lymphocyte responses of the domestic fowl to corticosterone infusion

Physiology &Behavior, Vol. 42, pp. 24%253. Copyright ©Pergamon Press plc, 1988. Printed in the U.S.A.

0031-9384/88 $3.00 + .00

Tonic Immobility and Heterophil/Lymphocyte Responses of the Domestic Fowl to Corticosterone Infusion R. B R Y A N J O N E S , * G E R A R D B E U V I N G t

A N D H A R R Y J. B L O K H U I S t

*AFRC Institute o f Animal Physiology and Genetics Research Edinburgh Research Station, Roslin, Midlothian EH25 9PS, Scotland, U.K. tSpelderholt Centre for Poultry Research and Extension Spelderholt 9, 736l DA Beekbergen, The Netherlands R e c e i v e d 3 A u g u s t 1987 JONES, R. B., G. BEUVING AND H. J. BLOKHUIS. Tonic immobility and heterophil/lymphocyte responses of the domestic fowl to cortieosterone infusion. PHYSIOL BEHAV 42(3) 24%253, 1988.--The tonic immobility (TI) fear reactions, plasma corticosterone concentrations and heterophil/lymphocyte (H/L) ratios of adult laying hens were measured before and at intervals of 4 and 11 days after the subcutaneous implantation of osmotic minipumps delivering either corticosterone solution (15/~g/hr) or only polyethylene glycol vehicle. The dummy pumps exerted no apparent behavioral or endocrine effects, whereas tonic immobility was significantly prolonged and circulating corticosterone concentrations significantly elevated at 4 and 11 days after implantation of the corticosterone minipumps. H/L ratios were significantly elevated from pre-treatment levels in both groups. However, H/L ratios were considerably higher at both post-treatment points among birds receiving corticosterone rather than vehicle. The present findings suggest that chronic elevations of plasma corticosterone not only alter the haematological profile but may also predispose birds to react more fearfully to alarming stimulation. Corticosterone Tonic immobility

Domestic fowl

Fear

Heterophil/lymphocyte ratio

IT is becoming increasingly apparent that animals generally exhibit relatively well integrated behavioral and hormonal reactions to aversive stimulation [5, 19, 34]. Thus, amongst a variety of physiological effects, exposure to a potentially frightening situation may initially stimulate increased activity of the chicken's sympathetic nervous system and the secretion of catecholamines from the adrenal medulla. This component of the stress response may be followed by activation of the hypothalamo-pituitary-adrenocortical axis (HPA) and a resultant increase in the levels of circulating corticosterone [1, 11, 19]. Fear-related behavior patterns, such as immobility, fight or flight, may be shown during one or both of these stages. Such behavioral modifications may eliminate the threatening properties of the situation and thus reduce both fear intensity and the degree of neuroendocrine activation [19,23]. Fear, which is regarded here as an ~'adaptive psychophysiological response to perceived danger" [23,25], is, therefore, an important component of stress. Indeed, it has even been suggested that physical stressors such as heat, cold or starvation do not activate the HPA if emotional arousal is carefully avoided [5, 19, 34]. Procedures intuitively regarded as frightening, such as

Osmotic minipumps

crating, handling, immobilization, noise and systematic reductions of group size, significantly elevated plasma corticosterone concentrations in chickens [2, 12, 27, 40]. However, the influence of circulating corticosterone levels on avian fear responding is less well understood. For example, reduced fearfulness in ducks was associated with elevated plasma corticosterone concentrations after domestication [32] or transection of the archistriatal efferents [33]. Conversely, a maturational increase in circulating corticosterone was thought to provide the basis for increased fearfulness and consequent interference with the imprinting process in ducklings [30,39]. Similarly, domestic chicks genetically selected for high activity in a novel environment [10] were not only considered less fearful in a variety of situations than the corresponding "inactive" line but they also showed lower resting and stress-induced levels of plasma corticosterone [9]. Tonic immobility (TI) is a fear-potentiated catatonic-like state of reduced responsiveness to external stimulation induced by physical restraint and it is considered a useful behavioral index of fear [13,22]. The effects of subcutaneously implanting osmotic infusion minipumps allowing controlled release at a constant rate of either corticosterone solution or

250

JONES, B E U V I N G AND B L O K H U I S TABLE 1 TONIC IMMOBILITY RESPONSES OF HENS IMPLANTED WITH MINIPUMPS DELIVERING EITHER CORTtCOSTERONE SOLUTION (C) OR VEHICLE (V) (MEANS ~ SEM)

One Day Prior to Implantation C

4 Days PostImplantation

1l Days Post Implantation

V

C

V

C

V

Inductions (no.)

1.31 -+_0.13

1.15 +-0.10

1.15 ±0.10

1.31 -_+0.17

I. 15 +_0.10

I.~ ±017

Head Movements/ 100 sec

1.58 +_0.50

3.02 +_1.16

0.68 ±0.17

2.33* +--0.59

0.32 ±0.10

1.81)~ +_0 38

Latency to first head movement(sec)

160.1 _+25. t

217.9 _+40.0

316.5 ±62.6

177.2 +72,2

414.4 +49,8

103.2 y~23.4

Duration of tonic immobility (see)

265.8 +-36.2

277.3 +-36.7

555.7 ±83.2

287.1" ±68.8

639.8 ±91.2

213.3~ +-41.~

(No.)=number (sec)=seconds. pvalues refer to comparison between C and V groups; *=p<0.02: t =p<0.01.

vehicle on the TI responses of adult laying hens were, therefore, examined in the present study. Minipump implants delivering corticosterone have been successfully used to elicit chronic elevation of circulating corticosterone levels in chickens [8]. Stress and corticosteroids may exert powerful influences on the immune system [19, 36, 37]. One of these effects, namely an increase in the ratio of circulating heterophils to lymphocytes has been proposed as a sensitive index of chronic stress in the chicken [16]. Indeed, the incorporation of progressively greater amounts of corticosterone in chickens' feed, although a somewhat imprecise delivery system, induced stepwise increases in their heterophil/lymphocyte (H/L) ratios [16,17]. Similarly, implanted corticosterone pellets reduced the number of circulating lymphocytes [6]. H/L ratios and plasma corticosterone concentrations were measured in the present birds before and after the implantation of minipumps. METHOD

Animals Used Twenty-six laying hens of a pure White Leghorn sire strain, maintained at Spelderholt, were used. They had been reared socially in floor pens before their transfer to communal cages at 16 wks of age. They were subsequently housed in individual cages (40x25x40 cm deep) when 31 weeks old. Variations in fearfulness between hens caged in different tiers of a multi-deck battery system have been reported [21]. The present cages were, therefore, arranged along three sides of a room on a single level. The photoperiod ran from 05.00 to 19.00 hr and food and water were supplied ad lib. All testing was completed between 35 and 37 weeks o f age.

Tonic Immobility Tests At 35 weeks of age, the tonic immobility responses of each of the 26 birds were measured. Each bird was removed from its home cage, carried approximately 10 m to a separate room and tested individually. Tonic immobility was induced

by placing the bird on its back and restraining it for 15 sec in a U-shaped wooden cradle covered with several layers of cloth [26]. The observer sat nearby within sight o f the b i r d and recorded the following behavior patterns: (1) the number of inductions 115 sec periods o f restraint) necessary t o attain T l lasting at least 10 sec, (2) the latency f r o m t h e e n d of induction until the first alert head movement, (3) the number of such head movements expressed as a function of the time between the first movement and the termination of TI and (4) the duration o f TI, i.e., until the bird righted itself. I f the bird remained in TI and showed no alert head movements over the 20 min observation period, maximum s c o r e s o f 1200 sec were given for latency and duration respectively. Tests were carried out between 08.30 and 14.30 hr and no time of day effects were apparent. All birds were ranked from low to high 11-26) according to the duration of their immobility response, i.e., the shortest scored I and the longest 26. They were assigned alternatively to one of the two treatment groups, i.e., for implantation with minipumps containing either corticosterone solution or vehicle. Each bird's tonic immobility response was measured again in similar fashion and at approximately the same time of day at intervals of 4 and 11 days after implantation o f the minipumps.

Implantation of Minipumps On the day after tonic immobility testing, osmotic minipumps (model 2ML2; Abnet Corporation, Palo Alto. CA) were implanted subcutaneously,_ under local anaesthesia, in the ventral cervical region of the neck of each of tla¢ 26 hens. The pumps had been ~lled with either corticosterone (Serva, Heidelberg, FDR) dissolved in p o l y e t t ~ l e n e gl,/col 400 (BDH Chemicals Ltd., Poole, E n g l a ~ ) or with t h e vehicle alone after their passage through a millipore fiRer(0.22 /~m). Thus 13 birds received corticosterone solution and 13 vehicle. A preliminary experiment (Beuving, unpublished observation) revealed Lhat minipumps releasing corticosterone at a calculated rate of 15 t~g/hr significantly increased

CORTICOSTERONE AND TONIC IMMOBILITY

251

TABLE 2 PLASMA CORTICOSTERONECONCENTRATIONS(ng/ml)AND HETEROPHIL/LYMPHOCYTE RATIOS IN BIRDS IMPLANTEDWITHMINIPUMPSDELIVERINGEITHER CORTICOSTERONE SOLUTION OR VEHICLE (MEANS --- S.E.M.) Plasma Corticosterone Concentration

Heterophil/Lymphocyte Ratio

Minipump Containing Blood Sample Taken

Corticosterone (C) (n= 13)

Vehicle (V) (n= 13)

Corticosterone (C) (n= 13)

Vehicle (V) (n= 13)

1 Day Prior to Implantation

1.16 ± 0.17

1.10 _+ 0.22

0.11 _+ 0.02

0.15 _+ 0.02

4 Days PostImplantation

2.82 ___0.26

0.85 _+ 0.13"

1.14 _+ 0.09

0.29 _+ 0.03*

11 Days PostImplantation

2.93 ± 0.36

0.87 _+ 0.15"

1.75 ± 0.23

0.48 _+ 0.05*

*=p<0.01 and refer to comparisons between C and V groups.

circulating corticosterone concentrations over at least 11 days in birds of a comparable age to those used here. After an initial peak, the levels remained relatively constant from 3 days onwards. However, these elevations were well within a physiological range, i.e., equivalent to or below levels previously induced by the application of stressors such as handling and the deprivation of food and water [2]. Minipumps with a corticosterone dilution of approximately 2.5 mg/ml which allowed the same release rate as above were, therefore, used here.

Blood Sampling and Heterophil/Lympho~3,te Ratios At I day prior to and 4 and 11 days after implantation of the minipumps each hen was taken to a separate room and restrained while blood was withdrawn from the wing vein. This procedure was completed within the 45 sec period thought to be necessary for any significant handling-induced adrenocortical activation [2]. Blood samples were always taken between I 1.30 and 12.30 hr and the birds' home-room companions did not witness the blood-sampling procedure. One ml of blood was retained for corticosterone assay (see below) and a further 2 drops were smeared onto a glass slide. The smears were stained within 2-3 hr of preparation with May-Grunwald-Giemsa stain. One hundred leukocytes, including heterophils, lymphoctyes, monocytes, eosinophils and basophils, were counted on each slide and the H/L ratios were determined by dividing the number of heterophils by that of lymphocytes. Two counts were made for each smear and the average H/L ratio was calculated.

Corticosterone Assay Following centrifugation of whole blood the plasma samples were stored deep frozen before analysis. After extraction and purification by column chromatography using Sephadex LH20, the corticosterone concentrations in the plasma samples were determined using a previously described radioimmunoassay [3].

Statistical Analysis Differences between treatment groups and between sam-

piing points within treatment groups were compared using the Wilcoxon matched-pairs signed-ranks test [38]. Comparisons across tests were made using the Friedman two-way analysis of variance. RESULTS The alternative allocation of birds to the 2 treatment groups ensured relative homogeneity of their initial TI responses and any systematic effect was considered minimal (Table I). Indeed the difference between groups in the duration of TI was very small in relation to the within-group variation. Apart from reduced latencies to the first head movement (p <0.05) there were no effects of repeated testing in the vehicle group. However, in comparison with the vehicle control group, corticosterone therapy for 4 days reduced the numbers of alert head movements and increased the durations of tonic immobility respectively. Other than a reduction in the number of head movements (p<0.02) there were no additional effects of a further 7 days of corticosterone therapy. However, differences between treatment groups remained significant. Plasma corticosterone concentrations did not differ between treatment groups prior to implantation of minipumps (Table 2). There were no changes from initial levels of circulating corticosterone after implantation of minipumps releasing vehicle only for 4 or 11 days. Conversely, plasma corticosterone concentrations were higher among birds receiving corticosterone rather than vehicle at both 4 and 11 days after implantation of minipumps. The levels of circulating corticosterone were similar after either 4 or 11 days of hormone therapy. Both groups showed similar H/L ratios prior to implantation of pumps (Table 2). Although elevations (/9<0.001) from pre-treatment levels were subsequently observed in both groups, H/L ratios were considerably higher among birds implanted with minipumps delivering corticosterone rather than vehicle at both post-treatment points. DISCUSSION The implantation of osmotic minipumps containing only

252

JONES, B E U V I N G AND B L O K H U I S

polyethylene glycol vehicle into the present hens failed to affect their circulating corticosterone levels. Conversely, plasma corticosterone concentrations were significantly and similarly elevated at 4 and 11 days after implantation of minipumps releasing corticosterone solution (15 ttg/hr). The latter findings support previous observations (Beuving, unpublished) that corticosterone minipumps remained active for up to 14 days after implantation and induced relatively constant levels of circulating corticosterone. The induced plasma corticosterone levels found here fell well within a physiological range, that is, equivalent to or below those previously reported after application of stressors such as deprivation of food and water, handling and crating [2]. Tonic immobility is considered positively related to fear because procedures intended to elicit fear prolong the reaction whereas fear reducers attenuate it [13.22. 23]. Its validity as a behavioral index of general, underlying fearfulness is supported by the close association between a bird's TI reaction and its responses in other potentially frightening situations [22, 24, 28]. In the present study, the immobility reactions of adult hens were considerably prolonged at both 4 and 11 days after implantation of minipumps releasing corticosterone solution, whereas dummy pumps containing only vehicle exerted no significant behavioral effects. These findings suggest that chronic elevations of circulating corticosterone increased general fearfulness, at least under the present conditions. Despite proposals that docility was correlated with heightened plasma corticosteroid levels after section of the archistriatal efferents in chicks and ducklings [33], the present findings support previous proposals that chronic pituitary-adrenal activity and fear may be positively related in birds and mammals [4, 5, 9. 31.39]. However, this does not necessarily imply that acute elevations of circulating corticosterone are capable of similarly modifying fearrelated responses. Chronic corticosterone therapy and/or naturally increased baseline pituitary-adrenocortical activity prolonged TI in the present study, inhibited ducklings' approach and following responses to an imprinting stimulus [30,39], depressed open-field activity in rats [29] and suppressed active reactions to aversive stimulation in pigs [5]. If chronically elevated plasma corticosterone is indeed associated with in-

creased fearfulness, the above findings are consistent with the proposal that fear exerts a progressively inhibitory effect on activity [20,23]. It is not yet known whether the exogenous corticosterone exerted its effect directly in the present study or via a negative feedback effect on either the HPA axis or on the release of other brain ACTH-like peptides. Heterophil/lymphocyte ratios were increased at 4 and again at 11 days after implantation of the corticosterone minipumps. This effect is consistent with previous observations that the incorporation of corticosterone into the diet [16] or the implantation of a corticosterone pellet [6] increased H/L ratios and reduced the numbers of circulating lymphocytes respectively. The increases in H/L ratios observed among birds implanted with dummy minipumps may reflect low-level immune reactions to discomfort and/or tissue damage caused by the operation. However, the haematological effects observed in this group may not be closely associated with their circulating corticosterone levels, which remained unaltered. There is no obvious explanation for this apparent dissociation. A previous study revealed that 5 consecutive daily trials were required before the tonic immobility reactions of adult Brown Leghorn hens were significantly reduced [15]. Thus. despite the existence of strain differences in tonic immobility [ 14, 24, 34] and the fact that White Leghorn hens were used here. the present finding that 3 inductions failed to elicit habituation of TI in the birds implanted with dummy minipumps was, therefore, not unexpected. In conclusion, chronic elevations of circulating corticosterone may not only cause weight loss [7] and suppress growth, food conversion, reproductive capacity and immune responses m chickens [16, 18. 19. 36], but they may also predispose the birds to react more fearfully to alarming stimuli.

ACKNOWLEDGEMENTS We are grateful to the IAPGR and to Spetderholt tbrjointty funding the visits made to the Netherlands by Dr. Jones. We would also like to thank Ing. J M Rommers. J W. van der Haar. F. A. T. Jansonius and F. F. Putirulan for their technical assistance. Miss. D. M Velner for typing the manuscript and trig. P. Vereijken for statistical advice.

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