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Tonic immobility and open field responses in domestic fowl chicks during the first week of life
Applied Animal Behaviour Science 60 Ž1998. 347–357
Tonic immobility and open field responses in domestic fowl chicks during the first week of life R. Heiblum ) , O. Aizenstein, G. Gvaryahu, H. Voet, B. Robinzon, N. Snapir Department of Animal Science, Faculty of Agriculture, The Hebrew UniÕersity of Jerusalem, P.O. Box 12, RehoÕot 76100, Israel Accepted 26 April 1998
Abstract The ontogeny of the tonic immobility ŽTI. response in domestic fowl chicks was studied during the first week of life. The TI response of naive White-Leghorn Gallus domesticus male chicks Ž N s 5–9., was tested at the age of 1, 2, 3, 5 and 7 days. TI was induced dorsally and its duration, the number of induction trials and the latency of peeping were recorded. The TI response was strongly affected by age. It was poorly developed during the first 3 days of life, when the median TI duration in control chicks was 10 s and the mean number of induction trials 2.3 " 0.3. After the third day of life, TI duration increased by up to 15 = and susceptibility by about two. Peeping latencies were very short throughout the first week and in many cases, peeping started long before the termination of TI. Immediately following recovery from TI, chicks were put in an open field and the latencies of walking and jumping and the number of steps, jumps and peeps were observed. No changes in either locomotion or vocalization in an open field were found between the third and fifth day. Furthermore, there was no correlation between any of the parameters of the TI and OF tests. The effect of habituation, which is known to attenuate the TI response, was studied by repeatedly subjecting chicks to TI and OF tests, once on each day of the experiment. Habituation prevented the increase in TI duration and susceptibility after the third day of life, but did not affect the OF response. The effect of aversive treatment, which was expected to increase TI, was examined by placing chicks in 5-cm deep tap water for 5 min, prior to testing. Treatment significantly attenuated TI on Day 1 and increased overall locomotion and peeping in open field. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Chicken—fearful behaviour; Tonic immobility; Open field; Ontogeny; Habituation
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1. Introduction Tonic immobility ŽTI. is a catatonic-like, death feigning, anti-predator response present in many species throughout the animal kingdom ŽRatner, 1967; Sargeant and Eberhard, 1975.. It can be easily induced by a brief period of physical restraint and has been extensively studied in domestic fowl ŽJones, 1986.. The reaction terminates spontaneously seconds to hours after induction, when the animal rights itself. The variable duration of TI is considered to be a measure of the animal’s innate fearfulness ŽJones, 1986.. Earlier reports regarding the ontogeny of the TI response stated that it could not be elicited by dorsal restraining, before the second week of life ŽRatner and Thompson, 1960; Salzen, 1963.. This is in contrast to other fear-related behaviours Že.g., imprinting, freezing in response to a loud noise, eliminating in an open field., which appear earlier than dorsally induced TI ŽJaynes, 1957; Candland et al., 1963; Phillips and Siegel, 1966.. The curious delay reported in the onset of dorsally induced TI and other fear-related behaviours, led us to re-examine the development of the response during the first week of life as compared with another test for fearfulness, the open-field ŽOF. test. ‘Open field’ is a term for a novel arena, in which an animal is placed alone and its behaviour Žlocomotion and vocalization among others., is recorded ŽHarro, 1993.. Although fear of novelty is one variable that affects OF behaviour, it is only one of many other factors, such as social motivation, exploration and territorial marking ŽJones, 1989.. Nevertheless, the OF test has been widely used to estimate fearfulness in chickens ŽJones, 1989.. It is generally accepted that lower locomotion and vocalization indicate higher fearfulness ŽJones, 1977.. We examined how locomotion and vocalization developed with age and how they were related, if at all, to the TI response. What characterizes TI as a fear response is the fact that it is attenuated by procedures that reduce fear and enhanced by ones that increase it. For example, habituation inhibits TI in chicks ŽRatner and Thompson, 1960; Salzen, 1963. and older fowls ŽNash and Gallup, 1976; Gentle et al., 1985.. The inhibition increases with the number of testing stimuli, and can last for over 2 months ŽNash and Gallup, 1976.. In contrast, procedures that increase fear, such as electric shock, loud noise and simulated predation, enhance TI reaction ŽGallup, 1977.. Corticosterone, which is released during emotional or physical stress, potentiates the response ŽJones et al., 1988.. We investigated the effect of habituation and aversive treatment on the TI and OF responses during the first week of life.
2. Methods For the experiment, we obtained 100 White-Leghorn, just-hatched male chicks of similar weights from a local commercial hatchery. The chicks were not immunized, to avoid additional unnecessary stress. They were housed in a space-heated room at 308C with continuous lighting ŽNorth and Bell, 1990.. Commercial food and water were provided ad lib. To ensure uniform rearing conditions, chicks were divided into five groups of 20 each and placed in five cages in the top tier of a three tier battery. The
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experiment was conducted at 1, 2, 3, 5 and 7 days of age. Chicks from only one of the cages were used for each day of the experiment.
3. Procedure All chicks were marked with chicken marks. Half of the chicks from each cage were treated individually by standing them for 5 min in a pint-size cup containing tap water Ž18–208C., 5-cm deep ŽT—treated group.. No holding was necessary. The remaining chicks served as controls ŽC group.. The number of chicks in each group was five to nine. T Žall ages. and C chicks ages 2, 3, 5 and 7 days were tested once. C chicks, which were tested on the first day, were repeatedly tested once on every consecutive day of the experiment ŽDays 2, 3, 5 and 7. and re-named H Žhabituated. group. Treatment and tests were conducted in a room adjacent to the rearing one at a distance of 5 m. Following testing, chicks were marked with a black marker on their head and returned to their cage before the next chick was removed. 3.1. TI test Chicks were tested in a glass terrarium measuring 50 = 30 = 25 cm and lined with newspaper. Each chick was laid on its back, with one hand resting on the sternum and fingers capped lightly over its beak for 15 s. Towards the end of the 15-s period, hand pressure was gradually lifted so that if the chick still moved, another induction period of 15 s is started, until movement ceased. When the chick lay still with one or both legs extended Žalthough sometimes still peeping., it was then released and observed from a position a meter away, out of its sight ŽGallup, 1977.. Latency of vocalization and TI duration was recorded. When duration was less than 3 s, additional induction trials of 15 s each were made, up to a maximum of four trials. Scores of zero for both latency of vocalization and TI duration were given to chicks after four unsuccessful induction trials. These tests lasted until spontaneous recovery from TI occurred. Control and treated chicks were tested alternately throughout the day. 3.2. Open-field test Immediately after the TI test, chicks were placed in the corner of a white plastic box measuring 80 = 80 = 20 cm with a black plastic 0.5 cm mesh net on its floor and a wire net as a cover, and observed for a period of 5 min. Latencies of first step and first jump and the number of step, jumps and peeps were recorded. 3.3. Statistical analysis Values which were widely scattered over two orders of magnitude ŽTI duration, latency of peeping during TI, and latency of walking in OF. are presented as medians with their respective ranges. Other parameters, are presented as means " SEM. Analyses of variance using the General Linear Model procedure ŽSAS, 1994., were performed on
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the ranks of all the variables, because the residuals of the non-transformed values were not normally distributed. One-way ANOVAs were performed for the C and T groups to determine the age effect followed by Tukey’s studentized range Ž a s 0.05. for multiple comparisons. Repeated measures analysis was performed to determine the effect of age in the H group, followed by paired Student’s t-tests and Holm’s modified Bonferroni correction ŽAickin and Gensler, 1996.. T group was compared to C using two-way ANOVA on the ranks. The comparison between H and C included only values for ages 2, 3, 5 and 7 days, and was done by repeated measures two-way ANOVA using a chick Žgroup. nested design. Between-groups comparison to C, for each age were done using one-way ANOVA on the ranks. Correlations were calculated between parameters of coupled TI and OF tests for each chick on each day Ž N s 92..
4. Results 4.1. TI response A chick was considered to be in a TI state when he lay still while the hand was gradually lifted during the last 5 s of the induction period and remained still for at least 3 s. Peeping usually ceased when the chick entered TI, although in very young chicks, it resumed shortly thereafter, intensifying towards the end of TI. LegŽs. extension and tremor were typically seen. The TI response was poorly developed in the first three days of life but, in non-habituated chicks, it was much enhanced on Days 5 and 7. Between Day 3 and Day 5, the median TI duration increased from 10 to 145 s in the C group and from 18 to 59 s in the T group ŽTable 1.. Concomitantly, the number of induction trials, Nt , needed to initiate TI, approximately halved ŽTable 1.. The age effect for TI duration and susceptibility was significant in both C and T groups. In C chicks, although the median TI duration on Day 5 was higher than on Day 7, all the values below and above the median were higher on Day 7. Therefore, TI duration on Day 7 and not the one on Day 5 was significantly higher than TI on the first three days. Habituation prevented the increase in TI duration and susceptibility with age, which resulted in non-significant age effect for these two parameters ŽTable 1.. TI duration in H chicks was significantly shorter than in C chicks on Day 7 and their susceptibility significantly lower on Day 5. Between-groups comparison of H to C Žages 2, 3, 5, and 7 days., showed that TI duration was significantly lower in H than in C Ž F Ž1,27. s 4.48, p - 0.0436., and the number of induction trials higher Ž F Ž1,27. s 8.83, p - 0.0062.. Treatment inhibited the TI response significantly on Day 1, when it was almost impossible to induce TI. Later on, median TI duration and the mean number of induction trials were similar in the T and C groups ŽTable 1.. Overall, the two groups did not differ significantly. The significant decrease in the number of induction trials in T, between Days 1 and 2, which was opposite to the trend in C chicks was reflected in significant age)group interaction Ž F Ž4,54. s 3.11, p - 0.022.. No significant age or group effects were found for peeping latencies ŽTp .. In all groups, peeping latencies were very short. In C chicks, median Tp was 1.5 s on Day 1 and 7 s on Day 7 and the range was 1 to 272 Žmeasured in a chick with TI duration of
Age in days
1 2 3 5 7 Age effect
TI duration Žmedian and range in seconds.
Number of induction trials, Nt ŽMean"SEM.
C
H
T
C
H
T
10Ž4–5.a 12Ž3–40.a 10Ž4–98.a 145Ž5–359.ab 92Ž26–1965.b F Ž4,27. s 4.89, P - 0.0042
10Ž4–15. 19Ž0–92. 11Ž3–30. 30Ž4–58. 18Ž3–240.)) F Ž4,20. s 0.69, P - 0.6090
0Ž0–25.a) 20Ž9–82.b 18Ž4–53.ab 59Ž4–136.b 88Ž16–149.b F Ž4,27. s6.82, P - 0.0006
1.9"0.48ab 2.6"0.38a 1.8"0.37ab 1.0"0.00b 1.2"0.2ab F Ž4,27. s 3.00, P - 0.0358
1.9"0.48 3.1"0.48 2.3"0.33 2.1"0.46) 1.9"0.46 F Ž4,20. s1.47, P - 0.2492
3.4"0.42a) 1.7"0.29b 2.2"0.49ab 1.0"0.00b 1.2"0.2b F Ž4,27. s6.93, P - 0.0006
Significant differences Ž a s 0.05. among ages within groups are indicated with different letters. Among groups, differences from C are marked by ) P - 0.05, )) P - 0.01. N s8 and for all groups Day 1 and H group on Day 2, N s9 for C and T groups on Day 2, N s 5 for C and T groups on days 3 and 5 and N s 7 for H group on Days 5 and 7.
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Table 1 TI response ŽTI duration and number of induction trials. at different ages in C s control, H s habituated and T s treated chicks
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Age in days
Latency of walking, Ts Žmedian and range in seconds.
Number of steps, S ŽMean"SEM.
C
H
T
C
H
T
C
H
T
1 2 3 5 7 Age effect
80Ž21–300.a 8Ž1–89.b 15Ž1–87.ab 20Ž1–77.ab 19Ž8–33.ab F Ž4,27. s 3.38, P - 0.023
80Ž21–300.a 2Ž1–270.ab 1Ž1–7.b) 15Ž2–300.a 1Ž1–300.ab F Ž4,20. s 5.06, P - 0.0055
9.5Ž1–300.) 18Ž2–90. 22Ž7–50. 37Ž1–183. 7Ž1–15. F Ž4,27. s 0.98, P - 0.4364
38"15 44"15 18"7 30"6 30"8 F Ž4,27. s 0.33, P - 0.8551
38"15 22"16 36"21 29"13 37"15 F Ž4,20. s 0.42, P - 0.7937
33"8a 91"15b) 82"23ab 74"11ab)) 36"5a F Ž4,27. s 5.66, P - 0.0019
408"79.4 301"50 303"36 428"27 256"51 F Ž4,27. s1.76, P - 0.165
408"79 273"80 331"74 341"85 212"64 F Ž4–20. s1.32, P - 0.2972
338"51a 496"34b)) 398"45ab 504"29b) 326"62ab F Ž4,27. s 4.57, P - 0.006
Significant differences Ž a s 0.05. among ages within groups are indicated with different letters. Among groups, differences from C are marked by ) P - 0.05, )) P - 0.01. See Table 1 for N.
Number of peeps, P ŽMean"SEM.
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Table 2 OF response at different ages: latency of the walking ŽTs ., number of steps Ž S . and peeps Ž P . in C s control, H s Habituated and T s treated chicks
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1096 s.. Treatment reduced Tp significantly on Day 1 Žfrom 1.5 to 0 s., but did not affect it in subsequent ages. 4.2. Open-field response Latency of walking, Ts , and the number of steps, S, and peeps P, are presented in Table 2. In the C chicks, Ts decreased sharply after Day 1. S and P, however, did not change significantly with age. Habituation reduced Ts significantly on Day 3, but overall, the difference in Ts between H and C was not quite significant Ž F Ž1,27. s 3.36, p - 0.0778.. Habituation did not affect the locomotion or vocalization. Treatment reduced Ts significantly on Day 1 compared with C and increased S and P on Day 2. Locomotion and vocalization remained higher in treated chicks until Day 7, when it decreased to Day 1 values. Overall, T chicks walked and peeped significantly more than C Ž F Ž1,54. s 13.7, p - 0.0005 and F Ž1,54. s 6.1, p - 0.0166, respectively.. Latency of jumping, Tj , and the number of jumps, J, in C chicks did not change significantly with age. In the H group, there was a significant age effect for Tj Ž F Ž4,20. s 3.1, p - 0.0388., due to a decrease from 283 " 11 s on Day 1 to 156 " 52 s on Day 7, when it was significantly lower than Tj of C chicks Ž300 " 0 s.. Although the overall mean number of jumps, J, in the H group was 5.9 " 2.2 compared with 1.3 " 0.65 in C, the difference was not significant in repeated measures ANOVA Ž F Ž1,27. s 2.37, p - 0.1352.. Treatment caused a significant decrease in Tj compared with C. Overall group averages of Tj were 187 " 22 s in T and 261 " 15 s in C Ž F Ž1,54. s 9.98, p - 0.0026.. The lack of correspondence between the development of the TI and the OF responses is further emphasized by the lack of correlation between any of the parameters of the two tests. While there was a significant correlation between almost all parameters of the same test, all correlations between TI and OF were not significant.
5. Discussion We investigated the early development of the TI response in relation to another fear-related behaviour, the OF response. Our findings show that although poorly developed during the first three days of life, TI could be elicited by dorsal induction as early as the first day of life and was much enhanced between the third and fifth day. Earlier reports claimed that the TI response is absent before the age of 7 to 10 days ŽRatner and Thompson, 1960; Salzen, 1963.. Our chicks were undoubtedly in TI because even on Day 1, half of them stayed immobile for 10–15 s Žin addition to a few more seconds during the induction period.. Moreover, like older fowls during TI, chicks in our study also had their legs extended with slight tremor. Genetic differences, which influence the development of fear ŽPhillips and Siegel, 1966. could account for our different findings. Ratner and Thompson used purebred male Cobb chicks, rather than the Leghorns that were used in the present study. Salzen Ž1963. used dorsal induction in
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Brown Leghorn= Light Sussex chicks. Although he claimed that TI did not appear until the eighth day, his results are actually in agreement with ours. His claim was based on the fact that TI duration of more than 5 min was not obtained before the eighth day. However, he did not check the TI response between the third and fourth days and the eighth day, so that an earlier rise in TI could have occurred within that time interval. Indeed, about 20% of Salzen’s chicks between the ages of 1 to 4 days remained immobile for at least 1 min. These findings support our contention that the response can be induced dorsally as early as the first day of life. The sharp increase in TI response between the third and fifth day coincided with the end of the sensitive period for imprinting ŽJaynes, 1957.. Salzen Ž1963. concluded that fear expressed by TI was due to separation from the chick’s companions to which it had been imprinted. As discussed above, due to the way he measured TI, he did not see it earlier than Day 8 and had a problem explaining the difference in the time of onset of fear as shown by the immobility reaction and by the fear responses recorded in imprinting situations. Our study shows that indeed, the two responses coincide and that at the end of sensitive period for imprinting the TI response is markedly increased. Unlike older fowls that peep right before awakening from TI, very young chicks peeped long before. The fact that vocalization is very prominent at an early age can explain it. We did not detect any significant increase after the third day that would correspond to the increase in TI duration and susceptibility. Although peeping is present during the TI response, it might reflect various other impulses aside from fearfulness. Indeed, Montevecchi et al. Ž1973. showed that in contrast to the TI response, peeping in response to loud noise, was not affected by tranquilizers or by proximity of the experimenter ŽGallup, 1977.. We examined the behaviour in OF that has been used to assess fearfulness in domestic fowl ŽJones, 1989.. We did not find any changes with age, in any of the parameters checked that coincided with the increase in TI. There was no correlation between locomotion or vocalization and TI, which is contrary to findings of studies on older fowls ŽJones et al., 1991; Jones and Waddington, 1992; Moriarty, 1995.. Other studies, though, found an increase in locomotion and vocalization with no change in TI ŽJones and Andrew, 1992; Jones et al., 1995.. An explanation to the difference between TI and OF responses could be that activity in OF reflects underlying social motivation rather than fearfulness. Kruijt Ž1964. emphasized that alert posture, head shaking, peeping, and running are present in approach as well as in avoidance behaviour of young chicks. Jones Ž1989. stated that OF response represents a compromise between opposing tendencies to regain contact with the chick’s companions and to minimize detection by potential predators. There was a decrease in the latency of walking in OF after the first day of life. This is in agreement with Montevecchi et al. Ž1973. who found a decrease in latency of peeping and locomotion from Day 1 to Day 2. We also observed a significant increase in walking between the first and second day of life in T chicks as did Kruijt Ž1964., who saw an increase in running in Jungle fowl at the same age. But the fact that we did not see a similar increase in C chicks remains unclear. In agreement with what has been reported in young chicks ŽRatner and Thompson, 1960; Salzen, 1963. and adult fowls ŽGallup, 1977; Nash, 1978; Gentle et al., 1985., we
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found that habituation attenuated the TI response. Our study indicates that three one-a-day tests were sufficient to inhibit the increase in TI duration and susceptibility on the fifth day Žlatency of peeping during TI was not affected.. Ratner and Thompson Ž1960. also observed an attenuation of TI duration at the onset of the TI response Žin their study, on the ninth day post-hatch. after five one-a-day tests. Moreover, in that same study, chicks that were tested just once at the age of 10 days showed habituation when re-tested a week later. Thus, habituation to the testing conditions easily counteracts the fear that they initially elicit. Habituation reduced latencies of walking in OF but did not affect locomotion or vocalization. Jones and Waddington Ž1992. showed that regular handling of domestic fowl chicks did not affect OF behaviour, but we were unable to find comparable reports on the effect of repeated OF testing in birds. In mice ŽFrances et al., 1996. and rats ŽMolinengo, 1995. habituation reduced locomotion, due mainly to reduction in exploratory activity. If we assume that reduction of fearfulness increases OF activity and habituation reduces exploratory activity, then the lack of change is understandable. Chicks were subjected to social Žisolation. and physical Žcold. stress to investigate its effects on the fear-related behaviours of the young chick. Isolation of 3-week-old chicks increased their TI response ŽEddy and Gallup, 1990.. Cold stress leads to an increase in corticosterone levels ŽNir et al., 1975., which also potentiates the TI response ŽJones et al., 1988.. Thus, we expected to see an increase of the TI response but found, instead, inhibition on Day 1 and no significant differences thereafter. As in the C group, TI duration and susceptibility of T chicks increased after the third day but was significant only compared with Day 1. One possible explanation is that the two components of stress trigger opposite responses. While isolation may enhance TI, cold stress might attenuate it. Immobility is hardly the proper response to cold stress, when extra muscular activity is needed for heat production. This is especially true for very young chicks, who are essentially ectothermic ŽWhittow, 1986.. Moreover, although Rovee-Collier et al. Ž1991. reported a rise in core temperature during immobility, Nash et al. Ž1976. and Eddy and Gallup Ž1990. found that it drops during the TI response. Naturally, this reduction in temperature represents an extra burden on thermoregulation under cold stress. The reason for the strong inhibition of TI in day-old chicks might be that they are even more susceptible to cold stress than the slightly older chicks. Indeed, in their study of the effect of temperature on TI, Rovee-Collier et al. Ž1991. found that TI is inhibited at temperatures that markedly differ from pre-adapted rearing temperature. They concluded that when immobilization and behavioural thermoregulation present conflicting survival demands, the control of core temperature assumes behavioural priority. Because young chicks are very sensitive to cold, it is likely that the overriding response would be to keep warm. Increased need for heat production can also explain the increased locomotion and vocalization of T chicks in OF between ages 2 and 5 days. At the age of 7 days, there was no difference in the number of steps and peeps, possibly indicating improved thermoregulation. This study emphasizes the importance of the first 3 days of life, as far as being a low-fearfulness period, as well as the sensitive period for imprinting. Exposing chicks at this time to a new environment or any fear-eliciting factors, such as human handlers or loud noise can be of great consequence.
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The fact that TI is well developed at the age of 7 days makes it a useful tool in assessing the effect of various pre and peri-hatch treatments on the fearful behaviour of chicks, as well as for early screening for selection of low-fear birds.
Acknowledgements This study was supported by a donation by the late Mrs. Della Maiden. We wish to thank Mr. Sasson Ya’ish for his assistance during the study and Ms. N. Heiblum for her critical reading of the manuscript.
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Moriarty, D.D., 1995. Anxiogenic effects of a beta-carboline on tonic immobility and open field behaviour in chickens Ž Gallus gallus .. Pharmacol. Biochem. Behav. 51, 795–798. Nash, R.F., 1978. Habituation and tonic immobility in chicken: strain comparisons. Psychol. Rec. 28, 109–114. Nash, R.F., Gallup, G.G. Jr., 1976. Habituation and tonic immobility in domestic chickens. J. Comp. Physiol. Psychol. 90, 870–876. Nash, R.F., Gallup, G.G. Jr., Czech, D.A., 1976. Psychophysiological correlates of tonic immobility in the domestic chicken Ž Gallus gallus .. Physiol. Behav. 17, 413–418. Nir, I., Yam, D., Perek, M., 1975. Effects of stress on the corticosterone content of the blood plasma and adrenal gland of intact and bursectomized Gallus domesticus. Poult. Sci. 54, 2101–2110. North, M.O., Bell, D.D., 1990. Commercial Chicken production Manual. 4th edn., Chapman & Hall, New York, p. 416. Phillips, R.E., Siegel, P.B., 1966. Development of fear in chicks of two closely related genetic lines. Anim. Behav. 14, 84–88. Ratner, S.C., 1967. Comparative aspects of hypnosis. In: Gordon, E. ŽEd.., Handbook in Clinical and Experimental Hypnosis. Macmillan, New York, pp. 550–587. Ratner, S.C., Thompson, R.W., 1960. Immobility reactions Žfear. of domestic fowl as a function of age and prior experience. Anim. Behav. 8, 186–191. Rovee-Collier, C.K., Kupersmidt, J., O’Brien, I., Collier, G., Tepper, V., 1991. Behavioural thermoregulation and immobilization: conflicting demands for survival. J. Comp. Psychol. 105, 232–242. Salzen, E.A., 1963. Imprinting and the immobility reactions of domestic fowl. Anim. Behav. 11, 66–71. Sargeant, A.B., Eberhard, L.E., 1975. Death feigning in response to predation by red foxes Ž Vulpes fulÕa.. Am. Midland Naturalist 94, 108–119. SAS Institute, 1994. SASrSTAT w User’s Guide. Version 6, 4th edn., Cary, NC, SAS Institute. Whittow, G.C., 1986. Regulation of body temperature. In: Sturkie, P.D. ŽEd.., Avian Physiology, 4th edn. Springer-Verlag, New York, pp. 246–248.
Tonic immobility and open field responses in domestic fowl chicks during the first week of life R. Heiblum ) , O. Aizenstein, G. Gvaryahu, H. Voet, B. Robinzon, N. Snapir Department of Animal Science, Faculty of Agriculture, The Hebrew UniÕersity of Jerusalem, P.O. Box 12, RehoÕot 76100, Israel Accepted 26 April 1998
Abstract The ontogeny of the tonic immobility ŽTI. response in domestic fowl chicks was studied during the first week of life. The TI response of naive White-Leghorn Gallus domesticus male chicks Ž N s 5–9., was tested at the age of 1, 2, 3, 5 and 7 days. TI was induced dorsally and its duration, the number of induction trials and the latency of peeping were recorded. The TI response was strongly affected by age. It was poorly developed during the first 3 days of life, when the median TI duration in control chicks was 10 s and the mean number of induction trials 2.3 " 0.3. After the third day of life, TI duration increased by up to 15 = and susceptibility by about two. Peeping latencies were very short throughout the first week and in many cases, peeping started long before the termination of TI. Immediately following recovery from TI, chicks were put in an open field and the latencies of walking and jumping and the number of steps, jumps and peeps were observed. No changes in either locomotion or vocalization in an open field were found between the third and fifth day. Furthermore, there was no correlation between any of the parameters of the TI and OF tests. The effect of habituation, which is known to attenuate the TI response, was studied by repeatedly subjecting chicks to TI and OF tests, once on each day of the experiment. Habituation prevented the increase in TI duration and susceptibility after the third day of life, but did not affect the OF response. The effect of aversive treatment, which was expected to increase TI, was examined by placing chicks in 5-cm deep tap water for 5 min, prior to testing. Treatment significantly attenuated TI on Day 1 and increased overall locomotion and peeping in open field. q 1998 Elsevier Science B.V. All rights reserved. Keywords: Chicken—fearful behaviour; Tonic immobility; Open field; Ontogeny; Habituation
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1. Introduction Tonic immobility ŽTI. is a catatonic-like, death feigning, anti-predator response present in many species throughout the animal kingdom ŽRatner, 1967; Sargeant and Eberhard, 1975.. It can be easily induced by a brief period of physical restraint and has been extensively studied in domestic fowl ŽJones, 1986.. The reaction terminates spontaneously seconds to hours after induction, when the animal rights itself. The variable duration of TI is considered to be a measure of the animal’s innate fearfulness ŽJones, 1986.. Earlier reports regarding the ontogeny of the TI response stated that it could not be elicited by dorsal restraining, before the second week of life ŽRatner and Thompson, 1960; Salzen, 1963.. This is in contrast to other fear-related behaviours Že.g., imprinting, freezing in response to a loud noise, eliminating in an open field., which appear earlier than dorsally induced TI ŽJaynes, 1957; Candland et al., 1963; Phillips and Siegel, 1966.. The curious delay reported in the onset of dorsally induced TI and other fear-related behaviours, led us to re-examine the development of the response during the first week of life as compared with another test for fearfulness, the open-field ŽOF. test. ‘Open field’ is a term for a novel arena, in which an animal is placed alone and its behaviour Žlocomotion and vocalization among others., is recorded ŽHarro, 1993.. Although fear of novelty is one variable that affects OF behaviour, it is only one of many other factors, such as social motivation, exploration and territorial marking ŽJones, 1989.. Nevertheless, the OF test has been widely used to estimate fearfulness in chickens ŽJones, 1989.. It is generally accepted that lower locomotion and vocalization indicate higher fearfulness ŽJones, 1977.. We examined how locomotion and vocalization developed with age and how they were related, if at all, to the TI response. What characterizes TI as a fear response is the fact that it is attenuated by procedures that reduce fear and enhanced by ones that increase it. For example, habituation inhibits TI in chicks ŽRatner and Thompson, 1960; Salzen, 1963. and older fowls ŽNash and Gallup, 1976; Gentle et al., 1985.. The inhibition increases with the number of testing stimuli, and can last for over 2 months ŽNash and Gallup, 1976.. In contrast, procedures that increase fear, such as electric shock, loud noise and simulated predation, enhance TI reaction ŽGallup, 1977.. Corticosterone, which is released during emotional or physical stress, potentiates the response ŽJones et al., 1988.. We investigated the effect of habituation and aversive treatment on the TI and OF responses during the first week of life.
2. Methods For the experiment, we obtained 100 White-Leghorn, just-hatched male chicks of similar weights from a local commercial hatchery. The chicks were not immunized, to avoid additional unnecessary stress. They were housed in a space-heated room at 308C with continuous lighting ŽNorth and Bell, 1990.. Commercial food and water were provided ad lib. To ensure uniform rearing conditions, chicks were divided into five groups of 20 each and placed in five cages in the top tier of a three tier battery. The
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experiment was conducted at 1, 2, 3, 5 and 7 days of age. Chicks from only one of the cages were used for each day of the experiment.
3. Procedure All chicks were marked with chicken marks. Half of the chicks from each cage were treated individually by standing them for 5 min in a pint-size cup containing tap water Ž18–208C., 5-cm deep ŽT—treated group.. No holding was necessary. The remaining chicks served as controls ŽC group.. The number of chicks in each group was five to nine. T Žall ages. and C chicks ages 2, 3, 5 and 7 days were tested once. C chicks, which were tested on the first day, were repeatedly tested once on every consecutive day of the experiment ŽDays 2, 3, 5 and 7. and re-named H Žhabituated. group. Treatment and tests were conducted in a room adjacent to the rearing one at a distance of 5 m. Following testing, chicks were marked with a black marker on their head and returned to their cage before the next chick was removed. 3.1. TI test Chicks were tested in a glass terrarium measuring 50 = 30 = 25 cm and lined with newspaper. Each chick was laid on its back, with one hand resting on the sternum and fingers capped lightly over its beak for 15 s. Towards the end of the 15-s period, hand pressure was gradually lifted so that if the chick still moved, another induction period of 15 s is started, until movement ceased. When the chick lay still with one or both legs extended Žalthough sometimes still peeping., it was then released and observed from a position a meter away, out of its sight ŽGallup, 1977.. Latency of vocalization and TI duration was recorded. When duration was less than 3 s, additional induction trials of 15 s each were made, up to a maximum of four trials. Scores of zero for both latency of vocalization and TI duration were given to chicks after four unsuccessful induction trials. These tests lasted until spontaneous recovery from TI occurred. Control and treated chicks were tested alternately throughout the day. 3.2. Open-field test Immediately after the TI test, chicks were placed in the corner of a white plastic box measuring 80 = 80 = 20 cm with a black plastic 0.5 cm mesh net on its floor and a wire net as a cover, and observed for a period of 5 min. Latencies of first step and first jump and the number of step, jumps and peeps were recorded. 3.3. Statistical analysis Values which were widely scattered over two orders of magnitude ŽTI duration, latency of peeping during TI, and latency of walking in OF. are presented as medians with their respective ranges. Other parameters, are presented as means " SEM. Analyses of variance using the General Linear Model procedure ŽSAS, 1994., were performed on
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the ranks of all the variables, because the residuals of the non-transformed values were not normally distributed. One-way ANOVAs were performed for the C and T groups to determine the age effect followed by Tukey’s studentized range Ž a s 0.05. for multiple comparisons. Repeated measures analysis was performed to determine the effect of age in the H group, followed by paired Student’s t-tests and Holm’s modified Bonferroni correction ŽAickin and Gensler, 1996.. T group was compared to C using two-way ANOVA on the ranks. The comparison between H and C included only values for ages 2, 3, 5 and 7 days, and was done by repeated measures two-way ANOVA using a chick Žgroup. nested design. Between-groups comparison to C, for each age were done using one-way ANOVA on the ranks. Correlations were calculated between parameters of coupled TI and OF tests for each chick on each day Ž N s 92..
4. Results 4.1. TI response A chick was considered to be in a TI state when he lay still while the hand was gradually lifted during the last 5 s of the induction period and remained still for at least 3 s. Peeping usually ceased when the chick entered TI, although in very young chicks, it resumed shortly thereafter, intensifying towards the end of TI. LegŽs. extension and tremor were typically seen. The TI response was poorly developed in the first three days of life but, in non-habituated chicks, it was much enhanced on Days 5 and 7. Between Day 3 and Day 5, the median TI duration increased from 10 to 145 s in the C group and from 18 to 59 s in the T group ŽTable 1.. Concomitantly, the number of induction trials, Nt , needed to initiate TI, approximately halved ŽTable 1.. The age effect for TI duration and susceptibility was significant in both C and T groups. In C chicks, although the median TI duration on Day 5 was higher than on Day 7, all the values below and above the median were higher on Day 7. Therefore, TI duration on Day 7 and not the one on Day 5 was significantly higher than TI on the first three days. Habituation prevented the increase in TI duration and susceptibility with age, which resulted in non-significant age effect for these two parameters ŽTable 1.. TI duration in H chicks was significantly shorter than in C chicks on Day 7 and their susceptibility significantly lower on Day 5. Between-groups comparison of H to C Žages 2, 3, 5, and 7 days., showed that TI duration was significantly lower in H than in C Ž F Ž1,27. s 4.48, p - 0.0436., and the number of induction trials higher Ž F Ž1,27. s 8.83, p - 0.0062.. Treatment inhibited the TI response significantly on Day 1, when it was almost impossible to induce TI. Later on, median TI duration and the mean number of induction trials were similar in the T and C groups ŽTable 1.. Overall, the two groups did not differ significantly. The significant decrease in the number of induction trials in T, between Days 1 and 2, which was opposite to the trend in C chicks was reflected in significant age)group interaction Ž F Ž4,54. s 3.11, p - 0.022.. No significant age or group effects were found for peeping latencies ŽTp .. In all groups, peeping latencies were very short. In C chicks, median Tp was 1.5 s on Day 1 and 7 s on Day 7 and the range was 1 to 272 Žmeasured in a chick with TI duration of
Age in days
1 2 3 5 7 Age effect
TI duration Žmedian and range in seconds.
Number of induction trials, Nt ŽMean"SEM.
C
H
T
C
H
T
10Ž4–5.a 12Ž3–40.a 10Ž4–98.a 145Ž5–359.ab 92Ž26–1965.b F Ž4,27. s 4.89, P - 0.0042
10Ž4–15. 19Ž0–92. 11Ž3–30. 30Ž4–58. 18Ž3–240.)) F Ž4,20. s 0.69, P - 0.6090
0Ž0–25.a) 20Ž9–82.b 18Ž4–53.ab 59Ž4–136.b 88Ž16–149.b F Ž4,27. s6.82, P - 0.0006
1.9"0.48ab 2.6"0.38a 1.8"0.37ab 1.0"0.00b 1.2"0.2ab F Ž4,27. s 3.00, P - 0.0358
1.9"0.48 3.1"0.48 2.3"0.33 2.1"0.46) 1.9"0.46 F Ž4,20. s1.47, P - 0.2492
3.4"0.42a) 1.7"0.29b 2.2"0.49ab 1.0"0.00b 1.2"0.2b F Ž4,27. s6.93, P - 0.0006
Significant differences Ž a s 0.05. among ages within groups are indicated with different letters. Among groups, differences from C are marked by ) P - 0.05, )) P - 0.01. N s8 and for all groups Day 1 and H group on Day 2, N s9 for C and T groups on Day 2, N s 5 for C and T groups on days 3 and 5 and N s 7 for H group on Days 5 and 7.
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Table 1 TI response ŽTI duration and number of induction trials. at different ages in C s control, H s habituated and T s treated chicks
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352
Age in days
Latency of walking, Ts Žmedian and range in seconds.
Number of steps, S ŽMean"SEM.
C
H
T
C
H
T
C
H
T
1 2 3 5 7 Age effect
80Ž21–300.a 8Ž1–89.b 15Ž1–87.ab 20Ž1–77.ab 19Ž8–33.ab F Ž4,27. s 3.38, P - 0.023
80Ž21–300.a 2Ž1–270.ab 1Ž1–7.b) 15Ž2–300.a 1Ž1–300.ab F Ž4,20. s 5.06, P - 0.0055
9.5Ž1–300.) 18Ž2–90. 22Ž7–50. 37Ž1–183. 7Ž1–15. F Ž4,27. s 0.98, P - 0.4364
38"15 44"15 18"7 30"6 30"8 F Ž4,27. s 0.33, P - 0.8551
38"15 22"16 36"21 29"13 37"15 F Ž4,20. s 0.42, P - 0.7937
33"8a 91"15b) 82"23ab 74"11ab)) 36"5a F Ž4,27. s 5.66, P - 0.0019
408"79.4 301"50 303"36 428"27 256"51 F Ž4,27. s1.76, P - 0.165
408"79 273"80 331"74 341"85 212"64 F Ž4–20. s1.32, P - 0.2972
338"51a 496"34b)) 398"45ab 504"29b) 326"62ab F Ž4,27. s 4.57, P - 0.006
Significant differences Ž a s 0.05. among ages within groups are indicated with different letters. Among groups, differences from C are marked by ) P - 0.05, )) P - 0.01. See Table 1 for N.
Number of peeps, P ŽMean"SEM.
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Table 2 OF response at different ages: latency of the walking ŽTs ., number of steps Ž S . and peeps Ž P . in C s control, H s Habituated and T s treated chicks
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1096 s.. Treatment reduced Tp significantly on Day 1 Žfrom 1.5 to 0 s., but did not affect it in subsequent ages. 4.2. Open-field response Latency of walking, Ts , and the number of steps, S, and peeps P, are presented in Table 2. In the C chicks, Ts decreased sharply after Day 1. S and P, however, did not change significantly with age. Habituation reduced Ts significantly on Day 3, but overall, the difference in Ts between H and C was not quite significant Ž F Ž1,27. s 3.36, p - 0.0778.. Habituation did not affect the locomotion or vocalization. Treatment reduced Ts significantly on Day 1 compared with C and increased S and P on Day 2. Locomotion and vocalization remained higher in treated chicks until Day 7, when it decreased to Day 1 values. Overall, T chicks walked and peeped significantly more than C Ž F Ž1,54. s 13.7, p - 0.0005 and F Ž1,54. s 6.1, p - 0.0166, respectively.. Latency of jumping, Tj , and the number of jumps, J, in C chicks did not change significantly with age. In the H group, there was a significant age effect for Tj Ž F Ž4,20. s 3.1, p - 0.0388., due to a decrease from 283 " 11 s on Day 1 to 156 " 52 s on Day 7, when it was significantly lower than Tj of C chicks Ž300 " 0 s.. Although the overall mean number of jumps, J, in the H group was 5.9 " 2.2 compared with 1.3 " 0.65 in C, the difference was not significant in repeated measures ANOVA Ž F Ž1,27. s 2.37, p - 0.1352.. Treatment caused a significant decrease in Tj compared with C. Overall group averages of Tj were 187 " 22 s in T and 261 " 15 s in C Ž F Ž1,54. s 9.98, p - 0.0026.. The lack of correspondence between the development of the TI and the OF responses is further emphasized by the lack of correlation between any of the parameters of the two tests. While there was a significant correlation between almost all parameters of the same test, all correlations between TI and OF were not significant.
5. Discussion We investigated the early development of the TI response in relation to another fear-related behaviour, the OF response. Our findings show that although poorly developed during the first three days of life, TI could be elicited by dorsal induction as early as the first day of life and was much enhanced between the third and fifth day. Earlier reports claimed that the TI response is absent before the age of 7 to 10 days ŽRatner and Thompson, 1960; Salzen, 1963.. Our chicks were undoubtedly in TI because even on Day 1, half of them stayed immobile for 10–15 s Žin addition to a few more seconds during the induction period.. Moreover, like older fowls during TI, chicks in our study also had their legs extended with slight tremor. Genetic differences, which influence the development of fear ŽPhillips and Siegel, 1966. could account for our different findings. Ratner and Thompson used purebred male Cobb chicks, rather than the Leghorns that were used in the present study. Salzen Ž1963. used dorsal induction in
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Brown Leghorn= Light Sussex chicks. Although he claimed that TI did not appear until the eighth day, his results are actually in agreement with ours. His claim was based on the fact that TI duration of more than 5 min was not obtained before the eighth day. However, he did not check the TI response between the third and fourth days and the eighth day, so that an earlier rise in TI could have occurred within that time interval. Indeed, about 20% of Salzen’s chicks between the ages of 1 to 4 days remained immobile for at least 1 min. These findings support our contention that the response can be induced dorsally as early as the first day of life. The sharp increase in TI response between the third and fifth day coincided with the end of the sensitive period for imprinting ŽJaynes, 1957.. Salzen Ž1963. concluded that fear expressed by TI was due to separation from the chick’s companions to which it had been imprinted. As discussed above, due to the way he measured TI, he did not see it earlier than Day 8 and had a problem explaining the difference in the time of onset of fear as shown by the immobility reaction and by the fear responses recorded in imprinting situations. Our study shows that indeed, the two responses coincide and that at the end of sensitive period for imprinting the TI response is markedly increased. Unlike older fowls that peep right before awakening from TI, very young chicks peeped long before. The fact that vocalization is very prominent at an early age can explain it. We did not detect any significant increase after the third day that would correspond to the increase in TI duration and susceptibility. Although peeping is present during the TI response, it might reflect various other impulses aside from fearfulness. Indeed, Montevecchi et al. Ž1973. showed that in contrast to the TI response, peeping in response to loud noise, was not affected by tranquilizers or by proximity of the experimenter ŽGallup, 1977.. We examined the behaviour in OF that has been used to assess fearfulness in domestic fowl ŽJones, 1989.. We did not find any changes with age, in any of the parameters checked that coincided with the increase in TI. There was no correlation between locomotion or vocalization and TI, which is contrary to findings of studies on older fowls ŽJones et al., 1991; Jones and Waddington, 1992; Moriarty, 1995.. Other studies, though, found an increase in locomotion and vocalization with no change in TI ŽJones and Andrew, 1992; Jones et al., 1995.. An explanation to the difference between TI and OF responses could be that activity in OF reflects underlying social motivation rather than fearfulness. Kruijt Ž1964. emphasized that alert posture, head shaking, peeping, and running are present in approach as well as in avoidance behaviour of young chicks. Jones Ž1989. stated that OF response represents a compromise between opposing tendencies to regain contact with the chick’s companions and to minimize detection by potential predators. There was a decrease in the latency of walking in OF after the first day of life. This is in agreement with Montevecchi et al. Ž1973. who found a decrease in latency of peeping and locomotion from Day 1 to Day 2. We also observed a significant increase in walking between the first and second day of life in T chicks as did Kruijt Ž1964., who saw an increase in running in Jungle fowl at the same age. But the fact that we did not see a similar increase in C chicks remains unclear. In agreement with what has been reported in young chicks ŽRatner and Thompson, 1960; Salzen, 1963. and adult fowls ŽGallup, 1977; Nash, 1978; Gentle et al., 1985., we
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found that habituation attenuated the TI response. Our study indicates that three one-a-day tests were sufficient to inhibit the increase in TI duration and susceptibility on the fifth day Žlatency of peeping during TI was not affected.. Ratner and Thompson Ž1960. also observed an attenuation of TI duration at the onset of the TI response Žin their study, on the ninth day post-hatch. after five one-a-day tests. Moreover, in that same study, chicks that were tested just once at the age of 10 days showed habituation when re-tested a week later. Thus, habituation to the testing conditions easily counteracts the fear that they initially elicit. Habituation reduced latencies of walking in OF but did not affect locomotion or vocalization. Jones and Waddington Ž1992. showed that regular handling of domestic fowl chicks did not affect OF behaviour, but we were unable to find comparable reports on the effect of repeated OF testing in birds. In mice ŽFrances et al., 1996. and rats ŽMolinengo, 1995. habituation reduced locomotion, due mainly to reduction in exploratory activity. If we assume that reduction of fearfulness increases OF activity and habituation reduces exploratory activity, then the lack of change is understandable. Chicks were subjected to social Žisolation. and physical Žcold. stress to investigate its effects on the fear-related behaviours of the young chick. Isolation of 3-week-old chicks increased their TI response ŽEddy and Gallup, 1990.. Cold stress leads to an increase in corticosterone levels ŽNir et al., 1975., which also potentiates the TI response ŽJones et al., 1988.. Thus, we expected to see an increase of the TI response but found, instead, inhibition on Day 1 and no significant differences thereafter. As in the C group, TI duration and susceptibility of T chicks increased after the third day but was significant only compared with Day 1. One possible explanation is that the two components of stress trigger opposite responses. While isolation may enhance TI, cold stress might attenuate it. Immobility is hardly the proper response to cold stress, when extra muscular activity is needed for heat production. This is especially true for very young chicks, who are essentially ectothermic ŽWhittow, 1986.. Moreover, although Rovee-Collier et al. Ž1991. reported a rise in core temperature during immobility, Nash et al. Ž1976. and Eddy and Gallup Ž1990. found that it drops during the TI response. Naturally, this reduction in temperature represents an extra burden on thermoregulation under cold stress. The reason for the strong inhibition of TI in day-old chicks might be that they are even more susceptible to cold stress than the slightly older chicks. Indeed, in their study of the effect of temperature on TI, Rovee-Collier et al. Ž1991. found that TI is inhibited at temperatures that markedly differ from pre-adapted rearing temperature. They concluded that when immobilization and behavioural thermoregulation present conflicting survival demands, the control of core temperature assumes behavioural priority. Because young chicks are very sensitive to cold, it is likely that the overriding response would be to keep warm. Increased need for heat production can also explain the increased locomotion and vocalization of T chicks in OF between ages 2 and 5 days. At the age of 7 days, there was no difference in the number of steps and peeps, possibly indicating improved thermoregulation. This study emphasizes the importance of the first 3 days of life, as far as being a low-fearfulness period, as well as the sensitive period for imprinting. Exposing chicks at this time to a new environment or any fear-eliciting factors, such as human handlers or loud noise can be of great consequence.
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The fact that TI is well developed at the age of 7 days makes it a useful tool in assessing the effect of various pre and peri-hatch treatments on the fearful behaviour of chicks, as well as for early screening for selection of low-fear birds.
Acknowledgements This study was supported by a donation by the late Mrs. Della Maiden. We wish to thank Mr. Sasson Ya’ish for his assistance during the study and Ms. N. Heiblum for her critical reading of the manuscript.
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