**2. The Ascites syndrome: Its causes and etiology in broiler chickens**

#### *Ascites physiology and etiology:*

The AS involves accumulation of fluids in the abdominal cavity [79], which prompted the common name of "water belly" to describe the syndrome; it occurs when broilers fail to supply sufficient oxygen to support their metabolic demands [80]. In the late 1970s AS was observed only at high altitudes [81], but since then it has been found also at low altitudes [82], mainly in broilers reared at low ambient temperatures and/or fed pelleted feed with high energy content.

Ascites Syndrome in Broiler Chickens – A Physiological Syndrome Affected by Red Blood Cells 249

growing broilers [30,87]. These results suggested that some meat-type chickens were not fully oxygenating their hemoglobin, even at low altitude. This might have been the result of increased blood flow rate through the lung capillary bed, which would reduce the time available for hemoglobin to be oxygenated in the lung interface [32,88], or to presence of immature red blood cells in the system [89]. In order to overcome this situation an increase in erythropoiesis takes place. However, such an increase, if not coupled to plasma volume expansion would increase blood viscosity, followed by increased bloodflow resistance [90]. The back pressure in the veins causes venous congestion, dilation and prominent vessels [50]. The lack of O2 in the heart muscle results in hypoxic damage and, finally, right-ventricular hypertrophy. As cardiac output is reduced and tissue hypoxia becomes worse, the left ventricle loses muscle mass, the wall thins (because of hypoxia and disuse atrophy), the valves thicken and the chamber enlarges. Heart muscle damage is caused by the excess workload and by the tissue hypoxia associated with circulatory failure, not by the tissue hypoxia that increases cardiac output and triggers pulmonary

**Altitude**: The partial pressure of oxygen becomes lower with increasing altitude. The ability of chickens to oxygenate their hemoglobin fully as the erythrocytes pass through the lung depends on the transit time in the lung, hemoglobin- O2 affinity, the thickness of the air– hemoglobin barrier and, especially, the partial pressure of O2 in the air [62]. The effects of high altitude or hypoxia on ascites and heart disorders in broilers were reported as early as the 1950s and 1960s [91-97]. Those reports indicated that birds raised at high altitudes died because of right ventricular hypertrophy, congested and edematous lungs, and accumulation of fluid in the abdominal cavity. Significant microscopic damage to the heart, lungs and kidneys was also found in birds reared at high altitude [95,97], as well as in 1 week-old broilers raised at high altitude [98] and in birds exposed to simulated high altitude

Because AS was first noticed in birds raised at high altitude, the use of natural or simulated high-altitude conditions was one of the first experimental protocols to be used [see, e.g., 47,97]. The hypobaric chamber has been shown to be an effective tool for simulating high altitude and consistently inducing AS [102-106]; it simulates high altitude conditions by generating a partial vacuum, thereby reducing the partial pressure of O2. Anthony and Balog [106], by simulating an altitude of 2,900 m above sea level, successfully induced 66% AS in a commercial sire line. In six lines of commercial broilers that were reared in the same

When birds are exposed to low atmospheric O2 levels pulmonary blood vessels constrict and pulmonary vascular resistance increases [108]. This immediate increase in pulmonary arterial pressure can, over time, cause right ventricular hypertrophy and eventually result in the ascites syndrome [81,89,109-111]. Additionally, hypoxemia leads to an increase in hematocrit, which, in turn, increases blood viscosity and results in increased resistance to

hypertension.

[99-101].

*Environmental causes of ascites syndrome:* 

hypobaric chamber, 47% of the birds developed AS [107].

blood flow through the pulmonary blood vessels [90,112-116].

The general pathogenesis of AS has been well documented [52,54,83,84]. Rapid growth requires a high resting metabolic rate, which requires adequate O2 supply and utilization. The broiler chicken probably has more genetic potential for growth than it has potential to provide O2 to sustain that growth, and in some broilers the demand for O2 might exceed the cardiopulmonary capacity to supply sufficient O2, ultimately leading to an O2 deficit [85]. The heart responds by increasing its output of (deoxygenated) blood to the lungs for oxygenation. This increased blood flow causes an increase in the blood pressure required to push the blood through the capillaries in the lung which, in turn, causes pulmonary hypertension. This increase in work load results in an enhanced pressure load on the right ventricular muscle wall, to which the muscle cells respond by adding parallel sarcomeres, causing thickening (hypertrophy) of the right ventricular wall. The muscular right ventricular wall increases the pressure in the pulmonary arteries, arterioles and capillaries of the lung. This process continues, causing additional hypertrophy. Meanwhile, the right atrio-ventricular valve thickens and starts to leak, partly because the thicker valve is now less effective and partly because of the increasing back pressure from the pulmonary arteries and right ventricular chamber [86]. The leaking valve aggravates the excess pressure problem by admitting excess volume, the right ventricle dilates, and the wall-muscle cells lengthen by producing longitudinally arranged sarcomeres.

The increased blood volume raises the pressure overload until valve deficiency occurs, causing a drop in cardiac output and pulmonary hypertension, but marked pressure increases in the right atrium, sinus venosus, vena cava and portal vein. This increased pressure in the sinusoids of the liver causes leakage of plasma from the liver into the hepatoperitoneal spaces, i.e., ascites. The leaking valve and increased venous pressure result in hypoxemia and tissue hypoxia, and the kidney responds by producing erythropoietin in an attempt to increase the blood's O2 carrying capacity by intensively producing more red blood cells.

Domestication had introduced several other insufficiencies into the cardiovascular system; among them is a thicker respiratory membrane than that in other birds, i.e., broilers have a thicker respiratory membrane than Leghorn-type laying fowl. This leads to: a. lower efficiency of O2 transfer through the respiratory membrane; and b. lower hemoglobin oxygenation capability [62]. Research focusing on hemoglobin O2 saturationin meat-type chickens indicated that fast-growing broilers have lower saturation than slowgrowing broilers [30,87]. These results suggested that some meat-type chickens were not fully oxygenating their hemoglobin, even at low altitude. This might have been the result of increased blood flow rate through the lung capillary bed, which would reduce the time available for hemoglobin to be oxygenated in the lung interface [32,88], or to presence of immature red blood cells in the system [89]. In order to overcome this situation an increase in erythropoiesis takes place. However, such an increase, if not coupled to plasma volume expansion would increase blood viscosity, followed by increased bloodflow resistance [90]. The back pressure in the veins causes venous congestion, dilation and prominent vessels [50]. The lack of O2 in the heart muscle results in hypoxic damage and, finally, right-ventricular hypertrophy. As cardiac output is reduced and tissue hypoxia becomes worse, the left ventricle loses muscle mass, the wall thins (because of hypoxia and disuse atrophy), the valves thicken and the chamber enlarges. Heart muscle damage is caused by the excess workload and by the tissue hypoxia associated with circulatory failure, not by the tissue hypoxia that increases cardiac output and triggers pulmonary hypertension.

#### *Environmental causes of ascites syndrome:*

248 Blood Cell – An Overview of Studies in Hematology

*Ascites physiology and etiology:* 

high energy content.

blood cells.

**2. The Ascites syndrome: Its causes and etiology in broiler chickens** 

The AS involves accumulation of fluids in the abdominal cavity [79], which prompted the common name of "water belly" to describe the syndrome; it occurs when broilers fail to supply sufficient oxygen to support their metabolic demands [80]. In the late 1970s AS was observed only at high altitudes [81], but since then it has been found also at low altitudes [82], mainly in broilers reared at low ambient temperatures and/or fed pelleted feed with

The general pathogenesis of AS has been well documented [52,54,83,84]. Rapid growth requires a high resting metabolic rate, which requires adequate O2 supply and utilization. The broiler chicken probably has more genetic potential for growth than it has potential to provide O2 to sustain that growth, and in some broilers the demand for O2 might exceed the cardiopulmonary capacity to supply sufficient O2, ultimately leading to an O2 deficit [85]. The heart responds by increasing its output of (deoxygenated) blood to the lungs for oxygenation. This increased blood flow causes an increase in the blood pressure required to push the blood through the capillaries in the lung which, in turn, causes pulmonary hypertension. This increase in work load results in an enhanced pressure load on the right ventricular muscle wall, to which the muscle cells respond by adding parallel sarcomeres, causing thickening (hypertrophy) of the right ventricular wall. The muscular right ventricular wall increases the pressure in the pulmonary arteries, arterioles and capillaries of the lung. This process continues, causing additional hypertrophy. Meanwhile, the right atrio-ventricular valve thickens and starts to leak, partly because the thicker valve is now less effective and partly because of the increasing back pressure from the pulmonary arteries and right ventricular chamber [86]. The leaking valve aggravates the excess pressure problem by admitting excess volume, the right ventricle dilates, and the wall-muscle cells

The increased blood volume raises the pressure overload until valve deficiency occurs, causing a drop in cardiac output and pulmonary hypertension, but marked pressure increases in the right atrium, sinus venosus, vena cava and portal vein. This increased pressure in the sinusoids of the liver causes leakage of plasma from the liver into the hepatoperitoneal spaces, i.e., ascites. The leaking valve and increased venous pressure result in hypoxemia and tissue hypoxia, and the kidney responds by producing erythropoietin in an attempt to increase the blood's O2 carrying capacity by intensively producing more red

Domestication had introduced several other insufficiencies into the cardiovascular system; among them is a thicker respiratory membrane than that in other birds, i.e., broilers have a thicker respiratory membrane than Leghorn-type laying fowl. This leads to: a. lower efficiency of O2 transfer through the respiratory membrane; and b. lower hemoglobin oxygenation capability [62]. Research focusing on hemoglobin O2 saturationin meat-type chickens indicated that fast-growing broilers have lower saturation than slow-

lengthen by producing longitudinally arranged sarcomeres.

**Altitude**: The partial pressure of oxygen becomes lower with increasing altitude. The ability of chickens to oxygenate their hemoglobin fully as the erythrocytes pass through the lung depends on the transit time in the lung, hemoglobin- O2 affinity, the thickness of the air– hemoglobin barrier and, especially, the partial pressure of O2 in the air [62]. The effects of high altitude or hypoxia on ascites and heart disorders in broilers were reported as early as the 1950s and 1960s [91-97]. Those reports indicated that birds raised at high altitudes died because of right ventricular hypertrophy, congested and edematous lungs, and accumulation of fluid in the abdominal cavity. Significant microscopic damage to the heart, lungs and kidneys was also found in birds reared at high altitude [95,97], as well as in 1 week-old broilers raised at high altitude [98] and in birds exposed to simulated high altitude [99-101].

Because AS was first noticed in birds raised at high altitude, the use of natural or simulated high-altitude conditions was one of the first experimental protocols to be used [see, e.g., 47,97]. The hypobaric chamber has been shown to be an effective tool for simulating high altitude and consistently inducing AS [102-106]; it simulates high altitude conditions by generating a partial vacuum, thereby reducing the partial pressure of O2. Anthony and Balog [106], by simulating an altitude of 2,900 m above sea level, successfully induced 66% AS in a commercial sire line. In six lines of commercial broilers that were reared in the same hypobaric chamber, 47% of the birds developed AS [107].

When birds are exposed to low atmospheric O2 levels pulmonary blood vessels constrict and pulmonary vascular resistance increases [108]. This immediate increase in pulmonary arterial pressure can, over time, cause right ventricular hypertrophy and eventually result in the ascites syndrome [81,89,109-111]. Additionally, hypoxemia leads to an increase in hematocrit, which, in turn, increases blood viscosity and results in increased resistance to blood flow through the pulmonary blood vessels [90,112-116].

**Low temperature:** Temperature is the most-studied environmental cause of ascites [see, e.g., 117-125]. In endothermic animals (mammals and birds) body temperature (Tb) is the most physiologically protected parameter of the body; therefore, the thermoregulatory system in these animals operates at a very high gain, in order to hold Tb within a relatively narrow range, despite moderate to extreme changes in environmental conditions [126]. The ability to maintain a stable Tb springs from the mechanisms that control heat production and heat loss; mechanisms that changed in the course of evolution, to enable endothermia to replace ectothermia [127,128]. Birds mostly respond to acute or chronic cold exposure by increasing their metabolic rate and oxygen requirement [129,130]. It was reported that a drop in environmental temperature from 20 to 2C almost doubled the oxygen requirement of White Leghorn hens [131], and in another study there was a 32.7% increase in oxygen requirement in response to low temperatures [132].

Ascites Syndrome in Broiler Chickens – A Physiological Syndrome Affected by Red Blood Cells 251

**3. Cardiovascular functioning and responsiveness in ascitic broilers** 

The blood system provides the main systemic response to environmental changes and metabolic demands, either through the cardiovascular system or through alteration in O2-

Reduced O2 availability in the blood (hypoxemia), reduces the O2 partial pressure (PO2) of the arterial blood (PaO2). In such a situation the blood system must maintain an adequate delivery of O2 to the peripheral tissues, while maintaining an adequate PO2 at the vascular

Oxygen delivery can be enhanced by increasing the total cardiac output (Q) and by increasing the blood O2 capacitance coefficient (βbO2). The latter parameter is defined as the ratio (CaO2 – CvO2)/(PaO2 – PvO2), where CaO2 – CvO2 is the arterial–venous difference in O2

With regard to maintaining an adequate PO2 at the vascular supply source, the lower critical PO2 can be expressed as PvO2 = PaO2 – [βbO2 × (Q/VO2)] – 1, in which VO2 is the rate of O2 consumption by the tissues and the product βbO2 × (Q/VO2) is the specific blood O2 conductance [148,149]. Because PaO2 is determined by ventilation and O2 equilibration at the blood–gas interface, this equation shows that an increase in specific blood-O2 conductance minimizes the decline in PvO2 under hypoxia, thereby maintaining an adequate pressure

Under severe hypoxia, an increased blood-O2 affinity will tend to maximize βbO2. The resultant increase in the specific blood O2 conductance helps meet challenges of both delivery and supply: it minimizes the expected PO2 decrement in the tissue capillaries while preserving a constant CaO2 – CvO2 difference. Likewise, an increased hemoglobin concentration increases CaO2, thereby increasing blood O2 conductance if PaO2, Q and VO2 all remain constant. With excessive polycythemia, however, potential advantages of an increased Hb concentration for O2-carrying capacity might be more than offset by a

Several significant alterations to the blood system in AS broilers were well documented: increased red blood cell numbers, through increased erythropoietin production [96,100,150- 153]; elevation of hematocrit values and blood viscosity [54,72,154], and central venous blood congestion [50,155]. These findings raised the question of the association between the plasma and the fluid that accumulated in the abdominal cavity, and whether the increase in hematocrit resulted from a decline in plasma volume caused by plasma leakage out of the blood vessels, or from increased erythropoiesis that occurred as a compensatory reaction to the lack of oxygen in the tissue. In ascitic broilers the composition of the abdominal cavity fluid was fairly similar to that of the plasma, with regard to osmolality, and total protein and albumin concentrations, which suggests a deficiency in the selective permeability of the blood vessels [89]. These findings resemble those in cirrhotic human patients with ascites

supply source,, in order to permit O2 diffusion to the tissue mitochondria.

concentration and PaO2 – PvO2 is the arterial–venous difference in PO2.

head for O2 diffusion to the tissue mitochondria [2].

corresponding reduction in Q.

*Blood O2 transport, erythropoiesis and ascites* 

carrying capacity.

Low temperatures were found to increase ascites by increasing both metabolic O2 requirements and pulmonary hypertension [122,133]. This increase in pulmonary arterial pressure was attributed to a cold-induced increase in cardiac output, rather than to hypoxemic pulmonary vasoconstriction [134]. As a result, low ambient temperature has been widely used to induce AS in broilers [60,66,73,115,122,134-140]. Various protocols were developed, ranging from exposure to constant low temperatures [60,73,122,135,136,140], through gradual stepping down of ambient temperature [66,122,137,139], to episodic protocols under which the birds were exposed to natural fluctuations of winter temperatures [115,138]. The efficacy of a cold-exposure protocol depends upon its timing, duration and magnitude, as well as husbandry and the birds' genetic tendency to develop AS.

The effect of the timing of a cold-stress application on ascites development in broilers indicates that exposure to low temperatures during brooding has a long-lasting effect on ascites susceptibility [62,120,125,137,141,142]. The consensus appears to be that cold stress during the first two weeks of life affects the birds' metabolic rate for several weeks, and increases their susceptibility to ascites [62,120,125,137,141,142]. A novel AIC protocol for AS [72] involved rearing the tested birds in individual cages from 19 d of age, so that they could not escape the challenge of the environmental conditions, which comprised fan-induced air movement at about 2 m/s and moderately low ambient temperatures (18 to 20°C). The effects of the environmental conditions were augmented by early use of high-energy pelleted feed to enhance rapid growth and by lighting for 23 h/d. Under this combination of conditions, %AS among the broilers was 44% – much higher than those reported for coldstressed broilers on litter, and similar to or slightly lower than that among broilers challenged by hypobaric chamber.

The birds that developed ascites as a result of exposure to low temperatures exhibited the same pathological symptoms as those that developed it under low O2 partial pressure – symptoms including increased hematocrit, hemoglobin, heart weight, and rightventricle:total-ventricle ratios [70-72,122,124,143-147].
