**4. Genetic and physiological aspects of coping with the syndrome**

## *The genetic control of susceptibility to AS*

Recent reports [71,72,107] indicate that about 50% of the broilers in commercial stocks develop AS under experimental protocols of high-challenge AIC. The term "high challenge" is used for AICs that apparently induce AS in all AS-susceptible individuals, whereas "lowchallenge" AICs induce lower rates of AS, probably only in the AS-S individuals whose higher growth rates necessitate higher O2 demands. The rates of AS reported in recent years are similar to those found under high-challenge AIC in the 1990s [68,73]. In recent years, however, actual AS mortality in commercial flocks has been significantly reduced or even completely eliminated by management practices that reduce feed intake and growth rate and, consequently, reduce the physiological O2 demand [47,62]. The problem with this approach is that it compromises the efficiency of broiler production.

A better solution would be to select against AS susceptibility: once all the broilers were resistant to AS, a managed reduction in growth rate would no longer be needed. However, breeding is feasible only if there is an inherent susceptibility to AS and if effective selection against it can be applied.

Several studies have found the tendency of broilers to develop AS to be under genetic control, with estimates of heritability ranging from 0.1 to 0.7 [72-74,180,181]. Significant heritability of 0.5 to 0.6 has also been found for the ratio of right ventricle weight to total ventricle weight (RV:TV) – a postmortem indicator for AS development and severity [72- 74,182]. These data indicate the feasibility of selecting against susceptibility to AS, but only if all the genetically susceptible birds are identified at the phenotypic level. Mortality or morbidity caused by AS provides the ultimate identification of AS-S individuals.

However, actual development of AS in susceptible birds depends on environmental conditions that lead to hypoxemia, either by reducing O2 supply or increasing the O2 demand [62]. It was found that a hypobaric chamber with a reduced O2 partial pressure, equivalent to that at 2,900 m above sea level, successfully induced 66% AS in a commercial sire line, suggesting full exposure of genetic variation in AS susceptibility [103]. Surgical inactivation of one lung induced AS in all or most of the susceptible individuals [32,68,183,184]. The AIC protocol for broilers housed in individual cages, where the tested broilers could not avoid the environmental conditions that were based on movement of cool air driven by a fan, combined with high-energy pelleted feed and 23 h of light per day, resulted in about 50% AS among commercial broilers [70-72], suggesting that all or, at least, most of the susceptible broilers developed AS.

The successful induction of AS by means of any of these approaches suggests that breeding for AS resistance can be achieved by keeping all selection candidates under high-challenge AIC and awaiting mortality of all susceptible individuals. However, this direct-selection 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, which are not fully expressed under AIC.

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

254 Blood Cell – An Overview of Studies in Hematology

*The genetic control of susceptibility to AS* 

vasoactive mediators [179].

against it can be applied.

most of the susceptible broilers developed AS.

be related to an innate or acquired variability in their pulmonary vascular responsiveness to

Recent reports [71,72,107] indicate that about 50% of the broilers in commercial stocks develop AS under experimental protocols of high-challenge AIC. The term "high challenge" is used for AICs that apparently induce AS in all AS-susceptible individuals, whereas "lowchallenge" AICs induce lower rates of AS, probably only in the AS-S individuals whose higher growth rates necessitate higher O2 demands. The rates of AS reported in recent years are similar to those found under high-challenge AIC in the 1990s [68,73]. In recent years, however, actual AS mortality in commercial flocks has been significantly reduced or even completely eliminated by management practices that reduce feed intake and growth rate and, consequently, reduce the physiological O2 demand [47,62]. The problem with this

A better solution would be to select against AS susceptibility: once all the broilers were resistant to AS, a managed reduction in growth rate would no longer be needed. However, breeding is feasible only if there is an inherent susceptibility to AS and if effective selection

Several studies have found the tendency of broilers to develop AS to be under genetic control, with estimates of heritability ranging from 0.1 to 0.7 [72-74,180,181]. Significant heritability of 0.5 to 0.6 has also been found for the ratio of right ventricle weight to total ventricle weight (RV:TV) – a postmortem indicator for AS development and severity [72- 74,182]. These data indicate the feasibility of selecting against susceptibility to AS, but only if all the genetically susceptible birds are identified at the phenotypic level. Mortality or

However, actual development of AS in susceptible birds depends on environmental conditions that lead to hypoxemia, either by reducing O2 supply or increasing the O2 demand [62]. It was found that a hypobaric chamber with a reduced O2 partial pressure, equivalent to that at 2,900 m above sea level, successfully induced 66% AS in a commercial sire line, suggesting full exposure of genetic variation in AS susceptibility [103]. Surgical inactivation of one lung induced AS in all or most of the susceptible individuals [32,68,183,184]. The AIC protocol for broilers housed in individual cages, where the tested broilers could not avoid the environmental conditions that were based on movement of cool air driven by a fan, combined with high-energy pelleted feed and 23 h of light per day, resulted in about 50% AS among commercial broilers [70-72], suggesting that all or, at least,

The successful induction of AS by means of any of these approaches suggests that breeding for AS resistance can be achieved by keeping all selection candidates under high-challenge AIC and awaiting mortality of all susceptible individuals. However, this direct-selection

morbidity caused by AS provides the ultimate identification of AS-S individuals.

**4. Genetic and physiological aspects of coping with the syndrome** 

approach is that it compromises the efficiency of broiler production.

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

also for several parameters (e.g., RV:TV and HCT) that have been found to be associated with development of AS; however, the exact biochemical and physiological precursor factors related to the genetic propensity to develop AS are still not known. It is often difficult to determine whether a particular change is primary in nature, and therefore determinative, or is a subsequent secondary manifestation in the development of AS. If parameters to specifically predict AS susceptibility or resistance are sought, it is of paramount importance that the primary changes be determined and evaluated. Moreover, in order to assess their significance as criteria for selection, it is necessary to estimate the heritability of these parameters, and their genetic correlation with consequent AS development under AIC.

Ascites Syndrome in Broiler Chickens – A Physiological Syndrome Affected by Red Blood Cells 257

induced in a hypobaric chamber where oxygen content was reduced to the level equivalent to 2,900 m above sea level. After 10 generations of divergent sire-family selection, %AS increased to about 90% in the AS-susceptible line and decreased to about 20% in the ASresistant line, thus reaching a divergence of about 70% [78]. Similarly successful divergent selection was applied by Druyan et al. [70]: the 1st selection cycle was based on progeny testing for AS mortality under low-challenge AIC, and two further cycles of full-pedigree progeny testing were conducted under a high-challenge AIC protocol [70,72]. Two divergent lines were established: AS-susceptible (AS-S) and AS-resistant (AS-R), with, respectively, 95 and 5% AS incidence, i.e., a divergence of 90%, when reared together under

The very rapid genetic divergence between the selected lines, along with pedigree analysis of %ASF within the AS-S- and AS-R-selected lines implies that a single or a few major genes were responsible for the difference in %AS between the lines [70]. It was concluded that one or more genes was/were involved in the response to a two-cycle selection against AS susceptibility [68]. Single-gene inheritance was also suggested after a complex segregation analysis of data on oxygen saturation of the hemoglobin in arterial blood (SaO2) [188], a trait known to be closely related to the AS [30,72]. Data on SaO2 from 12,000 males in fully pedigreed populations of a male line that had been closed for 30 to 40 generations were available for that study. The results suggested that a single diallelic dominant locus was responsible for 90% of the genetic variation in SaO2, with high levels of SaO2 indicating AS resistance and low levels indicating AS susceptibility. Data from test-crosses between fully divergent AS-S and AS-R lines suggested a model of complementary interaction between

If, indeed, only a few genes are involved in genetic control of susceptibility to AS, and in light of the current rapid development and application of genomic tools, the AS genes seem likely to be detected and mapped in the near future. Once mapped, with the help of current and future genomic methodologies, the causative SNPs (or closely linked ones, used as markers) in these genes will be identified. High-throughput genomic assays may soon facilitate efficient genotyping of these marker SNPs, and their routine utilization in commercial breeding programs. With availability of such markers, high-challenge AIC will not be needed to effectively select against susceptibility to AS, because breeders will be able to easily detect and cull individual birds, within the elite lines, that carry the alleles for AS susceptibility. All major broiler-breeding companies have been heavily involved in R&D

Broilers, being highly productive birds, have difficulties in maintaining a dynamic steadystate balance between higher metabolic rate, on the one hand, and, on the other hand, the consequently higher demand for O2 – a demand that might exceed the cardiovascular

the same high-challenge AIC [70].

*Genomic selection against susceptibility to AS* 

the dominant alleles of two unlinked major genes [77].

efforts aimed at achieving this goal.

**5. Overall conclusions** 

In order to conduct advanced physiological and genomic research on AS, and to find the primary cause of AS, identification of all AS-susceptible individuals is crucial. This identification depends solely on mortality or morbidity under AIC. Under low- or mediumchallenge AIC, relatively slow-growing broilers or those that can better withstand cold stress, have a relatively lower demand for oxygen and, therefore, do not develop AS. Incorrect identification of AS-susceptible chicks as AS-resistant leads to biased findings regarding the true genetic association between the measured traits and the genetic difference in broilers' susceptibility to AS.

To effectively select against AS susceptibility without interfering with the normal expression of other selected traits, one has to identify the genes responsible for the primary cause of AS or measure their phenotypic expression. There is evidence that the primary cause of AS is manifested in the prenatal or very early postnatal phases, when the cardiovascular system is being developed and is starting to function [188-190]. Measurements of such a manifestation, especially at the embryonic stage, necessitate sacrificing the investigated individuals, rendering it impossible to later determine, under AIC, if these individuals were susceptible or resistant to AS. Therefore, to conduct advanced physiological and genomic research on AS, one needs a pair of selected lines in which all the individuals are either AS-S or AS-R. Comparisons of tissues or functions of individuals from the divergent lines can help to identify the primary cause of AS and thereby to provide an effective indicator for selection against susceptibility. Resource populations derived from crosses between such divergent lines might facilitate genomic research aimed at identifying the genes involved in susceptibility or resistance to AS.

#### *Direct selection against susceptibility to AS*

Successful selection against AS susceptibility was conducted in a fully pedigreed elite commercial broiler breeder line [68,184]. Only males and females that did not develop AS following AS-inducing surgery, i.e., unilateral pulmonary artery occlusion, were used for reproduction. After two cycles of such selection, %AS among males that were exposed to low temperatures (14ºC) from 17 to 49 d of age was reduced to 4%, from 31% in the base population and 15% after one cycle. That study demonstrated the feasibility of selection based on mortality of AS-susceptible individuals under a protocol of high-challenge AIC. Divergent selection for AS mortality was conducted by Anthony et al. [78]: the AS was induced in a hypobaric chamber where oxygen content was reduced to the level equivalent to 2,900 m above sea level. After 10 generations of divergent sire-family selection, %AS increased to about 90% in the AS-susceptible line and decreased to about 20% in the ASresistant line, thus reaching a divergence of about 70% [78]. Similarly successful divergent selection was applied by Druyan et al. [70]: the 1st selection cycle was based on progeny testing for AS mortality under low-challenge AIC, and two further cycles of full-pedigree progeny testing were conducted under a high-challenge AIC protocol [70,72]. Two divergent lines were established: AS-susceptible (AS-S) and AS-resistant (AS-R), with, respectively, 95 and 5% AS incidence, i.e., a divergence of 90%, when reared together under the same high-challenge AIC [70].

#### *Genomic selection against susceptibility to AS*

256 Blood Cell – An Overview of Studies in Hematology

development under AIC.

difference in broilers' susceptibility to AS.

susceptibility or resistance to AS.

*Direct selection against susceptibility to AS* 

also for several parameters (e.g., RV:TV and HCT) that have been found to be associated with development of AS; however, the exact biochemical and physiological precursor factors related to the genetic propensity to develop AS are still not known. It is often difficult to determine whether a particular change is primary in nature, and therefore determinative, or is a subsequent secondary manifestation in the development of AS. If parameters to specifically predict AS susceptibility or resistance are sought, it is of paramount importance that the primary changes be determined and evaluated. Moreover, in order to assess their significance as criteria for selection, it is necessary to estimate the heritability of these parameters, and their genetic correlation with consequent AS

In order to conduct advanced physiological and genomic research on AS, and to find the primary cause of AS, identification of all AS-susceptible individuals is crucial. This identification depends solely on mortality or morbidity under AIC. Under low- or mediumchallenge AIC, relatively slow-growing broilers or those that can better withstand cold stress, have a relatively lower demand for oxygen and, therefore, do not develop AS. Incorrect identification of AS-susceptible chicks as AS-resistant leads to biased findings regarding the true genetic association between the measured traits and the genetic

To effectively select against AS susceptibility without interfering with the normal expression of other selected traits, one has to identify the genes responsible for the primary cause of AS or measure their phenotypic expression. There is evidence that the primary cause of AS is manifested in the prenatal or very early postnatal phases, when the cardiovascular system is being developed and is starting to function [188-190]. Measurements of such a manifestation, especially at the embryonic stage, necessitate sacrificing the investigated individuals, rendering it impossible to later determine, under AIC, if these individuals were susceptible or resistant to AS. Therefore, to conduct advanced physiological and genomic research on AS, one needs a pair of selected lines in which all the individuals are either AS-S or AS-R. Comparisons of tissues or functions of individuals from the divergent lines can help to identify the primary cause of AS and thereby to provide an effective indicator for selection against susceptibility. Resource populations derived from crosses between such divergent lines might facilitate genomic research aimed at identifying the genes involved in

Successful selection against AS susceptibility was conducted in a fully pedigreed elite commercial broiler breeder line [68,184]. Only males and females that did not develop AS following AS-inducing surgery, i.e., unilateral pulmonary artery occlusion, were used for reproduction. After two cycles of such selection, %AS among males that were exposed to low temperatures (14ºC) from 17 to 49 d of age was reduced to 4%, from 31% in the base population and 15% after one cycle. That study demonstrated the feasibility of selection based on mortality of AS-susceptible individuals under a protocol of high-challenge AIC. Divergent selection for AS mortality was conducted by Anthony et al. [78]: the AS was The very rapid genetic divergence between the selected lines, along with pedigree analysis of %ASF within the AS-S- and AS-R-selected lines implies that a single or a few major genes were responsible for the difference in %AS between the lines [70]. It was concluded that one or more genes was/were involved in the response to a two-cycle selection against AS susceptibility [68]. Single-gene inheritance was also suggested after a complex segregation analysis of data on oxygen saturation of the hemoglobin in arterial blood (SaO2) [188], a trait known to be closely related to the AS [30,72]. Data on SaO2 from 12,000 males in fully pedigreed populations of a male line that had been closed for 30 to 40 generations were available for that study. The results suggested that a single diallelic dominant locus was responsible for 90% of the genetic variation in SaO2, with high levels of SaO2 indicating AS resistance and low levels indicating AS susceptibility. Data from test-crosses between fully divergent AS-S and AS-R lines suggested a model of complementary interaction between the dominant alleles of two unlinked major genes [77].

If, indeed, only a few genes are involved in genetic control of susceptibility to AS, and in light of the current rapid development and application of genomic tools, the AS genes seem likely to be detected and mapped in the near future. Once mapped, with the help of current and future genomic methodologies, the causative SNPs (or closely linked ones, used as markers) in these genes will be identified. High-throughput genomic assays may soon facilitate efficient genotyping of these marker SNPs, and their routine utilization in commercial breeding programs. With availability of such markers, high-challenge AIC will not be needed to effectively select against susceptibility to AS, because breeders will be able to easily detect and cull individual birds, within the elite lines, that carry the alleles for AS susceptibility. All major broiler-breeding companies have been heavily involved in R&D efforts aimed at achieving this goal.

#### **5. Overall conclusions**

Broilers, being highly productive birds, have difficulties in maintaining a dynamic steadystate balance between higher metabolic rate, on the one hand, and, on the other hand, the consequently higher demand for O2 – a demand that might exceed the cardiovascular system's capacity to satisfy the O2 needs. This non-steady-state situation leads to the development of the physiological syndrome – ascites.

Ascites Syndrome in Broiler Chickens – A Physiological Syndrome Affected by Red Blood Cells 259

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Following exposure to AIC of birds from various backgrounds, birds that manifested AS were found to differ significantly from their healthy counterparts, in traits that were measured after initiation of the various AIC protocols, e.g., RV:TV ratio, hematocrit, erythrocyte counts, SaO2, heart rate, weight gain (WG). These differences are consistent with findings of numerous reports; they represent changes in secondary manifestations of AS and, therefore, could be useful in diagnosis of birds that are developing AS, but not in prediction of AS susceptibility.

Only Druyan's lines that were divergently selected for AS were found to differ significantly in heart rate during the first week of life, when reared under standard brooding conditions (SBCs). Heart rate was significantly higher in the AS-S line than the AS-R line, but before the manifestation of the syndrome no such differences were found between the sick and healthy birds from commercial flocks that were kept under SBCs. Therefore, it appears that higher heart rate cannot be used as a general indicator to identify AS-susceptible broiler chicks.

It is expected that the problem of AS will be solved by genetic eradication of the alleles for AS susceptibility. However, manifestation of AS by genetically susceptible individuals depends on environmental conditions as well as genetic variation in growth rate. Therefore genomic information is required for effective integration of selection against AS susceptibility into breeding programs of commercial broiler stocks.
