Effect of intermingling chicks and bird density on fear and stress responses in chickens

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1 Arch.Geflügelk., 69 (5). S , 2005, ISSN Verlag Eugen Ulmer KG, Stuttgart Effect of intermingling chicks and bird density on fear and stress responses in chickens Einfluss des Vermischens der Küken und der Besatzdichte auf die Furcht und Stressreaktion von Hühnern J.L. Campo, M.G. Gil and S.G. Davila Manuskript eingegangen am 26. Februar 2004, angenommen am 1. Mai 2004 Introduction Housing environment can be a source of social stress. While there has been substantial work on the effect of intermingling chicks of diverse genetic background on ingestive behaviour (SAVORY 1982; NOBLE et al. 1993; MAHAGNA et al. 1994), there is a lack of information on the effect on social behaviour and welfare of the birds. Previous studies have shown that chronic social stress can be induced in chickens by intermingling individuals from different strains (GVARYAHU et al. 1996). SAVORY (1975) found that intermingling different strains, the behaviour pattern shown by one strain did not affect the other. MEUNI- ER-SALAÜN and FAURE (1984) reported that whether or not the birds had been raised intermingled or separate had no clear effect on their welfare. ZULKIFLI et al. (1998) found that commercial broilers were stressed when intermingled with jungle fowl. The intermingled flock, while had no influence on the stress response of jungle fowl, elevated heterophil to lymphocyte ratio of their commercial broiler counterparts. On the other hand, results on the effect of bird density on indicators of fear and stress are contradictory. LEE (1989) reported that floor space allowance, group size, or area density (a combination of the two) had no effect on fearfulness of the birds housed in litter floor pens. Similarly, cage density did not affect fear-related response of the birds (CUNNINGHAM and OSTRANDER 1982; CRAIG et al. 1986; RAMOS et al. 1986; LEE and MOSS 1995). On the contrary, KUJIYAT et al. (1983) reported that the social environment in a cage with large group size increased fearfulness, and ANDREWS et al. (1997) found that the broiler chickens that had been stocked at the high density showed the longest tonic immobility. Besides, BILCIK et al. (1998) reported that duration of tonic immobility increased with group size in laying hens raised in floor pens. On the other hand, HESTER et al. (1996), and PATTERSON and SIEGEL (1998) reported that cage density had no significant effect on the heterophil to lymphocyte ratio, and similarly, no significant differences in plasma corticosterone concentration between population sizes were observed by DAVAMI et al. (1987). Although MASHALY et al. (1984) found that corticosterone concentrations were consistently higher in White Leghorn hens housed five per cage than in hens housed at three or Dep. de Genetica Animal, Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentatia, Madrid, Spain four per cage, PESTI and HOWARTH (1983) reported that increasing cage density significantly decreased plasma corticosterone. The purposes of the present study were to evaluate the effects of intermingling chicks and bird density on the duration of the tonic immobility reaction and the heterophil to lymphocyte ratio, the two more adequate measures of fear and stress in chickens (GALLUP 1979; GROSS and SIEGEL 1983). Relative to the existing literature, Experiment 1 analyses for the first time the relationship between intermingling and fear, and complements the only previous experiment to analyse the relationship between intermingling and heterophil to lymphocyte ratio with meat animals. On the other hand, Experiment 2 complements, with rearing birds, the experiments analysing the relationship between bird density and fear (two with layers in floor pens, five with caged layers, and one with broilers), and the two experiments analysing the relationship between bird density and heterophil to lymphocyte ratio in caged layers. Materials and Methods In both experiments birds from each breed were grouped from 1 d of age, in intermingled (1:1) or separated floor pens with straw litter and a group size of 150 chicks (Experiment 1), and in separated floor pens with a group size ranging from 150 to 750 chicks (Experiment 2). They were reared with a density of 10 chicks/ m 2 until 8 wk of age. Artificial light was only provided during the first week of age (23L: 1D). Temperature was controlled with gas heaters (33-35ºC at the chick level during the first week followed by a reduction of 3ºC each week until to reach 18-20ºC in the sixth week of age). Birds were fed standard rearing diet, containing 19% CP, 2,800 kcal ME/kg, 1% Ca, and 0.5% P until 8 wk, and 15% CP, 2,700 kcal ME/kg, 0.9% Ca, and 0.4% P until 20 wk of age. Four different Spanish breeds of chickens (Black Castellana, Red Villafranquina, Barred Red Vasca, and Buff Prat), and a synthetic breed (Quail Castellana) were used. The Black Castellana is a white-shelled egg layer, whereas the Buff Prat is a tinted shell egg layer; and the Barred Red Vasca and Red Villafranquina lay brown- and dark brown-shelled eggs, respectively. The Quail Castellana originated from a F 2 cross between Black Castellana and Buff Prat (CAMPO and OROZCO 1986; CAMPO 1991). All these breeds are maintained at the Experimental Station of El Encín (Madrid, Spain) in a conservation program of genetic resources started in 1975 (CAMPO and OROZCO 1982) and have been

2 200 Campo et al.: Fear and stress responses in chickens described by CAMPO (1998). They are used in free-range systems for the commercial production of quality eggs. On two different days, birds were tested for heterophil to lymphocyte ratio and tonic immobility duration at 20 wk of age. Tonic immobility was tested on the day following the test for blood sampling. Birds were gentle caught with a hand hook. In Experiment 1, there were three replicates at intervals of seven days. In replicate 1, 18 birds were randomly sampled from the intermingled floor pen (seven from the Black Castellana and 11 from the Quail Castellana breed) and then 18 birds were randomly sampled from the two separate floor pens (seven and 11 from that of each breed). One bird from the intermingled Quail Castellana died before tonic immobility was tested so the final of birds was seven and 10, respectively. Replicate 2 was similar to replicate 1. In replicate 3, 19 birds were randomly sampled from the intermingled floor pen (six from the Black Castellana and 13 from the Quail Castellana breed) and then 19 birds were randomly sampled from the two separate floor pens (six and 13 from that of each breed). Two birds from the intermingled Quail Castellana died before tonic immobility was tested so the final of birds was six and 11, respectively. In Experiment 2, there were two replicates at an interval of seven days. In replicate 1, five birds were randomly sampled from each of the four floor pens of the Black Castellana, Red Villafranquina, Barred Red Vasca, and Buff Prat breeds reared with a density of 4 birds/ m 2 from 8 wk of age. Similarly, five birds were randomly sampled from each of the four floor pens of the Black Castellana, Red Villafranquina, Barred Red Vasca, and Buff Prat breeds reared with a density of 8, 12, 16 and 20 birds/ m 2 from 8 wk of age. Replicate 2 was similar to replicate 1 except that there were five birds randomly sampled from each breed reared with a density of 4 birds/m 2 and six birds from each breed reared with a density of 8, 12, 16 and 20 birds/ m 2 To obtain the heterophil to lymphocyte ratio, birds were carried to a separate room and collection of blood was made immediately. Two drops of blood were taken from a small puncture in the comb of each bird, one drop being smeared on each of two glass slides. The smears were stained using May-Grünwald and Giemsa stains (LUCAS and JAMROZ 1961), approximately 2 to 4 h after preparation with methyl alcohol fixation. One hundred leukocytes, including granular (heterophils, eosinophils, and basophils) and nongranular (lymphocytes and monocytes), were counted on one slide of each bird, and the heterophil to lymphocyte ratio was calculated. Eosinophils, basophils, and monocytes were counted as a remaining group. Tonic immobility was induced in a separate room, as soon as a bird was randomly sampled and caught, by placing the animal on its back with the head hanging in a U-shaped wooden cradle (JONES and FAURE 1981). The bird was restrained for 10 s. The observer sat in full view of the bird, about 1 m away, and fixed his eyes on the bird because of the fear-inducing properties of eye contact. If the bird remained immobile for 10 s after the experimenter removed his hands, a stopwatch was started to record latencies until the bird righted itself. If the bird righted itself in less than 10 s, then it was considered that tonic immobility had not been induced, and the restraint procedure was repeated (3 times maximum). If the bird did not show a righting response over the 10-min test period, a maximum score of 600 s was given for righting time. A three-way analysis of variance was used in both experiments (SOKAL and ROHLF 1981), according to the following model: x ijkl = µ + T i + B j + TB ij + R k + TR ik + BR jk + TBR ijk + ijkl. In this model, x ijkl is the analyzed measurement (heterophil to lymphocyte ratio, or tonic immobility duration), µ is the overall mean, T i is the effect of the intermingled or separated treatment in Experiment 1 (i = 1...2) and the bird density treatment in Experiment 2 (i = 1...5), B j is the effect of breed (j = in Experiment 1, and j = in Experiment 2), TB ij is the interaction of treatment and breed, R k is the effect of replicate (k = in Experiment 1, and k = in Experiment 2), and ijkl is the residual (l ranged from 6 to 13 in Experiment 1, and was 5 or 6 in Experiment 2). Treatment and breed were considered fixed effects and replicates were assumed to be a random effect. Replicates, replicate by treatment, replicate by breed, and replicate by treatment by breed interactions were not significant for either experiment, and they were pooled with the residual to give a two-way factorial model of treatment and breed effects (x ijk = µ + T i + B j + TB ij + ijk.). Significant differences among treatments and among breeds in Experiment 2 were evaluated using the Student-Newman-Keuls multiple range test (SNEDECOR and COCHRAN 1980). Square root (heterophil to lymphocyte ratio) or logarithmic (tonic immobility duration) transformations were used for calculations. Results Treatment by breed interaction was significant in Experiment 1 for the heterophil to lymphocyte ratio and for heterophil and lymphocyte s (Table 1). The intermingled and separate flocks differed, and they did not keep their rankings in both breeds (qualitative interaction; Figure 1). Mean values indicating the effect of treatment and breed are summarised in Table 2. The heterophil to lymphocyte ratio was significantly higher in the separate than in the intermingled Black Castellana, but the more elevated ratio observed in the intermingled Quail Castellana did not differ significantly from that in the separate Quail Castellana. Quail Castellana had higher ratio than Black Castellana in the intermingled flock, although both breeds did not differ when they were reared in separate flocks. In Table 1. F-values and significance for sources of variation in Experiment 1 F-Werte und Signifikanzen für die Variationsursachen in Versuch 1 Heterophil: lymphocyte ratio Heterophil Lymphocyte Tonic immobility duration Sources of variation F-values Pr > F F-values Pr > F F-values Pr > F F-values Pr > F Treatment (T) Breed (B) T x B Mean square error ,762.79

3 Campo et al.: Fear and stress responses in chickens 201 0,7 0,6 0,5 0,4 Intermingled Separate 0,3 0,2 0,1 0 Black Quail Figure 1. Treatment by breed interaction for the heterophil to lymphocyte ratio; Experiment 1 Wechselwirkung zwischen Behandlung und Rasse für das Verhältnis von Heterophilen zu Lymphozyten, Versuch 1 Table 2. Mean heterophil:lymphocyte ratio, of heterophils, of lymphocytes, and tonic immobility duration (s) in birds from two breeds intermingled or separate (n = 106). Experiment 1 Mittleres Verhältnis von Heterophilen zu Lymphozyten, Anzahl der Heterophilen, Anzahle der Lymphozyten und Dauer der tonischen Immobilität (s) bei Hühner von zwei Rassen, die entweder einzeln oder vermischten gehalten wurden (N=106), Versuch 1 Breed Treatment Trait Black Quail (Treatment) Intermingled Heterophil:lymphocyte ratio 0.32 b 0.56 a (0.46) Heterophil b b (23.49) Lymphocyte a ab (65.17) Tonic immobility duration a b (224.39) Separate Heterophil:lymphocyte ratio 0.59 a 0.41 ab (0.48) Heterophil a b (28.22) Lymphocyte b a (63.74) Tonic immobility duration ab a (287.96) (Breed) Heterophil:lymphocyte ratio (0.46) (0.47) Heterophil (27.05) (25.52) Lymphocyte (63.70) (64.80) Tonic immobility duration (293.55) (235.45) a-b Means within the same trait with no common superscript differ significantly (P < 0.05). birds from the Black Castellana breed reared separately there was significant heterophilia and lymphopenia in relation to the intermingled Black Castellana flock. Separate Black Castellana had significant heterophilia and lymphopenia compared to separate Quail Castellana. The heterophil to lymphocyte ratios presented in Table 2 differ from those calculated with the presented means for heterophils and lymphocytes (0.30, 0.41, 0.57 and 0.38 in intermingled black, intermingled quail, separate black, and separate quail groups, respectively) due to the effect of variance and covariance on the value of the ratio: x/y = m x /m y + 2m x s 2 y/m y 3 s xy /m y 2, being m x and m y the mean values of the numerator and denominator, s xy the covariance between them, and s 2 y the variance of the denominator. The same is true for the Experiment 2. Treatment by breed interaction was significant for the tonic immobility duration (Table 1), and it was also due to a change in ranking and not to a change in scale (qualitative interaction; Figure 2). The intermingling, whereas had no influence on the fear response of Black Castellana, reduced fearfulness of Quail Castellana (Table 2). Tonic immobility duration was shorter in the intermingled than in the separate Quail Castellana, while there was similar tonic immobility duration in intermingled or separate Black Castellana. Quail Castellana had shorter duration than Black Castellana when they were intermingled, but not when they were separate. Mean values indicating bird density effects on heterophil to lymphocyte ratio, heterophil and lymphocyte are indicated in Table 3. There was a significant difference among bird densities and among breeds for the heterophil to lymphocyte ratio, and treatment by breed interaction was not significant (Table 4). There was a consistent trend to elevate the heterophil to lymphocyte ratio when bird density was increased, mean value in the highest density (20 birds/m 2 ) differing significantly from those in the two lowest densities (4 and 8 birds/m 2 ). In birds reared in high densities, there was also a consistent in-

4 202 Campo et al.: Fear and stress responses in chickens Intermingled Separate Black Quail Figure 2. Treatment by breed interaction for the tonic immobility duration; Experiment 1 Wechselwirkung zwischen Behandlung und Rasse für die Dauer der tonischen Immobilität, Versuch 1 Table 3. Mean heterophil:lymphocyte ratio, of heterophils, of lymphocytes, and tonic immobility duration (s) in birds from four breeds reared with five different bird densities (n = 216). Experiment 2 Mittleres Verhältnis von Heterophilen zu Lymphozyten, Anzahl der Heterophilen, Anzahle der Lymphozyten und Dauer der tonischen Immobilität (s) bei Hühner von vier Rassen, die unter fünf verschiedenen Besatzdichten gehalten wurden (N=216), Versuch 2 Heterophil: lymphocyte Heterophil Lymphocyte Tonic immobility Variable ratio duration Bird density (birds/m 2 ) b ab a a b b a a ab ab ab a ab ab ab a a a b a Breed Castellana 0.42 a a b a Prat 0.27 b b a a Vasca 0.34 ab ab ab a Villafranquina 0.31 b b ab a a-bmeans within the same variable and column with no common superscript differ significantly (P < 0.05). crease in heterophil, together with a corresponding decrease in lymphocytes, the difference among bird densities being significant for both traits (P < 0.05). Birds in a density of 20 birds/m 2 showed significantly more heterophils than those in a density of 8 birds/m 2, and significantly less of lymphocytes than those in densities of 4 and 8 birds/m 2. The Castellana breed showed a significant higher heterophil to lymphocyte ratio than the Prat and Villafranquina breeds. The effect of breed was significant for heterophil and heterophil s, and treatment by breed interaction was not significant (Table 4). The Castellana had significant heterophilia in comparison to the Prat and Villafranquina breeds, and significant lymphopenia compared with the Prat breed. Neither treatment, breed, nor treatment by breed interaction were significant for the tonic immobility duration (Table 4). There was a trend for reduced duration of tonic immobility in the higher bird densities, although this trend was not significant, and the treatment did not affect the level of fear. Discussion The intermingling by breed interaction was significant for either analysed measurement (heterophil to lymphocyte ratio, its numerator or denominator, and tonic immobility duration), differences associated with intermingling being not the same for both breeds. Similarly, differences associated with breeds were not the same for intermingled or separate groups. The intermingled Black Castellana birds appeared to be less stressed than the separate Black Castellana birds, as indicated by the heterophil to lymphocyte ratio, the intermingled group of birds having only 54% of the heterophil to lymphocyte ratio of the separate group. Birds in the intermingled group of this breed had significant heteropenia and lymphophilia, heterophils representing 62% compared with those in the separate group. Lymphocyte was 18% greater than in birds from the separate group. Intermingling had no effect on the stress level of birds from the Quail Castellana breed, because the higher

5 Table 4. F-values and significance for sources of variation in Experiment 2 F-Werte und Signifikanzen der Variationsursachen in Versuch 2 Campo et al.: Fear and stress responses in chickens 203 Sources of variation Heterophil: lymphocyte ratio Heterophil Lymphocyte Tonic immobility duration F-values Pr > F F-values Pr > F F-values Pr > F F-values Pr > F Treatment (T) Breed (B) T x B Mean square error , heterophil to lymphocyte ratio found in the intermingled birds of this breed did not differ significantly from that in the separate birds. The intermingled Quail Castellana birds appeared to be less fearful than the separate Quail Castellana birds, as indicated by the tonic immobility duration, the intermingled group of birds having only 55% of the tonic immobility duration of the separate group. Intermingling had no effect on the fear level of birds from the Black Castellana breed, because the higher tonic immobility duration found in the intermingled birds of this breed did not differ significantly from that in the separate birds. The difference between breeds was significant under the intermingling condition, both for the heterophil to lymphocyte ratio and the tonic immobility duration, but not under the separate condition. These findings agree with those of ZULKIFLI et al. (1998), who reported a significant genotype by flock interaction for heterophil to lymphocyte ratio in broilers and red jungle fowl reared intermingled or separately. Broilers were stressed when intermingled with jungle fowl (higher heterophil to lymphocyte ratio) but there was no influence on the stress response of jungle fowl. GVARYAHU et al. (1996) also reported that in broilers intermingled with White Leghorn, heart weight (indicator of chronic stress) was heavier than in the controls. It might be possible that Black Castellana birds reacted to the breast pheomelanin pigmented feathers of the Quail Castellana birds, whereas the latter reacted to the extended black plumage of the former, these reactions being associated with stress and fear responses, respectively. Domestic fowl use plumage colour and pattern to gain information about conspecifics, variations in both of them providing assessment before agonistic interactions (PRESCOTT and WATHES 1999). ZULKIFLI et al. (1998) indicated that intermingling was associated with an increase in bird-to-bird pecking behaviour in jungle fowl, particularly towards broilers, and GVARYAHU et al. (1996) found that feather condition (an indicator of pecking) was poorer among the intermingled broilers than in controls. There were no apparent differences in pecking behaviour in the current study. The mean value for heterophil to lymphocyte ratio increased significantly with bird density, suggesting that increased competition between birds might lead to greater stressfulness and higher heterophil to lymphocyte ratio. Mean values in the 4 and 8 birds/m 2 densities were 63-68% of the heterophil to lymphocyte ratio in the 20 birds/m 2 density. This increase was due to both significant heterophilia and lymphopenia. This finding disagrees with that of PATTERSON and SIEGEL (1998), who reported no significant effect of cage density on the heterophil to lymphocyte ratio, heterophil or lymphocyte in White Leghorn pullets. Similarly, HESTER et al (1996) did not find significant effect of cage density on the heterophil to lymphocyte ratio in White Leghorn hens of three lines at 44 wk of age and in the selected line at 33 wk of age. However, they found significant effect of cage density in the control and commercial lines at 33 wk of age on the heterophil to lymphocyte ratio and heterophil, in agreement with the results of our study. In the current work, there was no significant effect of bird density on the tonic immobility duration. This fact agrees with the results reported by LEE (1989) in White Leghorn hens at 52 wk housed in floor pens with bird densities ranging from 2.5 to 5 birds/ m 2. On the contrary, AN- DREWS et al. (1997) found that broilers stocked at high density (35 birds/ m 2 ) at 2 wk showed the longest tonic immobility duration, the low stock density being of 7 birds/ m 2, approximately. BILCIK et al. (1998) studied the effect of group size (15, 30, 60, and 120 birds) in White Leghorn hens housed in floor pens with the same bird density (5 birds/ m 2 ). Tonic immobility duration at 36 wk was significantly different between group sizes 15 and 120, suggesting that increased competition between birds in larger groups might lead to greater fearfulness and longer tonic immobility duration. In a majority of other experiments with cages, and in agreement with the results of the current study, increasing bird density did not significantly increase fearfulness as measured by tonic immobility reaction (CRAIG et al. 1986; LEE and MOSS 1995), by moving a pencil in front of the cage (CUNNINGHAM and OSTRANDER 1982), or by latency of feeding (RAMOS et al. 1986). However, KUJIYAT et al. (1983) reported that duration of tonic immobility was greater for hens kept in cages with large group size. In conclusion, the results of this study suggest that intermingling the birds in a group had no consistent effect on the heterophil to lymphocyte ratio (as a measure of stressfulness) and on the tonic immobility duration (as a measure of fearfulness), being significant and qualitative the treatment by breed interaction. The density of the flock in which the birds were raised had a clear and significant effect on the heterophil to lymphocyte ratio but not on the tonic immobility duration. Summary The purpose of the present study was to evaluate the effects of intermingling chicks and bird density on stressfulness and fearfulness of chickens. There were two different experiments. The first experiment measured heterophil to lymphocyte ratio and tonic immobility duration in 20-wk-old birds (n = 106) of two Spanish breeds (Black Castellana and Quail Castellana) which had been reared

6 204 Campo et al.: Fear and stress responses in chickens intermingled or separately. There was significant treatment by breed interaction (P < 0.01) for the heterophil to lymphocyte ratio, the ratio being significantly higher (heterophilia and lymphopenia) in the separate than in the intermingled Black Castellana, whereas treatment had no significant effect on the stress level of birds from the Quail Castellana breed. There was also significant qualitative treatment by breed interaction for the tonic immobility duration, intermingling reducing significantly fearfulness of Quail Castellana whereas had no influence on the fear response of Black Castellana.. The second experiment measured heterophil to lymphocyte ratio and tonic immobility duration in 20-wk-old birds (n = 216) of four Spanish breeds (Black Castellana, Red Villafranquina, Barred Red Vasca, and Buff Prat) which had been reared with five different bird densities: 4, 8, 12, 16, and 20 birds/m 2, respectively. There was significant difference among bird densities for the heterophil to lymphocyte ratio. The ratio increased significantly with bird density, mean value in the 20 birds/m 2 density being significantly higher (heterophilia and lymphopenia) than those in the 4 and 8 birds/m 2 densities. There was no significant effect of bird density on the tonic immobility duration. Key words Chicken, intermingling, stocking density, fear, stress Zusammenfassung Einfluss des Vermischens der Küken und der Besatzdichte auf die Furcht und Stressreaktion von Hühnern Das Ziel der vorliegenden Studie war die Untersuchung des Einflusses des Vermischens der Tiere und der Besatzdichte auf die Furcht und die Ängstlichkeit von Hühnern. Es wurden zwei verschiedene Versuche durchgeführt. Im ersten Versuch wurden das Verhältnis von Heterophilen zu Lymphozyten und die Dauer der tonischen Immobilität bei 20 Wochen alten Hühnern (N = 106) der spanischen Rassen Schwarze Kastilianer und Gesperberte Kastilianer untersucht, die separat oder vermischt aufgezogen wurden. Für das Verhältnis von Heterophilen zu Lymphozyten wurde eine signifikante (P<0,01) Interaktion zwischen der Behandlung und der Rasse ermittelt. Das Verhältnis war bei den Schwarzen Kastilianer bei separater Haltung signifikant höher als bei vermischter Haltung. Demgegenüber hatte bei den Gesperberten Kastilianern die Behandlung keinen signifikanten Einfluss auf das Stressniveau. In ähnlicher Weise wurde auch ein signifikanter Interaktionseffekt von Behandlung und Rasse auf die Dauer der tonischen Immobilität beobachtet. Bei den Gesperberten Kastilianern reduzierte das Vermischen die Furchtsamkeit signifikant, während bei den Schwarzen Kastilianern kein Effekt vorlag. Im zweiten Versuch wurden das Verhältnis von Heterophilen zu Lymphozyten und die Dauer der tonischen Immobilität bei 20 Wochen alten Hühnern (N=216) von vier spanischen Rassen (Schwarze Kastilianer, Rote Villafranquina, Gestreifte Rote Vasca, Buff Prat) bestimmt, die unter fünf verschiedenen Besatzdichten (4, 8, 12, 16, 20 Tiere/m 2 ) aufgezogen wurden. Für die verschiedenen Besatzdichten wurden signifikante Unterschiede im Verhältnis der Heterophilen zu Lmyphozyten ermittelt. Das Verhältnis nahm signifikant mit der Besatzdichte zu. Bei der höchsten Besatzdichte wurde ein signifikant höheres Verhältnis im Vergleich zu den beiden geringsten Besatzdichten beobachtet. Die Besatzdichte hatte sich nicht auf die Dauer der tonischen Immobilität ausgewirkt. Stichworte Huhn, Vermischen, Besatzdichte, Furcht, Stressreaktion References ANDREWS, S.M., H.M. OMED and C.J.C. 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