**1. Introduction**

Reduced oxygen availability to the tissues (hypoxia) poses numerous challenges to animal life. Hypoxia occurs as a result of diminished partial pressure of oxygen, such as occurs with increasing altitude, or reduced oxygen percentage in the air capillaries of the lung. The oxygen partial pressure drops by approximately 7 mm Hg, i.e, approximately 2.5% in the case of atmospheric oxygen, for each 1,000 m increase in altitude, and thereby reduces the amount of oxygen available to the hemoglobin in red blood cells as blood passes through the lung.

The hypoxia tolerance of birds has been suggested to be greater than that of mammals. Early studies found that lowland house sparrows (*Passer domesticus*) in a wind tunnel at a simulated altitude of 6100 m behaved normally and flew for short periods [1]. Such findings support the anatomical and physiological evidence that the O2 transport pathway of birds has several unique characteristics that help support energetic activity and aerobic metabolism during hypoxia.

The O2 cascade from inspired air to the tissue mitochondria includes several convective and diffusive steps at which physiological adjustments can preserve the rate of O2 flux in spite of hypoxia, thereby ensuring an uninterrupted supply of O2 to the energy-producing machinery of the cells [2]. These steps include ventilatory convection, diffusion across the blood–gas interface, circulatory convection, diffusion across the blood–tissue interface (including myoglobin-facilitated diffusion), and O2 utilization by the tissue mitochondria.

Breathing (ventilation) is stimulated when a decline in arterial PO2 is sensed by chemoreceptors in the carotid bodies. However, this hypoxic ventilatory response increases respiratory CO2 loss, causing a secondary hypocapnia (low partial pressure of CO2 in the

blood) and alkalosis (high pH) in the blood [3]. Hypocapnia reflexively inhibits breathing and causes an acid–base disturbance. It has been suggested that birds have a higher tolerance of hypocapnia than mammals [4], possibly because of an ability to rapidly restore blood pH in the face of CO2 challenges [5]. The significance of this tolerance is that it would enable birds to ventilate more deeply before depletion of CO2 in the blood impairs normal function, and thereby to enhance O2 transport to the gas-exchange surface. It seems that every step in the O2 transport pathway can be influential, and that the relative benefit of each step changes with the level of O2 availability.

Ascites Syndrome in Broiler Chickens – A Physiological Syndrome Affected by Red Blood Cells 245

increased transit time may not allow the red blood cells to pick up a full load of O2, so that

Hypoxia/hypoxemia directly stimulates the endothelial and smooth muscle cells in pulmonary blood vessels, causing vasoconstriction throughout the lungs and an increase in pulmonary blood pressure that can persist for a long time at high altitude [33,34]. This global vasoconstriction impairs O2 diffusion because it can divert blood flow away from the gas-exchange surface to pulmonary shunt vessels [35], and the resultant pulmonary hypertension can cause fluid leakage into the air spaces, which, in turn, causes a thickening of the O2 diffusion barrier [36,37]. Hypoxic pulmonary hypertension can also overburden the right ventricle of the heart and can contribute to pathophysiological conditions, such as

The commercial broiler of today represents the culmination of dramatic changes over the past 60 years. These changes were caused by genetic selection processes that focused mainly on production traits [39,40]; it has been reported that 85-90% of the changes in commercial broilers were directly related to genetic aspects [39-42]. Commercial broilers of 1991 were compared with the Athens-Canadian Random Bred Control Population, which represents the commercial broilers of 1957 [39,40]. Average daily weight gain of the 1957 and 1991 broilers were 10 and 31 g/d, respectively, from hatch to 3 weeks of age, and 19 and 68 g/d, respectively, from 3 to 6 weeks. The higher growth rate (GR) is driven by a higher feed intake per unit time and higher metabolic rate and, consequently, a higher demand for O2, from the embryonic stage onward [43-45]**.** However, it appears that the increase in growth rate occurred without concomitant development in the efficiency of the cardiovascular and

Thus, the increase in metabolic rate, coupled with exposure to environmental conditions such as temperature, lighting and ventilation, and nutritional factors such as feed form or content, all seem to promote the development of ascites [47]. The primary cause of the ascites syndrome, however, is believed to be hypoxia/hypoxemia [48,49], when the bird's demand for O2 exceeds its cardiopulmonary capacity and causes pulmonary hypertension

The etiology of the syndrome was well documented previously [52,54,55], and is characterized phenotypically by increased pulmonary hypertension, right-ventricle hypertrophy, fluid accumulation in the pericardium and abdominal cavity, increased hematocrit that results from increased red blood cell production (erythropoiesis), and a

An international survey in commercial broiler flocks showed that AS affected 4.7% of broilers worldwide [58]. Likewise, it was found that over 25% of overall broiler loss in the United Kingdom was a result of AS [59]. It is, therefore, apparent that this syndrome is a serious economic concern in the broiler industry. As the syndrome appears mainly at ages greater than 4 weeks, even 1% of mortality from AS causes significant economic losses,

[50], which results in development of the ascites syndrome (AS) [51-53].

decline in arterial blood O2 saturation [41,52,56,57].

hemoglobin O2 saturation is not complete, which causes hypoxemia [32].

chronic mountain sickness or ascites in broilers [9,38].

*Ascites in fast-growing broilers:* 

the respiratory systems [41,46].

The acclimatization response to hypoxia generally involves increases in hematocrit (Hct) and in hemoglobin (Hb) concentration, but this adaptive erythropoietic response is complicated [6-9]. It is reasonable to expect that an increased Hct could confer a physiological advantage under hypoxia, by enhancing O2-carrying capacity, but experimental results do not support this [10,11]. A moderately increased Hct enhances arterial O2 content and therefore increases aerobic capacity [12-14], but the highest attainable Hct is not necessarily associated with the highest possible aerobic power output [15,16]. This is because the associated increase in blood viscosity increases the peripheral vascular resistance, and this might compromise cardiac output (Q), thereby reducing the O2 consumption rate (*V*O2) [17,18].

Another mechanism that can sustain/enhance O2 transport under hypoxia is alteration in the O2-binding properties of Hb in the blood. These alterations could be mediated by changes in the intrinsic Hb–O2 affinity, changes in the sensitivity of Hb to allosteric cofactors that modulate Hb–O2 affinity, and/or changes in the concentration of allosteric cofactors within the erythrocytes [19-22].

Numerous high-altitude birds, such as the bar-headed goose, the Andean goose [23], and the Tibetan chicken (*Gallus gallus*) [24], possess Hb with an increased O2 affinity. This can dramatically increase O2 delivery and pulmonary O2 loading in hypoxia by increasing the saturation of Hb and, consequently, the O2 content of the blood at a given O2 partial pressure. Thus it can greatly improve the O2 transport pathway [25].

Contrary to the hematological changes that are typically associated with the acclimatization response to hypoxia, genetically based changes in Hb structure that increase intrinsic O2 affinity or that suppress sensitivity to allosteric cofactors are more important to hypoxia tolerance in naturally high-altitude birds [21,22,26], because in lowland birds an increased Hb–O2 affinity may hinder O2 unloading in the tissue capillaries.

Although these distinctive characteristics of birds should enhance hypoxia tolerance by improving the overall capacity for O2 transport, being avian is not in itself sufficient for coping with hypoxia. Domesticated meat-type chickens (broilers) exhibit high O2 requirements because of their very fast growth and, consequently, they may have a reduced blood O2 level, i.e., hypoxemia [27-31] resulting from vigorous digestion and metabolism which have high O2 requirements. When O2 demand increases, heart rate and cardiac output increase, thereby increasing the flow of blood through the lung and the pressure required to force blood through the arterioles and capillaries of the lung. The increased flow rate and increased transit time may not allow the red blood cells to pick up a full load of O2, so that hemoglobin O2 saturation is not complete, which causes hypoxemia [32].

Hypoxia/hypoxemia directly stimulates the endothelial and smooth muscle cells in pulmonary blood vessels, causing vasoconstriction throughout the lungs and an increase in pulmonary blood pressure that can persist for a long time at high altitude [33,34]. This global vasoconstriction impairs O2 diffusion because it can divert blood flow away from the gas-exchange surface to pulmonary shunt vessels [35], and the resultant pulmonary hypertension can cause fluid leakage into the air spaces, which, in turn, causes a thickening of the O2 diffusion barrier [36,37]. Hypoxic pulmonary hypertension can also overburden the right ventricle of the heart and can contribute to pathophysiological conditions, such as chronic mountain sickness or ascites in broilers [9,38].

#### *Ascites in fast-growing broilers:*

244 Blood Cell – An Overview of Studies in Hematology

consumption rate (*V*O2) [17,18].

the erythrocytes [19-22].

each step changes with the level of O2 availability.

blood) and alkalosis (high pH) in the blood [3]. Hypocapnia reflexively inhibits breathing and causes an acid–base disturbance. It has been suggested that birds have a higher tolerance of hypocapnia than mammals [4], possibly because of an ability to rapidly restore blood pH in the face of CO2 challenges [5]. The significance of this tolerance is that it would enable birds to ventilate more deeply before depletion of CO2 in the blood impairs normal function, and thereby to enhance O2 transport to the gas-exchange surface. It seems that every step in the O2 transport pathway can be influential, and that the relative benefit of

The acclimatization response to hypoxia generally involves increases in hematocrit (Hct) and in hemoglobin (Hb) concentration, but this adaptive erythropoietic response is complicated [6-9]. It is reasonable to expect that an increased Hct could confer a physiological advantage under hypoxia, by enhancing O2-carrying capacity, but experimental results do not support this [10,11]. A moderately increased Hct enhances arterial O2 content and therefore increases aerobic capacity [12-14], but the highest attainable Hct is not necessarily associated with the highest possible aerobic power output [15,16]. This is because the associated increase in blood viscosity increases the peripheral vascular resistance, and this might compromise cardiac output (Q), thereby reducing the O2

Another mechanism that can sustain/enhance O2 transport under hypoxia is alteration in the O2-binding properties of Hb in the blood. These alterations could be mediated by changes in the intrinsic Hb–O2 affinity, changes in the sensitivity of Hb to allosteric cofactors that modulate Hb–O2 affinity, and/or changes in the concentration of allosteric cofactors within

Numerous high-altitude birds, such as the bar-headed goose, the Andean goose [23], and the Tibetan chicken (*Gallus gallus*) [24], possess Hb with an increased O2 affinity. This can dramatically increase O2 delivery and pulmonary O2 loading in hypoxia by increasing the saturation of Hb and, consequently, the O2 content of the blood at a given O2 partial

Contrary to the hematological changes that are typically associated with the acclimatization response to hypoxia, genetically based changes in Hb structure that increase intrinsic O2 affinity or that suppress sensitivity to allosteric cofactors are more important to hypoxia tolerance in naturally high-altitude birds [21,22,26], because in lowland birds an increased

Although these distinctive characteristics of birds should enhance hypoxia tolerance by improving the overall capacity for O2 transport, being avian is not in itself sufficient for coping with hypoxia. Domesticated meat-type chickens (broilers) exhibit high O2 requirements because of their very fast growth and, consequently, they may have a reduced blood O2 level, i.e., hypoxemia [27-31] resulting from vigorous digestion and metabolism which have high O2 requirements. When O2 demand increases, heart rate and cardiac output increase, thereby increasing the flow of blood through the lung and the pressure required to force blood through the arterioles and capillaries of the lung. The increased flow rate and

pressure. Thus it can greatly improve the O2 transport pathway [25].

Hb–O2 affinity may hinder O2 unloading in the tissue capillaries.

The commercial broiler of today represents the culmination of dramatic changes over the past 60 years. These changes were caused by genetic selection processes that focused mainly on production traits [39,40]; it has been reported that 85-90% of the changes in commercial broilers were directly related to genetic aspects [39-42]. Commercial broilers of 1991 were compared with the Athens-Canadian Random Bred Control Population, which represents the commercial broilers of 1957 [39,40]. Average daily weight gain of the 1957 and 1991 broilers were 10 and 31 g/d, respectively, from hatch to 3 weeks of age, and 19 and 68 g/d, respectively, from 3 to 6 weeks. The higher growth rate (GR) is driven by a higher feed intake per unit time and higher metabolic rate and, consequently, a higher demand for O2, from the embryonic stage onward [43-45]**.** However, it appears that the increase in growth rate occurred without concomitant development in the efficiency of the cardiovascular and the respiratory systems [41,46].

Thus, the increase in metabolic rate, coupled with exposure to environmental conditions such as temperature, lighting and ventilation, and nutritional factors such as feed form or content, all seem to promote the development of ascites [47]. The primary cause of the ascites syndrome, however, is believed to be hypoxia/hypoxemia [48,49], when the bird's demand for O2 exceeds its cardiopulmonary capacity and causes pulmonary hypertension [50], which results in development of the ascites syndrome (AS) [51-53].

The etiology of the syndrome was well documented previously [52,54,55], and is characterized phenotypically by increased pulmonary hypertension, right-ventricle hypertrophy, fluid accumulation in the pericardium and abdominal cavity, increased hematocrit that results from increased red blood cell production (erythropoiesis), and a decline in arterial blood O2 saturation [41,52,56,57].

An international survey in commercial broiler flocks showed that AS affected 4.7% of broilers worldwide [58]. Likewise, it was found that over 25% of overall broiler loss in the United Kingdom was a result of AS [59]. It is, therefore, apparent that this syndrome is a serious economic concern in the broiler industry. As the syndrome appears mainly at ages greater than 4 weeks, even 1% of mortality from AS causes significant economic losses,

because it occurs toward the end of the growing period [58] and, therefore, affects heavy birds which have absorbed a considerable investment of labor and feed [60,61]. Two management approaches have been applied in order to minimize the actual AS mortality in commercial flocks: (1) increasing the broiler house temperature by means of heating and insulation, which are costly; and (2) reducing the actual growth rate and, therefore, the metabolic rate and demand for oxygen, by providing fewer hours of light so as to reduce the quantity of feed consumed, and using low-energy mash feeds to reduce intake of dietary energy [47,62]. Thus, while the genetic potential for rapid growth of commercial broilers has been continuously improved by breeding companies [41], its full expression is not allowed at the farm level, specifically to avoid morbidity and mortality of AS-susceptible birds. Consequently production costs are increased because of the longer period of rearing to marketing body weight.

Ascites Syndrome in Broiler Chickens – A Physiological Syndrome Affected by Red Blood Cells 247

1986 broiler line 2002 broiler line 1986 broiler line 2002 broiler line

relatively high incidence of AS in the slow-growing line, indicated that there is very little, if any, direct genetic association between AS and genetic differences in potential GR, which suggests that AS-resistant broilers can be selected for higher GR and remain healthy, even

2002 experiment 2006 experiment

Mortality n1 (*N*= 91) % n1 (*N* = 42) % n1 (*N* = 78) % n1 (*N* = 97) % 28 0 0.0 1 2.4 0 0 a 2 2.1 a 35 5 5.5 2 4.8 3 3.8 b 10 10.3 a 42 10 11.0b 9 21.4a 12 15.4 b 26 26.8 a 542 22 24.2 -- -- 15 19.2 -- --

Morbidity3 42 or 54 7 7.6 4 9.5 11 14.1 20 20.6

Total AS incidence 29 31.8 13 31.0 26 33.3 46 47.4 a, b Mortality or morbidity percentages per line within rows (ages), within experimental year, without common

3 Birds that survived to the end of trial (Day 54 for the 1986 broiler line; Day 42 for the 2002 and 2006 broiler lines) but

These results, supported by several previous studies [68,70-78], suggest that there is no "true" genetic correlation between the potential GR of broilers and their propensity to develop AS. It seems that AS is not caused by the increased O2 requirement of a fast growth

Thus, a better solution would be to select against AS susceptibility, because if all broilers were resistant to AS, management-induced reduction of growth rate would no longer be needed. Breeding against AS susceptibility should aim at identifying and eliminating all the AS-susceptible individuals in the selected population and selecting for high GR among the

The questions raised by the last hypothesis concern what might cause broilers to be susceptible to ascites, and whether it is related to physiological disorders of the

This chapter will introduce readers to the physiological Ascites Syndrome and the complexity of the problems that highly productive broiler chickens face in coping with high-oxygen-demand conditions such as cold stress and high altitude. It will focus on: a. the ascites syndrome – its causes and etiology in broiler chickens; b. cardiovascular functioning and responsiveness in

ascitic broilers; and c. genetic and physiological aspects of coping with the syndrome.

**Table 1.** Cumulative mortality and morbidity due to the ascites syndrome (AS) at various ages in broiler lines of the years 1986, 2002 and 2006, all reared together under high-challenge ascites-inducing

rate, but by an impairment of the O2 supply needed to sustain the fast growth rate.

under AICs

Age (d)

Cumulative

test, *P* < 0.05)

2 The birds from the 1986 broiler line were kept under AIC through 54 d of age.

conditions (AICs) from Day 19 to end of trial (According to [71]).

1 n = number of birds with AS; *N* = total number of birds in the line.

superscript differ significantly (<sup>2</sup>

were diagnosed with AS.

AS-resistant ones.

cardiovascular system.

There are two alternative hypotheses regarding the association between GR of contemporary broilers and their susceptibility or resistance to AS. Many studies showed that AS does not develop in slow-growing chickens, egg-type Leghorns [see, e.g., 63,64], or slow-growing broilers [see, e.g., 65,66]. It has been suggested that high GR is the direct cause of AS, because of the consequent high demand for oxygen by tissues and organs of these birds. According to this hypothesis, alleles or genotypes that increase GR of broilers also increase their tendency to develop AS. Such a situation should be manifested in a symmetrical genetic correlation between GR and AS: genetic differences in GR – whether between lines or families, or between individuals within lines – should be associated with corresponding differences in %AS. Symmetrically, individuals that develop AS, or families with higher %AS, should have a genetic potential for a higher GR than their counterparts that remain healthy under the same rearing conditions.

The second hypothesis asserts that broilers do not have to be the fastest growing birds in a flock in order to develop AS, but simply need to have their weight-gain rate exceed the growth rate of their pulmonary vascular capacity [67-71]. According to this hypothesis, there should be high-GR broilers that do not develop AS despite their high O2 demand, because they are genetically resistant. Similarly, there should be broilers with genetically low GR that, nevertheless, are susceptible to AS, although they require special environmental conditions to express this susceptibility.

The hypotheses regarding an inherent association between AS and the genetic potential for high GR were tested by examining contemporary commercial broilers in 2002 and 2006, and an experimental low-GR slow-growing line [71]. All the lines were tested under the same experimental protocol, that allowed measurement of GR under standard brooding conditions (SBCs) up to d 19, and then efficiently distinguished between AS-susceptible and AS-resistant individuals, the latter being those that remained healthy under the same highchallenge, ascites-inducing conditions (AICs) – conditions based on exposure to low ambient temperatures while receiving different forms of diet [72]. Ascites syndrome incidence was 31 and 47% in the 2002 and 2006 birds respectively, and 32% in the 1986 slowgrowing line (Table 1). Most broilers that remained healthy under the high-challenge AICs exhibited the same early GR and BW as those that later developed AS. These results, and the relatively high incidence of AS in the slow-growing line, indicated that there is very little, if any, direct genetic association between AS and genetic differences in potential GR, which suggests that AS-resistant broilers can be selected for higher GR and remain healthy, even under AICs


a, b Mortality or morbidity percentages per line within rows (ages), within experimental year, without common superscript differ significantly (<sup>2</sup> test, *P* < 0.05)

1 n = number of birds with AS; *N* = total number of birds in the line.

246 Blood Cell – An Overview of Studies in Hematology

marketing body weight.

that remain healthy under the same rearing conditions.

conditions to express this susceptibility.

because it occurs toward the end of the growing period [58] and, therefore, affects heavy birds which have absorbed a considerable investment of labor and feed [60,61]. Two management approaches have been applied in order to minimize the actual AS mortality in commercial flocks: (1) increasing the broiler house temperature by means of heating and insulation, which are costly; and (2) reducing the actual growth rate and, therefore, the metabolic rate and demand for oxygen, by providing fewer hours of light so as to reduce the quantity of feed consumed, and using low-energy mash feeds to reduce intake of dietary energy [47,62]. Thus, while the genetic potential for rapid growth of commercial broilers has been continuously improved by breeding companies [41], its full expression is not allowed at the farm level, specifically to avoid morbidity and mortality of AS-susceptible birds. Consequently production costs are increased because of the longer period of rearing to

There are two alternative hypotheses regarding the association between GR of contemporary broilers and their susceptibility or resistance to AS. Many studies showed that AS does not develop in slow-growing chickens, egg-type Leghorns [see, e.g., 63,64], or slow-growing broilers [see, e.g., 65,66]. It has been suggested that high GR is the direct cause of AS, because of the consequent high demand for oxygen by tissues and organs of these birds. According to this hypothesis, alleles or genotypes that increase GR of broilers also increase their tendency to develop AS. Such a situation should be manifested in a symmetrical genetic correlation between GR and AS: genetic differences in GR – whether between lines or families, or between individuals within lines – should be associated with corresponding differences in %AS. Symmetrically, individuals that develop AS, or families with higher %AS, should have a genetic potential for a higher GR than their counterparts

The second hypothesis asserts that broilers do not have to be the fastest growing birds in a flock in order to develop AS, but simply need to have their weight-gain rate exceed the growth rate of their pulmonary vascular capacity [67-71]. According to this hypothesis, there should be high-GR broilers that do not develop AS despite their high O2 demand, because they are genetically resistant. Similarly, there should be broilers with genetically low GR that, nevertheless, are susceptible to AS, although they require special environmental

The hypotheses regarding an inherent association between AS and the genetic potential for high GR were tested by examining contemporary commercial broilers in 2002 and 2006, and an experimental low-GR slow-growing line [71]. All the lines were tested under the same experimental protocol, that allowed measurement of GR under standard brooding conditions (SBCs) up to d 19, and then efficiently distinguished between AS-susceptible and AS-resistant individuals, the latter being those that remained healthy under the same highchallenge, ascites-inducing conditions (AICs) – conditions based on exposure to low ambient temperatures while receiving different forms of diet [72]. Ascites syndrome incidence was 31 and 47% in the 2002 and 2006 birds respectively, and 32% in the 1986 slowgrowing line (Table 1). Most broilers that remained healthy under the high-challenge AICs exhibited the same early GR and BW as those that later developed AS. These results, and the 2 The birds from the 1986 broiler line were kept under AIC through 54 d of age.

3 Birds that survived to the end of trial (Day 54 for the 1986 broiler line; Day 42 for the 2002 and 2006 broiler lines) but were diagnosed with AS.

**Table 1.** Cumulative mortality and morbidity due to the ascites syndrome (AS) at various ages in broiler lines of the years 1986, 2002 and 2006, all reared together under high-challenge ascites-inducing conditions (AICs) from Day 19 to end of trial (According to [71]).

These results, supported by several previous studies [68,70-78], suggest that there is no "true" genetic correlation between the potential GR of broilers and their propensity to develop AS. It seems that AS is not caused by the increased O2 requirement of a fast growth rate, but by an impairment of the O2 supply needed to sustain the fast growth rate.

Thus, a better solution would be to select against AS susceptibility, because if all broilers were resistant to AS, management-induced reduction of growth rate would no longer be needed. Breeding against AS susceptibility should aim at identifying and eliminating all the AS-susceptible individuals in the selected population and selecting for high GR among the AS-resistant ones.

The questions raised by the last hypothesis concern what might cause broilers to be susceptible to ascites, and whether it is related to physiological disorders of the cardiovascular system.

This chapter will introduce readers to the physiological Ascites Syndrome and the complexity of the problems that highly productive broiler chickens face in coping with high-oxygen-demand conditions such as cold stress and high altitude. It will focus on: a. the ascites syndrome – its causes and etiology in broiler chickens; b. cardiovascular functioning and responsiveness in ascitic broilers; and c. genetic and physiological aspects of coping with the syndrome.
