
following electron transport chain inhibition with site-specific inhibitors
of mitochondria isolated from A) control broilers (CON) and broilers
with pulmonary hypertension syndrome (PHS) in Experiment 1 (n = 7
to 8), and B) Single Comb White Leghorn (SCWL) males in Experiment
2 (n = 5 to 6). Chemical inhibitors were malonate (Mal), 4,4,4-Trifluoro1-[2-thienyl]-1,3-butanedione (TTFA), antimycin-A (AA), myxothiazol
(Myx), and potassium cyanide (KCN). *Mean values in PHS mitochondria were higher than controls (P < 0.05).
tetramethyl-p-phenylenediamine and ascorbate to assess
cytochrome oxidase activity alone (Chandel et al., 1995)
(data not shown). Similar to control mitochondria in Experiment 1, SCWL liver mitochondria in Experiment 2
exhibited greater percentage activity remaining following
respiratory chain inhibition with Complex II inhibitors
but not with the other respiratory chain inhibitors (Figure
3B). Because succinate was used as an energy substrate,
we did not test chemical inhibition of Complex I in these
studies (See Figure 1).
Measurements of mitochondrial respiration in control
and PHS liver mitochondria are presented in Table 2.
There were no differences in State 2 (initial) or State 4
(resting) respiration rates between groups, but lower State
3 (active) respiration resulted in lower ACR and RCR
values in PHS mitochondria than in controls. Respiration
rates and mitochondrial function in SCWL liver mitochondria were comparable to that observed in control
broilers in Experiment 1 (Table 2).
An unexpected finding in this study was the improved
function in mitochondria not treated with chemical inhibitors that was observed following sequential additions of
ADP. After the second addition of ADP, control mitochondria exhibited large improvements in the ADP:O and
RCR (Figure 4A and B), whereas function improved but
to a lesser extent in PHS mitochondria. The marked improvement in control mitochondrial function was not due
to alterations in State 3 respiration that did not change
in either group with the sequential addition of ADP, although it was higher in controls (Figure 4C). The improved function noted in control mitochondria appears to
be a result of changes in State 4 respiration with sequential
additions of ADP. State 4 respiration declined in both
groups (Figure 4D) following the second addition of ADP,
and although there were no differences between groups,
the percentage decrease in State 4 respiration following
479
LIVER MITOCHONDRIAL DYSFUNCTION IN ASCITES
TABLE 2. Oxygen consumption (State 3 and State 4 respiration) and mitochondrial function in liver
mitochondria isolated from controls and broilers with pulmonary hypertension syndrome (PHS)
in Experiment 1 and from Single Comb White Leghorn (SCWL) males in Experiment 21
Experiment 1
Variable
Control
(n = 8)
PHS
(n = 8)
Experiment 2
SCWL
(n = 6)
Oxygen consumption
(natoms of O/min per mg protein)
State 2 respiration
State 3 respiration
State 4 respiration
10.0 ± 0.5
39.2 ± 0.8
9.2 ± 0.3
9.3 ± 0.5
33.6 ± 2.7a
8.8 ± 0.6
10.5 ± 0.7
42.3 ± 2.4
9.9 ± 0.8
3.91 ± 0.19
4.37 ± 0.07
1.64 ± 0.07
3.28 ± 0.19a
3.83 ± 0.21a
1.54 ± 0.07
4.04 ± 0.15
4.31 ± 0.21
1.59 ± 0.03
Mitochondrial function2
ACR (State 2/State 3)
RCR (State 3/State 4)
ADP:O (ADP/State 3)
(nmol ADP/nmol O)
Mean PHS values are lower than Control (P < 0.05).
Values represent the mean ± SE (n = 5 to 8).
2
ACR = acceptor control ratio; RCR = respiratory control ratio; ADP:O = adenosine diphosphate to oxygen
consumption during State 3 respiration.
a
1
the second addition of ADP was not significantly greater
(P < 0.06) in controls (Figure 4E). Sequential additions of
ADP to isolated SCWL liver mitochondria in Experiment
2 exhibited a similar effect on respiration and function
as was observed in control broilers in Experiment 1 (Figure 5).
DISCUSSION
A defect in mitochondrial electron transport can be
identified if oxygen consumption continues following
chemical inhibition of the respiratory chain due to interaction of electrons with oxygen to form ROS (Kristal et al.,
FIGURE 4. Function and respiration of mitochondria isolated from control broilers (CON; open bars) and broilers with pulmonary hypertension
syndrome (PHS; closed bars) following sequential additions of adenosine diphosphate (ADP, 155 and 310 µM) in Experiment 1. Measurements
included A) the respiratory control ratio (RCR), B) the ratio of adenosine diphosphate (ADP) to oxygen consumption during State 3 respiration
(ADP:O), C) State 3, and D) State 4 respiration (number of natoms oxygen/min per milligrams mitochondrial protein), and E) the percentage
decrease in State 4 respiration between the first and second additions of ADP. Each bar represents the mean ± SE (n = 7 to 8). a–cMeans with
different letters are different (P < 0.05). *PHS means are lower than control (P < 0.05). +PHS mean is lower than control (P = 0.059).
480
CAWTHON ET AL.
FIGURE 5. Function and respiration of mitochondria isolated from Single Comb White Leghorn (SCWL) males following sequential additions
of adenosine diphosphate (ADP, 155 and 310 µM) in Experiment 2. Measurements included A) the respiratory control ratio (RCR), B) the ratio of
ADP to oxygen consumption during State 3 respiration (ADP:O), C) State 3, and D) State 4 respiration (number of natoms oxygen/min per
milligram mitochondrial protein), and E) the percentage decrease in State 4 respiration between the first and second additions of ADP. Each bar
represents the mean ± SE (n = 5 to 6). *Means following 310 µM ADP are different from 155 µM ADP values (P < 0.05).
1997). In the presence of malonate and TTFA (Complex
II inhibitors), continued oxygen consumption (percentage
activity remaining) was greater in PHS than in control
hepatic mitochondria (Figure 3). These findings indicate
the presence of a site-specific defect located within the
Complex II/CoQ (succinate:ubiquinone oxidoreductase)
pathway within PHS hepatic mitochondria (Figure 1). As
discussed below, the increased electron leak may account
for the mitochondrial dysfunction and the prevalent oxidative stress that has been observed in broilers with PHS.
THe SCWL liver mitochondria in Experiment 2 exhibited greater percentage activity remaining following respiratory chain inhibition with Complex II inhibitors but
not with the other inhibitors (Figure 3B). The similarity
in response to respiratory chain inhibition between the
two experiments indicates that at least in these two lines
of poultry, there may be a greater inherent leakage of
electrons associated with Complex II than at other sites
of the respiratory chain under the conditions used in this
study. No direct conclusions between broilers and SCWL
can be made because the birds were raised under different
environmental conditions. Birds in each experiment were
provided the same diet, however.
The major indices of mitochondrial function, the RCR
and ADP:O ratios, are measures of electron transport
chain coupling and efficiency of oxidative phosphorylation, respectively (Estabrook, 1967). The initial RCR values were lower in PHS mitochondria (Table 2), indicating
that PHS liver mitochondria use oxygen less efficiently
compared to controls. The lower RCR in PHS mitochondria in this study is similar to that observed previously
(Cawthon et al., 1999). Although there was no significant
difference in the initial ADP:O ratio between groups (Table 2), Control mitochondria exhibited higher ADP:O following sequential additions of ADP (Figure 4). The net
result of the depressed function in PHS mitochondria
would be higher cellular oxygen demand and less efficient energy production in broilers with PHS.
An important and unexpected finding in this study was
the improvement in mitochondrial function following sequential additions of ADP (Figures 4 and 5). To our
knowledge, the ability to improve function by lowering
State 4 respiration in response to sequential additions of
ADP in broiler and SCWL liver mitochondria has not
been previously reported. If this observation in avian
mitochondria does indeed differ from that in mammalian
mitochondria (Kristal, personal communication), this
finding could provide valuable new insight into mitochondrial physiology. Although the exact mechanisms
responsible for the improvement are not apparent at this
time, it is significant that sequential additions of ADP
magnified the mitochondrial dysfunction in PHS mitochondria. Studies are currently underway to determine
the biochemical nature responsible for the decrease in
State 4 respiration with sequential additions of ADP. At
this point we do know that the decrease in State 4 respiration is not simply a response to the amount of ADP added
and does not appear to be due to differences in calcium
LIVER MITOCHONDRIAL DYSFUNCTION IN ASCITES
in the isolation or incubation media (unpublished observations).
It is possible that the decrease in State 4 respiration with
sequential additions of ADP may be due to a decrease in
proton leak. Leakage of protons across the mitochondrial
membrane other than through the f1f0 ATP synthase lowers ATP synthesis and is associated with higher State 4
respiration (Brand et al., 1994). Higher State 4 respiration
in PHS mitochondria has been reported previously (Cawthon et al, 1999). Although there were no differences in
State 4 respiration between groups, control hepatic mitochondria were able to lower State 4 respiration proportionally more than PHS mitochondria following sequential additions of ADP (Figure 5E). Similar findings have
been observed in PHS lung mitochondria (unpublished
observations). Thus, besides having a site-specific defect
in the electron transport chain, PHS liver mitochondria
may have another inefficiency in producing energy for
the cell due to greater proton leak or an inability to alter
proton leak with repeated energy demand.
Site-specific electron leak may account, in part, for the
decreased coupling of the electron transport chain (lower
RCR) and lower ADP:O observed with sequential additions of ADP in this study and in a recent report (Iqbal
et al., 2001). The lower ADP:O after the second addition
of ADP in PHS mitochondria indicates that a greater
amount of oxygen would be consumed to produce the
same amount of ATP for the cell. This inefficiency in
oxygen use would only serve to aggravate the existing
cardiopulmonary insufficiency in broilers (Wideman and
Kirby, 1995ab; Wideman et al., 1997) and accentuate the
development of systemic hypoxia. In this regard, relative,
but not absolute, hypoxia is especially detrimental to mitochondrial function and enhances mitochondrial production of ROS (Dawson et al., 1993). Thus, the systemic
hypoxia characteristic of broilers with PHS (Peacock et
al., 1990; Wideman and Bottje, 1993; Wideman and Kirby,
1995b) would likely enhance mitochondrial ROS generation. Yet, the defects observed in PHS mitochondria may
not just be a secondary response to onset of disease symptoms. Wideman and French (1999; 2000) have conclusively demonstrated that there is a genetic relationship
to PHS. Mitochondria obtained from broilers selected for
genetic resistance to PHS generated less hydrogen peroxide than did mitochondria obtained from the unselected,
base population of broilers indicating that there is a genetic predisposition to mitochondrial ROS production in
broilers (Iqbal et al., 2001). Lower ADP:O, such as observed with PHS mitochondria (Cawthon et al., 1999;
Iqbal et al., 2001), may represent functional damage to
mitochondrial oxidative phosphorylation resulting from
radical-mediated damage to the electron transport chain
(Nakahara et al., 1998).
As the coupling between ADP phosphorylation and
oxygen utilization is represented by the ADP:O, the values of ADP:O can be used to calculate the approximate
amount of oxygen consumed by respiratory chain activity
that is not used to support ATP synthesis (Kristal et al.,
1997). By using mean ADP:O ratios in this study, an esti-
481
mate of the amount of oxygen incompletely reduced to
ROS could be made (Table 3). These values were calculated with the following assumptions: 1) that there is no
electron leak in perfectly coupled mitochondria (theoretical ADP:O = 2.0 in mitochondria provided succinate as
an energy substrate), and 2) that 1 to 4% of the oxygen
consumed by control mitochondria occurs by univalent
reduction of oxygen to superoxide as a result of electron
leakage from the respiratory chain (Chance et al., 1979).
Thus, following the initial addition of ADP, PHS mitochondria would have 1.25 to 5% of the oxygen consumed
by mitochondria, resulting in radical production compared to the normal range of 1 to 4% in controls. Although
this difference may initially seem trivial, it would mean
that PHS mitochondria would have the potential to generate 25% more ROS than controls. The second addition
of ADP significantly increased the ADP:O in controls,
whereas only a numerical increase in the ADP:O was
observed in PHS mitochondria (Figure 4B). By using the
same assumption as presented earlier, the lower ADP:O
ratios would result in a decrease ROS generation to 0.25
to 1% in control and 0.875 to 3.5% of total oxygen consumption in PHS mitochondria. Despite the decrease in
calculated ROS production after the second addition of
ADP in PHS mitochondria, the level would now be 250%
higher than the level in control mitochondria. Over prolonged periods of time, the additional amount of ROS
production in PHS mitochondria would undoubtedly
raise the oxidative stress load in cells and tissues of broilers with PHS. The potential for higher oxidative stress is
confirmed by the lower levels of mitochondrial antioxidants in PHS mitochondria compared to controls (Table
1). Thus, the apparent inability of PHS mitochondria to
improve mitochondrial function with repeated energy
demand could have profound consequences with regard
to cellular oxidative stress as well as oxygen utilization.
The evidence shown in this study indicates that an
increase in ROS production in liver mitochondria is likely
due to leakage of electrons from Complex II in PHS mitochondria. A consequence of defective electron transport
in PHS mitochondria would be a self-perpetuating cycle
of oxidative damage due to the formation of toxic compounds such as malondialdehyde and hydroxyalkenals
(Esterbauer et al., 1991) that can in turn cause further
deterioration of mitochondrial function (Esterbauer et al.,
1991; Kristal et al., 1994, 1996; Chen et al., 1995). Oxidative
stress occurs when the balance between endogenous antioxidant protection becomes overwhelmed by the generation of oxidants (Yu, 1994). The major lipophillic (α-tocopherol) and hydrophillic (GSH) antioxidants that protect against free radical oxidation (Yu, 1994) were lower
in PHS mitochondria. The presence of oxidative stress in
PHS liver mitochondria concurs with recent findings in
PHS lung mitochondria (Iqbal et al., 2001) and with that
of Maxwell et al. (1996) who presented histological evidence of increased hydrogen peroxide in heart mitochondria obtained from broilers with PHS. Oxidative stress
has also been observed in tissues (Enkvetchakul et al.,
1993; Bottje et al., 1995; Diaz-Cruz et al., 1996), plasma
482
CAWTHON ET AL.
TABLE 3. Estimation of changes in oxygen radical production based on the adenosine diphosphate
(ADP):O ratio obtained with sequential additions of ADP in control and
pulmonary hypertension syndrome (PHS) liver mitochondria
Treatment group
Variable
Control
After first addition of ADP
ADP:O
% ROS
Increase in calculated ROS
production relative to controls
1.64 ± 0.07
1 to 4%1
...
After second sequential addition of ADP
ADP:O
% ROS
Increase in calculated ROS
1.91 ± 0.07
0.25 to 1%2
...
PHS
1.54 ± 0.07
1.25 to 5%2
25%
1.68 ± 0.06a
0.875 to 3.5%2
250%
production relative to controls
1
Based on estimates of Chance et al. (1979) that 1 to 4% of oxygen consumed by mitochondria is incompletely
reduced to reactive oxygen species (ROS) from electron leakage from the respiratory chain.
2
This is based on a theoretical ADP:O ratio of 2.0 having no ROS production and 1 to 4% ROS being produced
in control mitochondria following the initial addition of ADP.
(Bottje et al., 1995), and in lung lining fluid (Bottje et al.,
1998a,b) of broilers with PHS. Thus, a pervasive oxidative
stress has now been observed in subcellular, cellular, and
extracellular fluid compartments in broilers with PHS.
Coenzyme Q (CoQn, ubiquinone) is responsible for the
transfer of electrons from Complexes I and II to Complex
III (Figure 1). Lass et al. (1997) reported that animals with
high levels of CoQ9 (rat and mouse) had higher levels of
mitochondrial ROS production than other species (cow,
rabbit, and pig) that had relatively more mitochondrial
CoQ10. There was also a direct relationship between the
degree of binding of ubiquinone to proteins and mitochondrial radical production (Lass and Sohal, 1999). The
chemical inhibitors, TTFA and malonate, block electron
transfer at the Complex II/CoQ interface. Thus, a unifying
mechanism between the results in lung mitochondria (Iqbal et al., 2001), and those in the present study with liver
mitochondria could be a defect or insufficiency of ubiquinone in broiler mitochondria. To date, there is no information on the relative binding of ubiquinone to mitochondrial proteins or the proportions of CoQ9 and CoQ10 in
broiler mitochondria. The hypothesis of a defect in mitochondrial CoQ in broilers is supported by a report in
which the feeding of CoQ9 attenuated the incidence of
cold temperature-induced PHS mortality in broilers (Nakamura et al., 1996). Other than indicating that CoQ9 is
integral to mitochondrial membranes, no other biochemical mechanism was presented by these researchers to explain their findings.
The results of the present study when combined with
those of Cawthon et al. (1999) and Iqbal et al. (2001)
present an intriguing look at the pathophysiology of this
disease. From these studies, at least three separate mitochondrial defects have been identified. In all three studies,
one defect consists of reduced mitochondrial function and
poor oxygen utilization. A second defect involves electron
leakage either at Complex I and III in lung mitochondria
(Iqbal et al., 1999) or at Complex II (present study). It is
possible that electron leak may also occur at Complex
I in PHS liver mitochondria, but due to the substrate
(succinate) that was used, this aspect of electron transport
chain activity was not addressed in the present study. It
should be emphasized that with a recent report by Lass
et al. (1998) demonstrating that sites of electron leak by
mitochondria vary from tissue to tissue, it is not surprising that there may also be tissue specificity with regards
to electron leak in PHS mitochondria as well. It is entirely
possible that there is a genetic basis for this electron leak
in PHS mitochondria (Iqbal et al., 2001) that is simply
enhanced or accentuated by relative hypoxia (Dawson et
al., 1993). Finally, a third defect may be due to higher
proton leak that elevates State 4 respiration in PHS mitochondria. Regardless of the mechanism, it is now evident
that mitochondrial dysfunction is pervasive in the pathophysiology of PHS in broilers.
In summary, using a series of chemical inhibitors of
mitochondrial respiration, it was demonstrated that PHS
hepatic mitochondria exhibit a site-specific defect in electron transport associated with Complex II/CoQ of the
electron transport chain. This defect could manifest itself
by increased univalent reduction of oxygen and ROS generation in PHS mitochondria, raising the level of oxidative
stress in both mitochondria and tissue. A second defect
in hepatic mitochondrial function in broilers with PHS
appears to involve an impaired ability to alter State 4
respiration, and therefore proton leak, in response to sequential additions of ADP. The paradigm of sequential
additions of ADP might be considered to be somewhat
similar to continuing or alternating demand for energy
that would be experienced by mitochondria in vivo. Improved function with sequential ADP additions was also
observed in SCWL liver mitochondria, indicating that this
response is not unique to broiler mitochondria. Regardless of the mechanism, the sequential additions of ADP
magnified the dysfunction in PHS mitochondria relative
to controls, making it apparent that PHS mitochondria
are even less efficient in using oxygen than revealed with
single additions of ADP observed in this and a previous
LIVER MITOCHONDRIAL DYSFUNCTION IN ASCITES
study (Cawthon et al., 1999). The mitochondrial dysfunction demonstrated in this and other studies (Cawthon et
al., 1999; Iqbal et al., 2001) therefore establishes a cellular
basis for the oxidative stress and blood oxygenation problems associated with PHS.
ACKNOWLEDGMENTS
The authors thank R. McNew (Agriculture Statistics
Laboratory) for statistical consultation and Helen Brandenburger for technical editing. Parts of this manuscript
were presented at the Poultry Science Association Annual
Meeting, Fayetteville, AR, August 8 to 11, 1999. This
manuscript was selected to receive the Student Research
Manuscript Award at the 89th Annual Meeting of the
Poultry Science Association in Montreal (August 18 to 20,
2000). Research presented in this manuscript is published
with the support of the Director of Agriculture Experiment Station, University of Arkansas and by a USDANRI grants (No. 96-2327 and 99-2123) to W. Bottje.
REFERENCES
Bottje, W., B. Enkvetchakul, R. Moore, and R. McNew, 1995.
Effect of α-tocopherol on antioxidants, lipid peroxidation,
and the incidence of pulmonary hypertension syndrome (ascites) in broilers. Poultry Sci. 74:1356–1369.
Bottje W. G., S. Wang, F. J. Kelly, C. Dunster, A. Williams, and
I. Mudway, 1998a. Antioxidant defenses in lung lining fluid
of domestic fowl: Impact of poor ventilation conditions. Poultry Sci. 77:516–522.
Bottje, W. G., S. Wang, K. W. Beers, and D. Cawthon, 1998b.
Lung lining fluid antioxidants in broilers: Age related
changes under thermoneutral and cold temperature conditions. Poultry Sci. 77:1905–1912.
Boveris, A., E. Cadenas, and O. M. Stoppani, 1976. Role of
ubiquinone in the mitochondrial generation of hydrogen peroxide. Biochem. J. 153:435–441.
Brand, M. D., L. F. Chien, E. K. Ainscow, D.F.S. Rolfe, and R.
K. Porter, 1994. The causes and function of mitochondrial
proton leak. Biochim. Biophys. Acta 1187:132–139.
Burton, R. R., A. Joyce, and K. U. Ingold, 1968. Effect of chronic
hypoxia on the pulmonary arterial blood pressure of the
chicken. Am. J. Physiol. 214:1438–1442.
Cawthon, D., R. McNew, K. W. Beers, and W. G. Bottje, 1999.
Evidence of mitochondrial dysfunction in broilers with pulmonary hypertension syndrome (ascites): Effect of t-butyl
hydroperoxide on hepatic mitochondrial function, glutathione, and related thiols. Poultry Sci. 78:114–124.
Chance, B., H. Sies, and A. Boveris, 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59:527–605.
Chandel, N., G.R.S. Budinger, R. A. Kemp, and P. T. Schumacker, 1995. Inhibition of cytochrome-c oxidase activity
during prolonged hypoxia. Am. J. Physiol. 268:L918–L925.
Chen, J. J., H. Bertrand, and B. P. Yu, 1995. Inhibition of adenine
nucleotide translocator by lipid peroxidation products. Free
Rad. Biol. Med. 19:583–590.
Currie, R.J.W. 1999. Ascites in poultry: recent investigations.
Avian Pathol. 28:313–326.
Dawson, T. L., G. J. Gores, A. Nieminen, B. Herman, and J. J.
Lemasters, 1993. Mitochondria as a source of reactive oxygen
species during reductive stress in rat hepatocytes. Am. J.
Physiol. 264:C961–967.
Diaz-Cruz, A., C. Nava, R. Villanueva, M. Serret, R. Guinzberg,
and E. Pina, 1996. Hepatic and cardiac oxidative stress and
other metabolic changes in broilers with the ascites syndrome. Poultry Sci. 75:900–903.
483
Enkvetchakul, B., W. Bottje, N. Anthony, and R. Moore, 1993.
Compromised antioxidant status associated with ascites in
broilers. Poultry Sci. 72:2272–2280.
Esterbauer, H., R. J. Schaur, and H. Zollner, 1991. Chemistry and
biochemistry of 4-hydroxynonnenal, and related aldehydes.
Free Rad. Biol. Med. 11:81–128.
Estabrook, R. W., 1967. Mitochondrial respiratory control and
the polarographic measurement of ADP/O ratios. Methods
Enzymol. 10:41–47.
Farris, M. W., and D. J. Reed, 1987. High performance liquid
chromatography of thiols and disulfides: dinitrophenol derivitives. Methods Enzymol. 143:101–109.
Fiegel, R. J., and B. L. Shapiro, 1979. Mitochondrial calcium
uptake and oxygen consumption in cystic fibrosis. Nature
278:276–277.
Hagen, T. M., D. L. Yowe, J. C. Bartholomew, C. M. Wehr, K.
L. Do, J. Y. Park, and B. N. Ames, 1997. Mitochondrial decay
in hepatocytes from old rats: Membrane potential declines,
heterogeneity, and oxidants increase. Proc. Natl. Acad. Sci.
94:3064–3069.
Herrero, A., and G. Barja, 1998. Hydrogen peroxide production
of heart mitochondria and aging rates are slower in canaries
and parakeets than in mice; Sites of free radical generation
and mechanisms involved. Mech. Aging Develop. 103:133–
146.
Iqbal, M., D. Cawthon, R. F. Wideman, Jr., and W. G. Bottje, 2001.
Lung mitochondrial dysfunction in pulmonary hypertension
syndrome I. Site-specific defects in the electron transport
chain. Poultry Sci. 80:485–495
Kristal, B. S., J. J. Chen, and B. P. Yu, 1994. Sensitivity of mitochondrial transcription to different free radical species. Free
Rad. Biol. Med. 16:323–329.
Kristal, B. S., C. T. Jackson, H. Y. Chung. M. Matsuda, H. D.
Nguyen, and B. P. Yu, 1997. Defects at center P underlie
diabetes-associated mitochondrial dysfunction. Free Rad.
Biol. Med. 22:823–833.
Kristal, B. S., B. K. Park, and B. P. Yu, 1996. 4-Hydroxyhexenal
is a potent inducer of the mitochondria permeability transition. J. Biol. Chem. 271:6033–6038.
Lass, A., S. Agarwal, and R. S. Sohal, 1997. Mitochondrial ubiquinone homologues, superoxide radical generation, and longevity in different mammalian species. J. Biol. Chem.
272:19199–19204.
Lass, A., and R. S. Sohal, 1999. Comparisons of coenzyme Q
bound to mitochondrial membrane proteins among different
mammalian species. Free Rad. Biol. Med. 27:220–226.
Lass, A., B. H. Sohal, R. Weindruch, M. J. Forster, and R. S. Sohal,
1998. Caloric restriction prevents age-associated accrual of
oxidative damage to mouse skeletal muscle mitochondria.
Free Rad. Biol. Med. 25:1089–1097.
Lehninger, A. L., D. L. Nelson, and M. M. Cox, 1992. Oxidative
phosphorylation and photophosphorylation (Chapter 18).
Pages 542–592 in: Principles of Biochemistry. 2nd Edition.
Worth Publishers, New York, NY.
Maxwell, M., G. W. Robertson, and C. Farquharson, 1996. Evidence of ultracytochemical mitochondria-derived hydrogen
peroxide activity in myocardial cells from broiler chickens
with an ascites syndrome. Res. Vet. Sci. 61:7–12.
Nakahara, H., T. Kanno, Y. Inal, K. Utsumi, M. Hiramatsu, A.
Mori, and L. Packer, 1998. Mitochondrial dysfunction in the
senescence accelerated mouse (SAM). Free Rad. Biol. Med.
24:85–92.
Nakamura, K., K. Noguchi, T. Aoyama, T. Nakajima, and N.
Tanimura, 1996. Protective effect of ubiquinone (coenzyme
Q9) on ascites in broiler chickens. Br. Poult. Sci. 37:189–195.
Olafsdottir, K., and D. J. Reed, 1988. Retention of oxidized glutathione by isolated rat liver mitochondria during hydroperoxide treatment. Biochim. Biophys. Acta 964:377–382.
Peacock, A. J., C. Pickett, K. Morris, and J. T. Reeves, 1990.
Spontaneous hypoxaemia and right ventricular hypertrophy
484
CAWTHON ET AL.
in fast growing broiler chickens reared at sea level. Comp.
Biochem. Physiol. 97A:537–541.
SAS Institute, 1996. SAS User’s Guide. Version 6.12 edition.
SAS Institute Inc., Cary, NC.
Shigenaga, M. K., T. M. Hagen, and B. N. Ames, 1994. Oxidative
damage and mitochondrial decay in aging. Proc. Natl. Acad.
Sci. USA 91:10771–10778.
Turrens, J. F., and A. Boveris, 1980. Generation of superoxide
anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem. J. 191:421–427.
Warren, D. L., and D. J. Reed, 1991. Modification of hepatic
vitamin E stores in vivo: Alterations in plasma and liver
vitamin E content by methyl ethyl ketone peroxide. Arch.
Biochem. Biophys. 285:45–52.
Wideman, R. F. Jr., and W. Bottje, 1993. Current understanding
of the ascites syndrome and future directions. Pages 2–20 in:
Proceedings of the Nutritional and Technical Symposium.
Novus International, St. Louis MO.
Wideman, R. F., Jr., and H. French, 1999. Broiler breeder survivors of chronic unilateral pulmonary artery occlusion produce progeny resistant to pulmonary hypertension syn-
drome (ascites) induced by cool temperatures. Poultry Sci.
78:404–411.
Wideman, R. F., Jr., and H. French, 2000. Ongoing improvement
in the ascites resistance of progeny from the second generation of broiler breeders selected using the chronic unilateral
pulmonary artery occlusion technique. Poultry Sci. 79:396–
401.
Wideman, R. F., Jr., and Y. K. Kirby, 1995a. A pulmonary artery
clamp model for inducing pulmonary hypertension syndrome (ascites) in broilers. Poultry Sci. 74:805–812.
Wideman, R. F., Jr., and Y. K. Kirby, 1995b. Evidence of a ventilation-perfusion mismatch during acute unilateral pulmonary
artery occlusion in broilers. Poultry Sci. 74:1209–1217.
Wideman, R. F. Jr., Y. K. Kirby, M. Ismail, W. G. Bottje, R. W.
Moore, and R. C. Vardeman, 1995. Supplemental l-arginine
attenuates pulmonary hypertension syndrome (ascites) in
broilers. Poultry Sci. 74:323–330.
Wideman, R. F., Jr., Y. K. Kirby, R. L. Owen, and H. French, 1997.
Chronic unilateral occlusion of an extrapulmonary primary
bronchus induces pulmonary hypertension syndrome (ascites) in male and female broilers. Poultry Sci. 76:400–404.
Yu, B. P., 1994. Cellular defenses against damage from reactive
oxygen species. Physiol. Rev. 74:139–162.