Hematologic parameters in broilers subjected to cyclic heat stress

Broilers are highly sensitive to environmental mainly to high temperatures, resulting in heat stress (HS)(Dionello et al., 2002) that is rious for bird's physiology and performance (Furlan and Macari, 2002).
In poultry, chronic HS induces increased release of adrenal steroids to the bloodstream, which results in altered numbers of leukocytes (Hsturkie, 1998). This happens because of high corticoid concentrations can cause the involution of both the thymus and the bursa of Fabricius through apoptosis mechanisms leading to decreased lymphocyte proliferation (Morgulis, 2002), in addition of causing the sequestration of circulating lymphoid cells in their way to lymphoid tissues (WinkelHSein, 2000). Corticosteroids cause alterations in the function of monocytes (MO) in vitro, suppressing their phagocytic activity on bacteria and reducing the resistance of the host to infection, in addition to increasing the numbers of circulating neutrophils (WinkelHSein, 2000). Reports also exist about increased heterophil percent and reduced basophils due to HS (McFarlane et al., 1989). The heterophil/lymphocyte ratio (H/L) is a primary indicator of stress in the birds, since in these cases increased H/L is seen (Gross and Siegel, 1983). The changes that occur in the blood of HS birds, such as increased numbers of red blood cells, increased hemoglobin concentrations, increased packed cell volume (PCV) and mean cell hemoglobin concentration (MCHC) in addition to decreased mean cell volume (MCV) (Furlan et al., 1999) are a part of the temperature-regulating responses of birds exposed to HS (Yahav et al., 1997).
Most studies on the effects of HS on poultry physiology have used constant ambient temperatures, even though in natural conditions this is not the case. Therefore, the purpose of this research was to study the effects of cyclic heat stress on the hematologic parameters of male broilers.

Seventy (70) Avian male broilers were used. Birds were placed in cages measuring 160 x 50 x 60 cm each. On day 1 of age two groups of 35 birds each were randomly formed. The first group was maintained at the thermoneutral environment (TN) while the other group (HS) was subjected to an ambient temperature of 38ºC for 12 - 13 hours on days 1 to  27 of age, and 40ºC for one hour on days 28 to 42 of age. In order to heat the environment, four incandescent 200 W lamps were used in each cage. During the exposure of birds to chronic HS (CHS) both water and feed were suspended, but later water and feed were given ad libitum, while maintaining the birds at a thermal comfort temperature during the remaining 23 hours. A commercial feed and water were offered ad libitum, and the birds were subjected to 24 hours of natural and artificial light throughout the experimental period. The design was completely at random under a 7 x 2 factorial arrangement with 7 age factors (1, 7, 14, 21, 28, 35, and 42 days of age) and two ambient conditions (HS and TN).
From day 1 to day 42 of age, 2 mL blood samples were collected weekly by ulnar vein punction, and deposited in 10% EDTA (anticoagulant) containing-tubes for erythrogram or red blood cell analysis (number of cells, hemoglobin, hematocrit (PCV), MCV, MCH, and MCHC, and leukogram (hemocytometer method) including the differential leukocyte counts using Leishman-stained blood smears, considering heterophils (HT), eosinophils (EO), basophils (BA), lymphocytes (LI), monocytes (MO) and H/L ratios.
The Kolmogorov-Smirmov's test was applied to study the distribution of blood analysis data. The means of parameters with a normal data distribution were evaluated using t test with 5% probability, while those with an abnormal distribution were evaluated using Wilcoxon's test with 5% probability.

An effect of the age x ambient interaction was found on the numbers of red blood cells. At 42 days of age (P<0.05) HS birds had a higher mean than TN birds (Table 1). This findings disagree with some literature reports (Furlan et al., 1999) with decreased numbers of red blood cells in the birds subjected to acute HS.
Mean hemoglobin, PCV, MCV, and MCH were not influenced by CHS or bird age (P>0.05). These results are not consisted with those reported by Furlan et al. (1999) where hemoglobin was increased regardless of the presence or absence of HS on the birds. MCV was affected by the age, since values decreased as birds aged. MCH was not affected, but PCV was increased (McFarlane et al., 1989) as a result of acute HS.
MCHC was increased as birds aged, with or without HS, and it peaked at 28 days (P<0.05), consistent with Furlan et al. (1999), with an increase in MCHC at week 7 of age in both HS and non-HS birds, and the effect of HS resulted in increased MCHC by week 7 of age.

Table 1. Means of erythrogram variables in Avian male broilers at different ages, subjected (HS) or not (TN) to cyclic heat stress for one hour (38ºC and 40ºC)

An effect was seen of the age x ambient interaction on the numbers of LE, LY, and on the H/L ratio. On days 7, 14 and 21 of age, the HS birds showed decreased (P<0.05) number of LE, s compared to TN birds (Table 2). In the HS group, the numbers of LE were increased up to day 35 of age. In the TN group, increased numbers were seen up to day 14, and the means were maintained up to day 35. Both LY and H/L in the HS group showed smaller means than those in the TN group on day 28, and higher means in the H/L ratio on day 28. In the HS group, lower Ly means were seen on day 28 and this occurred in the TN group on day 21. The H/L ratio in the HS group showed higher means on day 28 of age, while in the TN group the H/L ratio remained constant at all ages. These results corroborate those reported by McFarlane and Curtis (1989) who found increased H/L ratios in their HS birds.
No effect of the age, the ambient or the age x ambient interaction on the number of BA (P>0.05) (data not showed in the Table), which disagrees with McFarlane et al. (1989). The disagreements found in this research regarding reports elsewhere, can be partly due to methodological differences. Nevertheless, in addition to high ambient temperature, differences in the magnitude,  duration and type of stress to which the birds were subjected, are important factors to be taken into account.
No effect was seen of the age x ambient interaction on % HT, EO or MO (P>0.05). Even though, age had a influence on %HT and EO, that were increased up to day 21 of age (P<0.05), regardless of subjecting the birds to HS or not. The ambient in which birds were kept influenced both %EO and MO since HS birds showed higher means than those in the TN group (P<0.05).
Results in Tables 1 and 2 show that HS had an influence on the hematopoiesis in broilers  thus compromising the immune system due to increased EO/MO numbers and decreased LE/LY numbers, making the birds more prone to disease than those maintained under TN conditions.
HS birds spent more energy for maintaining homoeostasis and consumed less feed, in an attempt to reduce the production of endogenous heat. In consequence, these birds have a lower nutrient intake, and many of these nutrients play direct roles in hematopoiesis.

Table 2. Means and standard deviations of leukogram variables in Avian male broilers at different ages, subjected (HS) or not (TN) to high, cyclic environmental temperatures for one hour (38ºC and 40ºC

Heat stress has adverse effects on the immune status of birds in its variables leukocytes, eosinophils, lymphocytes, monocytes, and in the heterophil to lymphocyte ratio. Regarding the erythrogram variable, heat stress causes alterations in the numbers of red blood cells during the lay productive cycle.

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