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

#### *Blood O2 transport, erythropoiesis and ascites*

250 Blood Cell – An Overview of Studies in Hematology

requirement in response to low temperatures [132].

AS.

challenged by hypobaric chamber.

ventricle:total-ventricle ratios [70-72,122,124,143-147].

**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

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

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

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 rightThe blood system provides the main systemic response to environmental changes and metabolic demands, either through the cardiovascular system or through alteration in O2 carrying capacity.

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

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

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

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 corresponding reduction in Q.

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

[156-158]. The escape of plasma fluid out of the blood vessels was probably due to increased pulmonary hypertension and central venous congestion – symptoms found both in humans [158] and in broilers [56]. As in the case of human ascitic patients [159], AS broilers exhibited conservation of plasma volume similar to that of the healthy ones. However, the PCV in the AS broilers increased significantly, by up to 80%, as a result of a significant increase in the number of erythrocytes, which also contributed to a significant elevation in blood volume. Thus, enhanced erythropoiesis, and not plasma volume reduction, was found to be involved in the hemodynamics of the ascitic broilers [89]. This finding could also account for the blood congestion and the increased blood viscosity [90] that contribute to the enhanced cardiac workload [103,134], blood pressure [103], and blood-flow resistance [111] in AS chickens.

Ascites Syndrome in Broiler Chickens – A Physiological Syndrome Affected by Red Blood Cells 253

Alterations in the electrocardiogram (ECG) are seen in conjunction with AS. Of most importance has been the finding that increased S-wave amplitude in standard limb lead II indicates increased susceptibility to AS [111]. However, there were no ECG readings indicative of primary pulmonary hypertension in most birds that developed ascites [165]. A slower heart rate (bradycardia) [55,116], as well as reduction in the pulse rate had been

Heart rate on days 1 and 7 was found to be significantly higher in the AS-susceptible (AS-S) genetic broiler line than in the AS-resistant (AS-R) broiler line, with only the lowest quartile of individual heart rates in the AS-S line overlapping the highest quartile in the AS-R line [57]. These results were in agreement with those of Druyan et al. [166], who found that generation S3 chicks from their AS-S line had a significantly higher heart rate on day of hatch than that of generation S3 chicks from the AS-R line. It was reported [167] that heart rate began to increase shortly after hatch, and reached a peak close to 4 wk of age; thereafter, it declined slowly [168]. The AS-S selected line exhibited increased heart rate only between d 1 and d 7, with a decline thereafter toward d 17, while the birds were kept under standard brooding condition [57]. Mild hypoxia was found to elicit an increase in heart rate [169,170], which suggests that the AS-S birds in that study experienced O2 shortage already at the time of hatch, even when kept under optimal conditions. A higher mean partial pressure of CO2 in broilers' venous blood (a marker for lung ventilation rate) on d 11 was found to be associated with increased ascites susceptibility [171,172]. Those results indicate that ASsusceptible birds suffer O2 shortage at an early age. However, it also suggests that as long as the susceptible birds are under SBC, higher heart rate can compensate for a mild hypoxemia,

Birds with ascites induced by either low ventilation or cold temperatures exhibited hypertrophy of the medial layer of arteriols, which was probably a response to primary pulmonary hypertension [173]. In low, ventilation-induced ascites, the broilers had significant inflammation or osseous-nodule formation in the lungs [174,175], whereas in cold-stress-induced ascites, birds showed no inflammation [173]. Wideman et al. [50] suggested that increases in pulmonary vascular resistance initiate increases in venous pressure by challenging the capacity of the right ventricle to thrust all the returning venous blood through the lungs. An acute reversal of systemic hypoxemia was reported to have no effect on pulmonary hypertension – a finding that discounted the influence of hypoxic pulmonary vasoconstriction [176]. It was hypothesized that this reversal of systemic hypoxemia increased total peripheral resistance and normalized arterial pressure and cardiac output, but could not decrease pulmonary hypertension because of the overwhelming influence of sustained pulmonary vascular resistance [176]. Development of techniques to measure changes in pulmonary arterial pressure and changes in wedge pressure helped to clarify that changes in pulmonary arterial pressure contribute to the mismatch between pulmonary vascular capacity and cardiac output, and that pulmonary hypertension is initiated as a consequence of excessive pulmonary arterial or arteriolar resistance [177,178]. The difference between individual broilers' susceptibility to ascites may

found in birds developing AS [55] and in acutely cold-exposed birds [116].

and no other physiological parameter would be affected.

*Effect on heart and blood vessels* 

In AS birds, the high PCV, on the one hand, and the significant decline in blood oxygen saturation, on the other hand [30,57,66] raised the possibility of an impairment of blood O2 carrying capacity. Increased erythrocyte rigidity appears to be another important factor in AS [54,62,113]: the nucleated erythrocytes will normally curl or fold to pass through lung capillaries [160], but hypoxemia and high hemoglobin concentrations decrease the deformability of erythrocytes [62]. Further calculations of hemoglobin content per 1,000 red blood cells revealed a significant reduction in the AS broilers compared with that in the healthy and control broilers [57,89]. These results suggest the possibility of inefficient enhancement of the erythropoiesis process.

Ascites-induction conditions elicited enhanced erythropoiesis, which resulted in an increased proportion of immature erythrocytes in the bloodstream. However, whereas in the healthy broilers only a moderate proportion (7.2%) of immature erythrocytes was observed, in the AS ones, immature erythrocytes contributed up to 23.5% to the total erythrocyte count [89]. The significant increase in immature erythrocytes, coupled with the significant decline in hemoglobin content, might provide the explanation for the decline of O2 saturation in the blood of AS broilers [30,57,72,134].

The differences between healthy and AS chickens in their production of erythrocytes in general, and of immature erythrocytes in particular, suggest that erythropoiesis regulation in the ascitic birds is defective.

#### *The heart*

The avian heart is different from that of mammals in that the right atrio-ventricular valve is composed of a muscle loop made up mainly of muscle fibers from the right ventricle wall. The anatomy of this valve makes birds very susceptible to valve insufficiency [52,161,162]: when the right ventricle responds to an increased workload it becomes hypertrophic and the valve hypertrophies along with the ventricle [161]. This thickening of the valve interferes with its effectiveness and may lead to rapidly developing valve failure and ascites [161]. Although litter oiling did not reduce the average ascites score, litter oiling improved air quality significantly in the pens and also improved heart morphology by reducing the right ventricle area from 0.44 to 0.36 cm2 in ad libitum birds [163, 164].

Alterations in the electrocardiogram (ECG) are seen in conjunction with AS. Of most importance has been the finding that increased S-wave amplitude in standard limb lead II indicates increased susceptibility to AS [111]. However, there were no ECG readings indicative of primary pulmonary hypertension in most birds that developed ascites [165]. A slower heart rate (bradycardia) [55,116], as well as reduction in the pulse rate had been found in birds developing AS [55] and in acutely cold-exposed birds [116].

Heart rate on days 1 and 7 was found to be significantly higher in the AS-susceptible (AS-S) genetic broiler line than in the AS-resistant (AS-R) broiler line, with only the lowest quartile of individual heart rates in the AS-S line overlapping the highest quartile in the AS-R line [57]. These results were in agreement with those of Druyan et al. [166], who found that generation S3 chicks from their AS-S line had a significantly higher heart rate on day of hatch than that of generation S3 chicks from the AS-R line. It was reported [167] that heart rate began to increase shortly after hatch, and reached a peak close to 4 wk of age; thereafter, it declined slowly [168]. The AS-S selected line exhibited increased heart rate only between d 1 and d 7, with a decline thereafter toward d 17, while the birds were kept under standard brooding condition [57]. Mild hypoxia was found to elicit an increase in heart rate [169,170], which suggests that the AS-S birds in that study experienced O2 shortage already at the time of hatch, even when kept under optimal conditions. A higher mean partial pressure of CO2 in broilers' venous blood (a marker for lung ventilation rate) on d 11 was found to be associated with increased ascites susceptibility [171,172]. Those results indicate that ASsusceptible birds suffer O2 shortage at an early age. However, it also suggests that as long as the susceptible birds are under SBC, higher heart rate can compensate for a mild hypoxemia, and no other physiological parameter would be affected.

#### *Effect on heart and blood vessels*

252 Blood Cell – An Overview of Studies in Hematology

enhancement of the erythropoiesis process.

blood of AS broilers [30,57,72,134].

in the ascitic birds is defective.

*The heart* 

chickens.

[156-158]. The escape of plasma fluid out of the blood vessels was probably due to increased pulmonary hypertension and central venous congestion – symptoms found both in humans [158] and in broilers [56]. As in the case of human ascitic patients [159], AS broilers exhibited conservation of plasma volume similar to that of the healthy ones. However, the PCV in the AS broilers increased significantly, by up to 80%, as a result of a significant increase in the number of erythrocytes, which also contributed to a significant elevation in blood volume. Thus, enhanced erythropoiesis, and not plasma volume reduction, was found to be involved in the hemodynamics of the ascitic broilers [89]. This finding could also account for the blood congestion and the increased blood viscosity [90] that contribute to the enhanced cardiac workload [103,134], blood pressure [103], and blood-flow resistance [111] in AS

In AS birds, the high PCV, on the one hand, and the significant decline in blood oxygen saturation, on the other hand [30,57,66] raised the possibility of an impairment of blood O2 carrying capacity. Increased erythrocyte rigidity appears to be another important factor in AS [54,62,113]: the nucleated erythrocytes will normally curl or fold to pass through lung capillaries [160], but hypoxemia and high hemoglobin concentrations decrease the deformability of erythrocytes [62]. Further calculations of hemoglobin content per 1,000 red blood cells revealed a significant reduction in the AS broilers compared with that in the healthy and control broilers [57,89]. These results suggest the possibility of inefficient

Ascites-induction conditions elicited enhanced erythropoiesis, which resulted in an increased proportion of immature erythrocytes in the bloodstream. However, whereas in the healthy broilers only a moderate proportion (7.2%) of immature erythrocytes was observed, in the AS ones, immature erythrocytes contributed up to 23.5% to the total erythrocyte count [89]. The significant increase in immature erythrocytes, coupled with the significant decline in hemoglobin content, might provide the explanation for the decline of O2 saturation in the

The differences between healthy and AS chickens in their production of erythrocytes in general, and of immature erythrocytes in particular, suggest that erythropoiesis regulation

The avian heart is different from that of mammals in that the right atrio-ventricular valve is composed of a muscle loop made up mainly of muscle fibers from the right ventricle wall. The anatomy of this valve makes birds very susceptible to valve insufficiency [52,161,162]: when the right ventricle responds to an increased workload it becomes hypertrophic and the valve hypertrophies along with the ventricle [161]. This thickening of the valve interferes with its effectiveness and may lead to rapidly developing valve failure and ascites [161]. Although litter oiling did not reduce the average ascites score, litter oiling improved air quality significantly in the pens and also improved heart morphology by reducing the right

ventricle area from 0.44 to 0.36 cm2 in ad libitum birds [163, 164].

Birds with ascites induced by either low ventilation or cold temperatures exhibited hypertrophy of the medial layer of arteriols, which was probably a response to primary pulmonary hypertension [173]. In low, ventilation-induced ascites, the broilers had significant inflammation or osseous-nodule formation in the lungs [174,175], whereas in cold-stress-induced ascites, birds showed no inflammation [173]. Wideman et al. [50] suggested that increases in pulmonary vascular resistance initiate increases in venous pressure by challenging the capacity of the right ventricle to thrust all the returning venous blood through the lungs. An acute reversal of systemic hypoxemia was reported to have no effect on pulmonary hypertension – a finding that discounted the influence of hypoxic pulmonary vasoconstriction [176]. It was hypothesized that this reversal of systemic hypoxemia increased total peripheral resistance and normalized arterial pressure and cardiac output, but could not decrease pulmonary hypertension because of the overwhelming influence of sustained pulmonary vascular resistance [176]. Development of techniques to measure changes in pulmonary arterial pressure and changes in wedge pressure helped to clarify that changes in pulmonary arterial pressure contribute to the mismatch between pulmonary vascular capacity and cardiac output, and that pulmonary hypertension is initiated as a consequence of excessive pulmonary arterial or arteriolar resistance [177,178]. The difference between individual broilers' susceptibility to ascites may be related to an innate or acquired variability in their pulmonary vascular responsiveness to vasoactive mediators [179].

Ascites Syndrome in Broiler Chickens – A Physiological Syndrome Affected by Red Blood Cells 255

approach has not been used by breeding companies, because it would force them to compromise the selection for more important traits, such as growth rate and meat yield,

Many studies focused on identifying reliable diagnostic indicators for AS in broilers. Hematocrit (HCT) is a marker for high rate of erythropoiesis in ascitic birds, therefore it is always significantly higher in AS broilers than in their healthy counterparts reared under the same conditions [30,54,60,115,124,125,139,154]. HCT values from broilers aged 35 and 44 d were used to screen one sire line and two dam lines for AS susceptibility [154]: they were used to select individuals that were considered the most (> 36%) and least (< 29%) AS susceptible, and the males and females with the highest and lowest HCT values, from the two dam lines, were selected and classified as high hematocrit (H) and low hematocrit (L) groups. These individuals were then reared under broiler breeder management conditions. Males and females within each group were mated, to create offspring that were HH, HM-no definition for HM, LM, and LL. The progeny underwent screening for hematocrit on days 6, 42, and 49, and from d 33 onward birds were subjected to cold stress. Differences in HCT values were seen at d 6: the HH chicks had significantly higher values than all other groups. On d 49 HCT values of the HH birds were significantly higher than those of the LL birds. Cold stress increased AS mortality in all combinations, but the HH birds had significantly higher AS mortality then the LL birds, which suggests that HCT value is heritable. It was also suggested that HCT screening and selection based on HCT values could be effective in developing resistant populations of broilers. However, later studies revealed that the variation in HCT was a secondary manifestation of developing AS, therefore it could not be used as an early indicator of AS sensitivity under normal conditions [57,72]. Heart rate (HR), measured by pulse oximetry or by encephalography, was found to be lower in broilers suffering from AS than in healthy ones [111,163,185]. At 35 days of age, HR in feedrestricted broilers was significantly higher than that in fast-growing broilers, and the HR of broilers suffering from congestive heart failure, which is associated with hypoxemia and AS, was significantly lower than that of feed-restricted, slow-growing broilers and healthy fastgrowing broilers [64]. Broilers with AS were found to have a significantly lower SaO2 than their healthy counterparts at the age of 6 weeks (62.1 and 86.0%, respectively) [30]. Broilers with AS induced by a pulmonary artery clamp had a significantly lower SaO2 and higher right-ventricle:total-ventricle weight ratio (hypertrophy of the right ventricle RV:TV) than those of healthy, non-AS broilers [32]. Therefore, low SaO2 was suggested to be a reliable genetic early indicator for AS susceptibility [186]. In recent years, some breeding companies have selected against broilers with low SaO2, as measured in selection candidates at 5 wk of age [187]. However, because of the low %AS in these unstressed flocks, high SaO2 levels are expected in susceptible individuals that do not develop AS; also, low heritability (0.15) was reported for SaO2 at 5 wk of age in commercial breeding lines [187]. Because of this low heritability and only moderate genetic correlation with actual manifestation of AS, the effectiveness of 5-wk SaO2 as an indicator for selection against AS susceptibility must be limited. All the cited findings suggest that there is a genetic component for AS mortality and

which are not fully expressed under AIC.

*Indirect selection against susceptibility to AS, cardiovascular indicators:* 
