Сельскохозяйственная биология, 2012, № 4, с. 62-68.

doi: 10.15389/agrobiology.2012.4.62eng

УДК 636.52/.58:636.081.4:57.04:591.111.1

FEATURES OF LEUKOCYTIC REACTION TO TRANSPORTATION IN CHICKEN WITH VARIOUS STRESS SENSITIVITY

A.V. Miftakhutdinov, A.I. Kuznetsov

The number of basophiles, eosinophiles, lymphocytes, monocytes and heterophiles in blood was compared before and after transportation of 37 day-old chicks of the Hubbard ISA F15 meat cross. For preliminary determination of individual stress sensitivity in tested poultry, we have proposed the technique, consisting in a modeling of local adaptive syndrome by means of intradermal injection of 70 % turpentine solution into a jowl. As it was confirmed experimentally, the transportation was a powerful irritant, causing the development of stress in chicks, that was manifested as a change in leukogram, in particular, the considerable decrease in lymphocytes number and the increase in heterophiles number. The individual sensitivity to stress depends directly on a chick’s reactivity. The reactivity was higher in stress resistant individuals, than in stress sensitive ones.

Keywords: individual stressful sensitivity of chickens, leukogram, stress, transportation of chickens.

Complete realization of high genetic potentials of modern chicken crosses is a key target of poultry industry, which though is often hindered by adverse effects of environmental factors and various stresses that reduce manifestation of traits associated with productivity (1). Pharmaceutical treatment just partially helps, and it could be more efficient to obtain the genotype with determinants for high resistance to technological stresses and enhanced adaptivity (2, 3). One of such strong technological stresses is transportation that combines joint action of several adverse factors (4-7).
Individual sensitivity of chickens to stress in conditions of poultry farm is manifested as different reaction of resistant and susceptible individuals to adverse factors leading to more or less useful adaptive effect assuming it in terms of improved viability and productivity potential. Stress sensitivity is yet insufficiently studied physiological feature. At the same time, stress and other nonspecific adaptive reactions are associated with specific changes in leukogram (8). For example, it was shown that 30-minutes transportation of poultry resulted in eosinopenia, reduce in count of basophils and lymphocytes, increase in proportion of segmented neutrophils, monocytes, and leukocyte indices; all these symptoms indicated the development of acute stress and forced adaptation of the organism (9).
The purpose of this work was to reveal adaptive responses to transportation in leukogram of chickens with different individual sensitivity to stress.
Technique. Experiments were performed in Argayash poultry farm (the enterprise “Uralbroiler”, Chelyabinsk province, 2011) on chickens the meat-producing cross Hubbard ISA F15. Feeding and keeping conditions corresponded to recommended for the cross. Individual stress sensitivity of chickens was determined by evaluation of a local adaptive syndrome induced by intradermal injection in wattle of  0,1 ml 70 % turpentine solution and following severe reaction of acute aseptic inflammation in 24 hours after the test (10, 11) (the method was improved and adapted to physiology of meat-producing chickens). Turpentine test was carried out on chickens aged 35 days, and 2 days later they were transported by road transport to the distance of 220 km with a total journey time of nearly 3 hours (the experiment was conducted in summer). During transportation, the chickens were crated in two cages of 2 m² area each one deprived from water and feed.
To assess manifestation of adaptive responses and stress impact, leukogram was determined before the transportation (in a state of relative rest), immediately after transportation and 1 day after; along with it, the ratio heterophils / lymphocytes (H/L) was calculated. The blood was sampled from comb puncture at the same time (13⁰⁰ to 14⁰⁰). The smears were stained according to Romanovsky-Giemsa, blood cells were counted under an immersion microscope Mikmed 6 (“LOMO”, Russia) using the three-field method of Filipchenko (each smear - 200 white blood cells) (12).
The data were computed in the program Statistica 6.0 with nonparametric statistics and determination of Spearman’s rank correlation coefficient between stress sensitivity and the value of H/L ratio (ρ). Data in tables show medians and quartiles Me (Q₁: Q₃).
Results. Preliminary testing of individual stress sensitivity of chickens by the own developed turpentine test has revealed in the tested group stress-sensitive and stress-resistant individuals.
Leucograms of chickens with different stress sensitivity before the transportation (i.e. in relative rest) showed the absence of any statistically reliable differences and symptoms of a stress: low count of eosinophils, heterophils and lymphocytes within the physiological norm. Probably, turpentine persisted in wattle tissues long after setting the test, and its general and local action could cause the observed eosinopenia.
The correlation coefficient r between stress sensitivity and the value of H/L ratio was equal to 0,26, so there wasn’t any  statistically significant relationship between these traits in chickens in the state of relative rest.
Transportation caused a significant stressful impact on the appearance and behavior of chickens. They refused food and water, showed weak reaction to stimuli or no reaction; they manifested muscle tremors, cyanosis of the mucous membranes, most of the chickens had ruffled feathers. One stress-sensitive chicken died during transportation.
The leukogram of stress-sensitive chickens had statistically reliable changes: the proportion of lymphocytes decreased (P = 0,043115), heterophils - increased (P = 0,043115), as well as H/L ratio (P = 0,043115). Stress-resistant individuals manifested similar patterns of leukogram though with lower degree of statistical significance: the count of lymphocytes was lower (P = 0,027709), heterophils - higher (P = 0,027709), the value of H/L ratio increased (P = 0,027709), as well as the count of eosinophils (P = 0,043115) (Table). Two groups of chickens with different sensitivity to stress had statistically reliable differences in counts of lymphocytes, heterophils, and H/L ratio.

The coefficient of correlation between stress sensitivity and H/L ratio (0,69) indicated a moderate statistically significant relationship between these characteristics immediately after transportation.
In 1 day after transportation the chickens began to recover appetite, normal state of muscles and color of mucous membranes, but they lost feathers in the neck and chest (more intense in stress-sensitive individuals). Changes in leukogram were recorded as well: is stress-sensitive individuals – the decrease in count of heterophils (P = 0,043115), while in stress-resistant ones – the reduce in value of H/L ratio (P = 0,043115). At the same time, stress-sensitive chickens manifested higher levels of basophils, eosinophils, and heterophils, but stress-resistant had lower level of lymphocytes.  In 1 day after transportation the shift of H/L value in stress-sensitive individuals was statistically more significant than in stress-resistant chickens.
The coefficient of correlation between stress sensitivity and the value of H/L ratio was equal to 0,74 – i.e. there was a strong statistically significant relationship in 1 day after transportation.
Thus, leukogram of chickens revealed a pronounced influence of transit stress. Major changes were associated with the increase in count of heterophils and reduce in count of lymphocytes, while H/L ratio reliably increased in both groups. In mammals, stress reactions accompanied by neutrophilia and lymphopenia are described in detail (13). Such manifestations were observed in humans and laboratory animals (14-16), in cattle (17), and horses (18). Changes of leukogram are based on the destruction of leukocytes or redistribution of lymphocytes in the body. The release of glucocorticoids is a signal for lymphocytes to move from the bloodstream to organs and tissues, in mammals - mainly to the lymph nodes, spleen, bone marrow, and skin (19). Simultaneously, glucocorticoids stimulate the release of neutrophils from the bone marrow and other tissues and organs into the bloodstream (20). Similar processes occur in other vertebrates - birds, amphibians, reptiles and fish.
This phenomenon was also observed in chicks (21). Later, the presence of correlation between H/L ratio and the blood level of glucocorticoids was confirmed in hens (22). Moreover, H/L ratio was used a key parameter in the method for prediction of chickens’ productivity and natural resistance in conditions of large-scale poultry farms (23, 24). It was also proved the presence of inherited genetic determination for patterns of H/L variation under the influence of changing environmental factors (25).
In this experiment, the chickens’ response to stress was directly correlated with H/L ratio. The relationship between stress sensitivity, the dynamics of H/L ratio, productivity and viability of poultry was observed by other authors as well (26).
In the relative rest, leukogram of chickens was almost independent of their individual stress sensitivity. However, after transportation, stress-sensitive individuals manifested the increase in H/L ratio in 4,4 times, stress-resistant ones – in 2,6 times. So, the arise of stress response was more pronounced in stress-sensitive chickens, as it can be seen from nearly 2-fold difference in the value of H/L ratio.
In 1 day after transportation the leukogram recovered to normal levels, which reflected high adaptive capacity of both groups of chickens. According to the authors’ data, the counts of basophils and eosinophils were statistically higher in stress-sensitive chickens compared with stress-resistant ones, which could be assumed as indirect symptom of a higher degree of glucocorticoid deficit in stress-resistant chickens due to weakened adrenocortical function. This assumption could be supported by more pronounced and prolonged inflammatory reaction to turpentine test in stress-sensitive chickens. However, the comparison of leukogram of chickens in relative rest and 1 day after transportation showed the absence of significant glucocorticoid dysfunction of the adrenal cortex. In other words, the observed changes in leukogram indicated lower degree of glucocorticoid activity in 1 day after transportation in stress-sensitive individuals compared with stress-resistant ones, which occurred against the background of normal physiological reactions to stress.
Despite the marked changes in levels of heterophils and lymphocytes, there wasn’t any adequate response in respect to eosinophils. Probably, this fact was associated with inflammation and necrosis of the wattle ridge after the turpentine test. As it was noted by D.A. Bass et al. (27), eosinopenia could be the result of migration of eosinophils to the site of inflammation and necrosis. It is also possible that eosinopenia was one of the long-term effects to stress after the turpentine test.
The following adaptation of chickens to transit stress revealed by changes in leukogram of both experimental groups can be classified as resistance phase described by H. Selye et al. (4).  According to the theory of L.H. Harkavy et al. (16), the response recorded after the impact of stressful factors corresponds to the realization of adaptive function at a high level of reactivity. The observed changes in leukograms indicate higher degree of stress reactivity in stress-resistant chickens than in stress-sensitive ones.
So, transportation causes in chickens a transit stress accompanied by the development of complex adaptive responses – the reduce in proportion of lymphocytes and the increase in relative count of heterophils in the leukogram. Changes in leukogram of experimental chickens were typical for the state of stress. Individual sensitivity of an organism to stress was proved as a feature associated with stress reactivity reflected by corresponding changes in leukogram. In this experiment, stress-resistant chickens showed more significant stress-reactivity then stress-sensitive individuals.

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