Ptlysiology & Behavior, Vol. 17, pp. 561-564. Pergamon Press and Brain Research Publ., 1976. Printed in the U.S.A.

Testosterone and Fear Behavior in Male Chicks

JOHN A R C H E R l

Ethology and Neurophysiology Group, School o f Biological Sciences, Universi O, o f Sussex, U. K.

(Received 24 Oc tober 1975)

ARCHER, J. Testosterone and fear behavior in male chicks. PHYSIOL. BEHAV. 17(4) 561-564, 1976. Testosterone-injected and control male chicks were tested either for (a) 3 rain a day on consecutive days in an open field containing a bell which was sounded for the first 5 sec, or (b) for 3 rain in a plain open field l'ollowed by a bell sounded from outside the field. In the first situation, hormone-injected chicks showed more freezing and immobility, bu! less active escape and avoidance responses. In the second situation, testosterone-treated and control chicks did not show these differences. It was suggested that the occurrence of a Iocatised aversive stimulus (the bell) in the centre of the open field had resulted in different types of fear behavior in the two types of chick because the hormone injected group attended more to the Iocalised stimulus.

Testosterone l:ear behavior Avoidance responding

T E S T O S T E R O N E is k n o w n to faci l i ta te a t t ack in a var iety of avian and m a m m a l i a n species [ 1 2 , 1 7 ] . Since fear behavior

def ined here as fleeing or immobi l i t y - is the c o m m o n al ternat ive response in aggression-evoking s i tua t ions [ 12], it seemed i m p o r t a n t to de t e rmine w he t he r t e s tos t e rone in- f luences the occur rence of fear behav ior itself, thus providing a possible indirect means of faci l i ta t ing at tack.

The y o u n g male chick has been used to s tudy the behavioral effects of t es tos te rone . Injected in the form of a long-act ing p repara t ion , t e s tos t e rone p roduces a n u m b e r of effects, inc luding the faci l i ta t ion of a t tack referred to above [1,3] and also an inf luence on the pers is tence of visual search dur ing feeding [ l , 5, 22] . These effects are k n o w n to occur in adul t male fowl (e.g., [ 2 2 , 2 6 ] , p resumably as a result of e n d o g e n o u s androgens [ 14,22] .

O the r changes in behavior induced by t e s tos t e rone t r e a t m e n t in chicks, bu t which have no t yet been studied in the adult , are more direct ly relevant to the ques t ion of fear responses. In a novel e n v i r o n m e n t , the peeping (distress call) rates of t e s tos te rone- t rea ted chicks declined more rapidly over several daily exposures than those of cont ro l s did I6,8] and in an open field, t e s tos t e rone - t r ea t ed chicks showed shor t e r la tencies to move f rom the i r initial posi t ion in the field [6,71. They also showed shor te r freezing dura t ions to a bell sounded from outside the open field [71.

A possible i n t e rp re t a t i on of these results is tha t teslo- s lerone lowered the t e n d e n c y to show any form of fear or emot iona l response but la ter f indings [81 on head posi t ions indicated that they could as readily be expla ined as an indirect consequence of a change in visual scanning [4 ,8 ] . Other findings, on t imes taken to emerge from the s ta r t -box of a runway [ 11], were incons i s ten t with an exp lana t ion in terms of emo t iona l responsiveness.

It is, the re fore , not clear w h e t h e r t e s tos t e rone does exer t a direct effect on the t endency to emit fear responses. The present expe r imen t was designed to invest igate the effect of t e s tos t e rone on fear behavior in young male chicks, by using a s i tua t ion in t ended to be more fear-evoking than the novel e n v i r o n m e n t s used previously [6, 7, 8[ . It involved placing the chick into a novel a rena con ta in ing a bell, sounded for the first five seconds of test ing: fear responses ( immobi l i t y and escape behavior ) and associated emot iona l behavior (e.g. distress calls) were recorded.

A pilot expe r imen t (n = 40) indica ted tha t whereas Ts showed more pro longed initial immobi l i t y ( p < 0 . 1 ) C s showed higher values for behav ior involving active avoid- ance and escape, par t icular ly j ump ing (p<0 .05 ) , bu t also for looking up (which of ten precedes the first few j u m p s ) and burrowing, an a l ternat ive form of escape behav io r ( p < 0 . 1 ) in bo th cases). ( 's also showed higher scores for looking away from the bell ( p < 0 . 0 0 2 ) , higher peeping scores ( p < 0 . 0 5 ) and more Cs than Ts defeca ted (p<0 .01) .

The first four measures are associated with active escape and avoidance, but in previous studies involving a novel e n v i r o n m e n t w i thou t a bell [6,8] there was no indica t ion that active escape responses were more f requen t in ('s, even though such responses were fairly c o m m o n in bo th Ts and Cs. Andrew [4] also found no di f ferences in . imnping or bur rowing be tween T and C chicks in a cold chamber . In these previous studies, it was also found that when a bell was sounded outs ide the open field, ( 's remained immobi le for s ignif icant ly longer than Ts [7 ] , an effect which is in the oppos i te d i rec t ion to the one found in the present pi lot study.

In view of these apparen t ly conf l ic t ing findings, and because substant ia l ba tch di f ferences were also found in the pilot s tudy, bo th this expe r imen t and the previous one

Presen! address: Division of Psychology, Preston Polytechnic, Prest(m, Lancashire, U. K.

561

562 AR( t t I R

involving a bell outs ide the open field were rel icated using the same ba tches of chicks for the two s i tua t ions .

METHOD

Male Warren sex-l inked chicks were ob ta ined f rom the ha t che ry ( S o u t h d o w n , Lewes) on the second day af ter ha tch ing , and in jec ted on the same day, e i the r wi th 12.5 mg of t e s t o s t e rone o e n a n t h a t e (a long-act ing androgen) in l).05 ml sesame oil (Ts) or wi th 0.05 sesame oil on ly (Cs). This dose of t e s t o s t e r o n e o e n a n t h a t e has previously been found to p r o d u c e max ima l changes in copu la t ion , a t tack and search behav ior in y o u n g male chicks [ 11.

The chicks were housed in individual cages at a mean t e m p e r a t u r e of 320( , . Two days af ter in jec t ion hall" were tes ted in an open field with a bell in the center , and the o the r ha l f in a ident ical open field w i t h o u t a belt. The firs ' group were tes ted on 3 consecut ive days for 3 rain and the second group for 1 day only. The open field was of the type descr ibed by Broadhurs t [ 1 3 ] , the f loor being marked in to 3 concen t r i c circles with the ou te r circle divided into g sect ions and the middle circle divided in to 4 sect ions. It was lit by a 40w Anglepoise lamp, 60 cm from the cen te r of the f loor of the open field, in an o therwise dark room.

For the group with the bell in the center , the bell was sounded for the first 5 sec the chick was placed in the arena, and behav io r was recorded dur ing the 3 min fol lowing the ch ick ' s en t ry to the field. During each successive 15 sec per iod the presence or absence of the fol lowing types of behav io r were recorded on a check-l is l : peeping, j umping , looking up, bu r rowing (vertical and ho r i zon ta l b o b b i n g m o v e m e n t s at the walls of the arena, see Andrew [4] who refers to this behav ior as t ry ing to get t h rough or TGT) , defaca t ion , immobi l i t y , c rouching, head shaking, crowing, pecking the g round , pecking the wall, sleeping, bill wiping, preening, and louch ing , pecking or a t t ack ing the bell. Measures of the ch ick ' s o r i en t a t i on in the open field were also recorded by the same sampling me thod . The arena was marked in to 3 concen t r i c circles, wi th equal d is tances be tween them, and thus en t r ies to these 3 par ts of the field (ou te r : near the walls; inner : near or on the bell; and i n t e r m e d i a t e ) could be quant i f ied . Whe the r the chick looked at or away from the bell was also recorded. Looking away involved look ing at the walt of the arena ( inc luding bu r rowing or j um p i ng at i t) and look ing up.

For the group wi thou t the bell in the center , the fol lowing measures were taken dur ing the first 3 rain of test ing: la tency to move a leg, and to move from the sect ion u p o n which they were first placed; t ime samples o t the fo l lowing mu tua l l y exclusive categories of behav io r (every 10 sec) - s tand and peep, walk, immobi le , j u m p i n g and burrowing. Af ter 3 min, a bell s i tua ted outs ide the open field was sounded for 5 sec and the fol lowing measures recorded: the n u m b e r of sect ions crossed af te r the bell had sounded ; la tency to (a) peep, (b) move head, ¢c) move a leg, and (d) move off the sect ion of the open field ( to a m a x i m u m of 3 min from when the bell began sounding) . This type of open field tes t ing was carried out on one day only.

Thus, ha l f the chicks f rom any one ba tch were tes ted for 3 days in an open field wi th a bell located at the cen te r and sounded for the first 5 sec, and the o t h e r half were tes ted only once in an open field no t con ta in ing a bell, but were exposed to a bell r inging af ter they had been in the open field for 3 min.

Al toge ther 80 chicks were used, 40 lot each x's! cond i t ion , ha l f of which were tesl~,s terone-injec[ed I T ) a n d half con t ro l - in jec ted (C). Four d i f fe rent ba tches e~l'chick,, were used, so tha t 20 birds were tested at any one l ime: tH these 10 were tested in one ope~ iield , . 'ondilion -rod It)ii7 the o ther , and half of each sample was t es los te ronc- in jec ted and the o the r half cont ro l - in jec lcd .

The chicks were tested in J ~oom near Io tilt' one in which lhey were housed , and the l empe ra lu r e of the tes l ing room was also ma in ta ined at 3 2 ( . f c s l s ~e re carried <ml be tween 14:00 and 17:00 hr.

Slat is t ical compar i sons were carrwd oul using !he Mann-Whi tney I! test or the \~ test [ 231.

kl. SOL I~,

l a b l e 1 shows the n u m b e r ,~i !5 ~,ec t ime per iods m which the most c o m m o n types of behav ior occurred (expressed as medians) , and the n u m b e r of birds showing less f r equen t types of behavior , for the open field con- ta in ing the bell. Immobi l i t y scores over all 3 days were s ignif icant ly higher in Ts than Cs, thus conf i rming the result of the pilot s tudy. Cs showed sig3fificantty higher overall scores for peeping and looking up. Signif icant ly more (N than Ts j u m p e d and more Ts than ( 's c rowed. There were no significant d i f ferences be tween the two groups in the n u m b e r of chicks defeca t ing (C , T in pilot s tudy, p< O.O1 t. not in the number s showing bur rowing (t" .~l in pilot s tudy, p~ 0.1). ( 's also showed looking away from the bell in more t ime per iods than did the Ts l m e d i a n s : Ts, 20.5, ( s 28.0, p = 0 .025) , a d i f ference which was present lo a more marked ex ten t in the pilot s tudy ( p < 0 . 0 0 2 ) . 'Fable 2 shows the results for the ()pen field wi thot l t the bell in the center , aml upon subsequen t r inging of the bell outs ide the open field, The d i f ferences be tween Ts and ( 's in la tencies to move arc in the same d i rec t ion as found before [71, but only one measure was s ta t is t ical ly s ignif icant (Ts showing shorteT latencies be tween thei r first leg m o v e m e n t and full move- ment off the sec t ion: see [71 ). There were no significanl d i f ferences in the t ime sample measures, and these were nol included in Table 2: the most commonly observed behavioT was s tand and peep, and immohi l i ty was in f requen t t.time sample means : 0.65 Ts, 2.9 ('~(L In bo th the pi lo t s tudy and this expe r imen t , several q~s a t tacked the bell where lhis was present in the cen te r of the open field. At tack involved runn ing towards the bell, fol lowed by repeated pecks al it, and most ly occur red on the th i ld day of testing. Over all 3 days, 5 Ts and 0 Cs a t t acked the bell (p = 0.05-0. I, x ~ tes~,. two-tai led) aml a similar d i f ference was apparen t m the n u m b e r s pecking the bell (also 5 Ts, 0 Cs). However, lhere were no di f ferences in the n u m b e r s of chicks tha t t ouched the bell t l l Ts, 10 ( s ) . In the pilol s tudy, only 2 birds a t lacked the bell (2 Ts, 0 Cs), 4 pecked it (3 Ts, i C), and 15 lonched it (7 Ts, 8 Cs). ( ' o m b i n i n g the 2 exper imen t s , r Ts and 0 ( 's a t t acked the hell (p = 0.(12 0 . 0 I , \~ lesl, two-tailed~, 8 T s a n d 1 ( ' p e c k e d il ( p = 0 . 0 5 0.{)2, x~ test, two-ta i led) but there was no d i f fe rence in the n u m b e r of birds t ouch ing the bell ( Ig Ts, I g {s) .

DlSCUSSI~)N

For the open field con ta in ing the bell in the center , fhis expe r imen t repl ica ted the main f indings of the pilot s tudy. ( 's showed more jumping , looking away f rom tile bell. looking up and peeping. These responses involve active escape, aw)idance of look ing at the bell, anti peeping, which

TESTOSTERONE AND FEAR BEHAVIOR 563

T A B L E 1

BEHAVIOR OF T AND C CHICKS IN OPEN FIELD WITH A BELL IN THE CENTER

T C Behavior IN - 20) {N = 20) p

Peeping* 33.5 36 0.001 Immobility* 8.0 4.0 <0.05 Look up* 0.5 2.5 <0.05 Jumps+ 1 7 <0.02 Crow+ ~, 0 <0.05 Defecate, 13 17 NS Burrow+ 5 9 NS

*Scored as presence or absence in each successive 15 sec period for 3 min periods on 3 successive days. Median values shown in table, p values calculated from Mann-Whitney U test (one-tailed, prediction beased on similar differences found in the pilot study: see Introduction).

+Scored as number of chicks showing particular behavior, p values calculated from X'-' test Cone-tailed, prediction based on similar differences found in the pilot study).

T A B L E 2

BEHAVIOR OF T AND C CHICKS IN AN OPEN FIELD, WITHOUT A B E L L , AND S U B S E Q U E N T RESPONSE TO A B E L L L O C A T E D

OUTSIDE THE OPEN FIELD

T C Behavior (N = 20) (N - 20t p*

Latency to move leg (sec) 30 35.5 NS

Latency to move from section of the open field (sect 38 77 NS

Time between first leg move and move off section of the open field (see} 3.5 19 <0.05

Latency to peep lifter bell Isec} 105.5 128.5 NS

Latency to move head after bell (sec) 145.5 172.5 NS

The chicks were observed for 3 min in the open field before the bell was sounded for 5 sec, and they were then observed for a further 3 min.

*Mann-Whitney U tesl, 2 tailed.

has previously been classed as emot ional or fear behavior [20 ,25] : (see also [41). These results suggest that whereas f s showed more f requent passive fear reponses, controls showed more active reponses. The results contras t with those found when chicks were placed into a plain open field and a bell sounded from outside after 3 min: in this case there were no differences suggesting an active-passive d i cho tomy in fear behavior; there was also a suggestion that ('s showed more prolonged immobi l i ty than Ts (similar differences were found to be statistically significant in a previous sludy [8] ) . Previous exper iments with an open field also fouml no differences be tween Ts and Cs in active escape responses such as jumping [6 ,8] .

Returning to the original ques t ion posed in the intro- duct ion, of whe the r t es tos te rone influences the tendency to emit fear responses, the answer from the present findings is

that it does, but that the influence is on the form of the fear responses rather than on the overall t endency to show fear behavior. Whether or not these differences occur also depends on the test s i tuat ion: they were not observed on initial exposure to a relatively uni form novel env i ronment , or subsequent ly when an audi tory stinnflus inducing pro- longed freezing was sounded from outside the novel envi ronment . From this second observat ion, it seems unlikely that the di f ferent results in the two si tuat ions can be a t t r ibu ted to d i f ferent intensit ies of fear responses (since freezing is often regarded as the highest intensity fear state 115,161).

One difference between the two si tuat ions is the 3 min period of exposure to the uniform open field, which occurred before the bell was sounded. There is, however, no indicat ion from previous studies [6, 7, 8] that T and (' chicks show different rates of habi tua t ion during initial exposure to a novel envi ronment . Nor is it clear how a difference in habi tua t ion be tween Ts and Cs could account for the aboli t ion of differences in lhe type of fear responding. (lt might have provided a more plausible explanat ion were the differences ones of degree or in- tensity).

It seems more likely that the bell in the center of the open field provided localisation of the major part of the stimulus configurat ion evoking fear behavior, and that the differences between the Ts and (Ts arise from their different responses to this localised stimulus, rather than to any difference in responses shown to fear-ew)king si tuat ions in general or in rates of habi tuat ion. A possible mechanism underlying these different responses is suggested by the findings of an a t tent ional effect of tes tos te rone (see In t roduct ion) : studies of food searching [1,221 and dis- tractabili ty in a learning task [9,211 indicate that testo- sterone induces more prolonged a t ten t ion to a st imulus source once a chick has begun a t tending to it [2,91. So far, this has only been clearly demons t ra ted where a particular type of st imulus has become the salient one li. e. as a result of prior training) although behavioral characterist ics of tes tos terone- t rea ted chicks in o ther si tuations are consis tent with similar a t tent ional changes occurring here too (e.g. fewer head movement s in a novel e n v i r o m n e n t [8} ). In the present s tudy, it is likely that the impact of the bell at the start of the test period is such that (al all birds initially a t tend to it, and tb) it is highly aversive. Since tes tos te rone would induce more prolonged a t tent ion once a bird had begun at tending, we might thus expect Ts to show fewer indications of overt escape and avoidance of 1he bell. The observed increase in immobil i ty might also be a direct effect of more prolonged a t tent ion lo the fear-evoking stimulus since it has been suggested [l~,*[ that immobi l i ty occurs in chicks when a strange, novel or surprising st imulus is fixated. An alternative possibili ty is that more prolonged periods of immobi l i ty represent a greater conflict in tes tos terone- t rea ted birds be tween the tendency to examine or a t tend to the st imulus and the t endency to avoid it: the st imulus would be equally aversive to both Ts and ('s but the former would tend to maintain a t ten t ion to it for longer whereas the latter would more readily show avoidance be- havior. Immobi l i ty can occur during conflicl [181 and its occurrence during ex t inc t ion of a food-rewarded response has been repor ted in mice [24] and in chicks [101. Tes tos terone was found to increase the f requency of such periods of immobi l i ty during ext inc t ion in chicks [ 101 and this finding might also be at t r ibutable to greater confl ict in

564 ARCHER

Ts be tween tendenc ies to a t t end to and to avoid an aversive st imulus ( the e m p t y food dish). There is also a higher incidence of behavior more c o m m o n l y associated with confl ict in t e s tos te rone- t rea ted chicks during ex t inc t ion , e.g. head shaking: [ 10] and adjunctive drinking (Andrew, in p repara t ion) and this would suppor t the confl ict theory for the ex t inc t ion results. Whether the same explanat ion is

appropr ia te for the present findings must await farthe~ studies.

A('KNOWLEDG|, M !,: N'I'S

Supported by a grant from the M.R.( . of the U.K. {o Prol)~so! R. J. Andrew. 1 thank professor Andrew. and Dr .P .1 B, Sla{cr for helpful comments in preparing this paper

REFERENCES

1. Andrew, R. J. Changes in search behavior in male and female 14. Davis, 1). E. and L. V. l)omm, lhe influence o~ hormones on chicks following different doses of testosterone. Anim. Behav. 20: 741-750, 1972.

2. Andrew, R. J. Recognition processes and behavior with special reference to effects of testosterone on persistence. In: Advances in the Study o f Behavior, Vol. 4, edited by D. S. Lehrman, R. A. Hinde and E. Shaw. New York: Academic Press, 1972, pp. 175- 208.

3. Andrew, R. J. Effects of testosterone on the behavior of the domestic chick: I. Effects present in males but not in females. Anita. Behav. 23: 139-155, 1975.

4. Andrew, R. J. Effects of testosterone on the calling o | the domestic chick in a strange environment. Anita. Behav. 23: 169-178, 1975.

5. Andrew, R. J. and L. Rogers. Testosterone, search behavior and persistence.Nature 237: 343-346, 1972.

6. Archer, J. The influence of testosterone on chick behavior in novel environments. Behav. BioL 8: 93- 108, 1973.

7. Archer, J. Effects of testosterone on immobility responses in the young male chick. Behav. Biol. 8:551 556, 1973.

8. Archer, J. A further analysis of responses to a novel environ- ment by testosterone-treated chicks. Behav. Biol. 9:389 396, 1973.

9. Archer, J. The effects of testosterone on the distractability of chicks by irrelevant and relevant novel stimuli. Anita. Behav. 22: 397-404, 1974.

10. Archer, J. Testosterone and behavior during extinction in chicks. Anita. Behav. 22: 650-655, 1974.

11. Archer, J. Emergence tests in testosterone-treated chicks. Physiol. Behav. 16: 513-514, 1976.

12. Archer, J. The organisation of aggression and tear in ver- tebrates. In: Perspectives in Ethology, Vol. 2, edited by P. P. G. Bateson and P. Klopfer. New York: Plenum Press, 1976, pp. 231-298.

13. Broadhurst, P. L. Determinants of emotionality in the rat. 1. Situational factors. Br. J. Psycholo 48: I -12 . 1957.

the sexual behavior of the domestic fowl. In: Essays" in Biology. University of California Press, 1943.

15. (,ray, J. and B. Laljee. Sex differences in emotional behavior in the rat: correlation between open field defecation and active avoidance. Anita. Behav. 22:856 861, 1974.

16, Ginsburg, H. J.. W. G. Braud and R. D. Taylor. Inhibition of distress vocalisation in the ()pen field as a function ,,t heightened fear or arousal in domestic fowl ( Gallus ¢allus). Anita, Behav. 22:745 749, 1974.

17.. Hart, B. L, Gonadal androgen and souiosexual behavior of male mammals: a comparative analysis Pwehol. Bull. 81 : 383.40tk 1974.

18. Hinde, R~ A. Animal Beltavz(~; Ne~ York and I ,ondon McGraw-ttill, 1970, p. 420.

19. Hogan. J. A. An experimental study of conflict and fear: an analysis of behavior of young chicks towards a mealworm. Behavior 25: 45-97, 1965.

20. Kaufman, I. C. and R. A. Hinde. t:actors influencing distress calling in chicks, with special reference to temperature changes and social isolation. An#n. Behav. 9:197 - 204, 196 I.

21. Messent, P. R. Distractability and persistence of chicks. Do Phil. Dissertation, University of Sussex, ! 973.

22. Rogers, L. Persistence and search influenced by natural levels of androgens in young and adull chickens. Physiol. Behav. 12: 197 204, 1974.

23. Siegel, S. Nonparamctrie Statistu s h)r the Beharioral Science;, McGraw-Hill, 1956.

24. Slater, E, P. Some aspects of exploration and lem-ning in three strains of mice. M. Phil. l)issertation. Universily of Sussex. 1972.

25. Sluckin, W., ('. l:ullerton and Ph. Guitun. The developlnent ol aversive responses in domestic chicks. Rev. Comp. Anita. 4: 73 79, 1972.

26. Wood-Gush, D. G. M. the agonisli~ and courtship behavior , t the brown leghorn cock. Bt; ,I. Ittim. Behar, 4 :133 142, 1956.