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Effects of Housing System and Cold Stress on Heterophil-to-Lymphocyte Ratio, Fluctuating Asymmetry, and Tonic Immobility Duration of Chickens
Effects of Housing System and Cold Stress on Heterophil-to-Lymphocyte Ratio, Fluctuating Asymmetry, and Tonic Immobility Duration of Chickens J. L. Campo,1 M. T. Prieto, and S. G. Da´vila Departamento de Mejora Gene´tica Animal, Instituto Nacional de Investigacio´n y Tecnologı´a Agraria y Alimentaria, Apartado 8.111, 28080 Madrid, Spain
Key words: housing system, cold stress, heterophil-to-lymphocyte ratio, fluctuating asymmetry, tonic immobility 2008 Poultry Science 87:621–626 doi:10.3382/ps.2007-00466
for measuring levels of stress, welfare, and fear in chickens (Gallup, 1979; Gross and Siegel, 1983; Parsons, 1990). Studies on the effect of housing system on these criteria are very scarce and only have compared the tonic immobility duration of hens housed in cages or on litter. Adult White Leghorn hens housed in cages showed significantly longer durations of tonic immobility than those housed in floor pens (Jones and Faure, 1981a; Kujiyat et al., 1983). Hansen et al. (1993) did not find significant differences in tonic immobility duration between White Leghorn hens housed in cages and in aviaries at 30 wk of age, although hens in cages showed tonic immobility of longer duration than hens in aviaries at 70 wk of age. On the other hand, although the degree to which animals suffer when experiencing cold stress is unknown (Curtis and Stricklin, 1991), Hangalapura et al. (2004b) indicated that during the winter season, thermoregulation in homeotherms is an important energy-demanding process and may be a constraint to immune function. They concluded that the phagocytes of the immune system responded immediately to cold stress. On the contrary, Han-
INTRODUCTION Under alternative outdoor or indoor housing systems resembling more natural conditions, chickens are provided with a richer environment and perform more behavioral repertoire than in cages, all of which may enhance animal welfare. However, they are exposed to several factors including infectious and parasitic diseases, social interactions, and adverse climatic conditions (in outdoor systems) that may increase both stress and fear reactions and reduce welfare. Environmental stressors such as infectious agents, temperature, light, air quality, environmental contaminants, and diet may influence health status of poultry (Dietert et al., 1994). Heterophil-to-lymphocyte ratio, fluctuating asymmetry, and duration of tonic immobility may be used as criteria
©2008 Poultry Science Association Inc. Received November 19, 2007. Accepted January 10, 2008. 1 Corresponding author: [email protected]
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tonic immobility between hens housed in deep litter or free range. Thus, hens with access to an outdoor area were less stressed than hens without access to an outdoor area, although the fearfulness was similar in both groups of birds. In experiment 2, cocks (n = 120; 36 wk old) from 4 Spanish breeds, a synthetic breed, and the White Leghorn population, which had been housed in cages with or without a cold stress (0 to 10°C) from 24 wk of age, were used. Cold × breed interaction was significant for heterophil-to-lymphocyte ratio (P < 0.05), differences between cold-stressed and control birds being significant in 2 breeds. In these breeds (Red-Barred Vasca and Buff Prat), heterophil-to-lymphocyte ratio was significantly greater in cold-stressed birds. Cold stress effect was significant for the relative asymmetry of toe length (P < 0.001) and the combined relative asymmetry (P < 0.05), the relative asymmetry of birds with cold stress being larger than that of control birds. Thus, cold stress seriously negatively affects the welfare of cocks.
ABSTRACT The purpose of this study was to analyze the effect of housing system and cold stress on the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration of chickens. In experiment 1, hens (n = 120; 36 wk old) from 5 Spanish breeds and a White Leghorn population, which had been housed in pens with or without access to an outdoor area from 20 wk of age, were used. The effect of housing system on heterophil-to-lymphocyte ratio varied from breed to breed, differences between housing systems being significant (P < 0.05) in 2 breeds. In these breeds (Red-Barred Vasca and Birchen Leonesa), heterophil-to-lymphocyte ratio was significantly greater in hens housed in deep litter. Housing effect was significant for the relative asymmetry of leg length (P < 0.01), wattle length (P < 0.05), and the combined relative asymmetry (P < 0.05), the relative asymmetry of hens housed in deep litter being larger. There was no significant difference for the duration of
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MATERIALS AND METHODS Five Spanish breeds of chickens (Red-Barred Vasca, Red Villafranquina, Birchen Leonesa, Black Menorca, and White-Faced Spanish) and a White Leghorn population originated by crossing 3 strains selected for egg number and egg weight (Campo and Jurado, 1982) were used in experiment 1. Four Spanish breeds of chickens (Black Castellana, Buff Prat, Red-Barred Vasca, and Red Villafranquina), a synthetic breed (Quail Castellana) that originated from an F2 cross between Black Castellana and Buff Prat (Campo and Orozco, 1986; Campo, 1991), and the White Leghorn population were used in experiment 2. All these breeds are maintained at the experimental station of El Encı´n (Madrid, Spain) in a conservation program of genetic resources started in 1975 (Campo and Orozco, 1982) and have been described by Campo (1998). They are used in free-range systems for the production of white (Black Castellana), tinted (Buff Prat), brown (Red-Barred Vasca), and dark brown (Red Villafranquina) quality eggs. Black Menorca and White-Faced Spanish are 2 classical ornamental breeds, and males of the Birchen Leonesa breed are used for fly tying. Birds were reared with a density of 10 birds/m2 until 8 wk of age (sexes mixed). Artificial light was only provided
during the first week of age (23L:1D). Temperature was controlled with gas heaters (33 to 35°C at the chick level during the first week followed by a reduction of 3°C each week until to reach 18 to 20°C in the sixth week of age). Birds were moved to another all-litter house and reared with a density of 6 birds/m2 from 8 to 20 wk of age. The lighting regimen was 8L:16D. Birds were fed standard rearing diets containing 19% CP, 2,800 kcal of ME/kg, 1% Ca, and 0.5% available P until 8 wk and 15% CP, 2,700 kcal of ME/kg, 0.9% Ca, and 0.4% available P until 20 wk. Two different replicates (hatches) separated by 14 d were used in experiments 1 and 2. Hatches were made in April 12 and April 26 (experiment 1) and in June 14 and June 28 (experiment 2). A total of 120 hens was used in experiment 1 (housing system), to study the effect of the housing system on the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration at 36 wk of age. Birds were housed at 20 wk of age in pens with a raised slatted floor covering a dropping pit and straw litter on the rest of the floor; the slatted area occupied onethird of the floor. Bird density was 4 birds/m2. Birds were fed a diet containing 16% CP, 2,700 kcal of ME/kg, 3.5% Ca, and 0.5% available P. Feed and water were supplied for ad libitum consumption. The room temperature was 16 to 20°C. Feeders, drinkers, and nest boxes were in the slatted floor area. Two housing systems (deep litter and free range) without any differences in the construction were used in this experiment; birds in the free-range system had access to an outdoor area providing 4 m2 per bird. The birds were equally divided into 2 groups. Group 1 consisted of 60 birds (randomly selected), 10 birds of each population in 2 replicates of 5 birds, housed in deep litter. Group 2 consisted of 60 additional birds (randomly selected), 10 birds of each population in 2 replicates of 5 birds, housed in the free-range system. Similarly, a total of 120 cocks was used in experiment 2 (temperature effect), to study the effect of a cold stress on the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration at 36 wk of age. Birds were housed at 24 wk of age in cages; bird density was 8 birds/m2. The birds were equally divided into 2 groups. Group 1 consisted of 60 birds (randomly selected), 10 birds of each population in 2 replicates of 5 birds, kept in cages with a cold stress for 12 wk, temperature being 0 to 10°C. Group 2 consisted of 60 additional birds (randomly selected), 10 birds of each population in 2 replicates of 5 birds, kept in cages without a cold stress (control), the room temperature being 16 to 20°C. On 2 different days, birds were tested for heterophil-tolymphocyte ratio and tonic immobility duration at 36 wk of age. To obtain the heterophil-to-lymphocyte ratio, hens were carried to a separate room, and blood was collected immediately. Two drops of blood were taken from a small puncture in the comb of each bird, 1 drop being smeared on each of 2 glass slides. The smears were stained using May-Gru¨nwald and Giemsa stains (Lucas and Jamroz, 1961), approximately 2 to 4 h after methyl alcohol fixation. One hundred leukocytes, including granular (heterophils, eosinophils, and basophils) and nongranular (lymphocytes
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galapura et al. (2003, 2004a) found that antibody responses of chickens were not significantly affected by cold stress. Studies on the effect of cold stress on those criteria of stress, welfare, and fear are lacking, too. Hester et al. (1996) found that caged hens exposed to a cold environment (0°C for 72 h) had higher heterophil-to-lymphocyte ratios than those of the control environment. Yalcin and Siegel (2003) found that relative asymmetry of wing, shank, tibia, and femur lengths increased in broiler embryos exposed to cooling. Campo and Carnicer (1994) found that the effect of cold stress (1°C for 24 h) on tonic immobility duration varied from breed to breed, the difference between treatments (cold stress vs. control) being significant in a breed (Red-Barred Vasca). The purpose of the present study was to analyze the effects of housing system (litter pens with or without access to an outdoor area) and a cold stress on the heterophil-tolymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration of different breeds of chickens. It would be hypothesized that housing in litter pens with access to an outdoor area would decrease the stress and fear levels of birds, whereas a cold stress would increase them. As far as the authors know, the relationships between the outdoor or indoor housing systems and the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, or the tonic immobility duration have not been studied yet, whereas the relationship between a cold stress and these criteria of stress and welfare has only been studied in 1 experiment each (Campo and Carnicer, 1994; Hester et al., 1996; Yalcin and Siegel, 2003). Duration of tonic immobility was not measured in the second experiment, because it had been previously analyzed by Campo and Carnicer (1994).
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Table 1. Mean heterophil-to-lymphocyte ratio (H:L), heterophil number (H), and lymphocyte number (L) in birds housed in deep-litter or freerange systems (n = 120) H:L Breed
Litter
Free range
Red-Barred Vasca Red Villafranquina Birchen Leonesa Black Menorca White-Faced Spanish White Leghorn
0.67a 0.68 0.54a 0.34 0.58 0.61
0.37b 0.52 0.37b 0.40 0.45 0.46
H
L
Litter
Free range
Litter
Free range
38a,x 39x 33a,x 24y 35x 36x
25b 32 25b 30 29 30
59b,y 59y 65b,y 73x 62y 61y
71a 65 73a 66 68 69
a,b Means within the same breed and trait with no common superscript differ (P < 0.05). x,y Means within the same treatment and trait with no common superscript differ (P < 0.05).
effect. Significant differences among breeds were estimated using the Student-Newman-Keuls multiple-range test (Snedecor and Cochran, 1980).
RESULTS There were no significant differences among replicates or their interactions for either experiment, and they were pooled with the residual to give a 2-way factorial model of treatment and breed effects (xijk = + Ti + Bj + TBij + εijk). Because housing system × breed interaction was significant for heterophil-to-lymphocyte ratio (P < 0.05), heterophil number (P < 0.05), and lymphocyte number (P < 0.05), subclass means are summarized in Table 1. The effects of housing system on heterophil-to-lymphocyte ratio, heterophil number, and lymphocyte number varied from breed to breed, differences between housing systems being significant in 2 breeds (Red-Barred Vasca and Birchen Leonesa). In these breeds, heterophil-to-lymphocyte ratio and heterophil number were significantly greater in hens housed in deep litter and smaller in free-ranged hens, with the opposite being true for lymphocyte number. In the deep-litter group of hens, there were no significant differences among breeds in terms of heterophil-to-lymphocyte ratio, although the Black Menorca breed had significant smaller heterophil number and greater lymphocyte number than the other 5 breeds. There were no significant differences among breeds in terms of heterophilto-lymphocyte ratio, heterophil number, and lymphocyte number in the free-range group of hens. The heterophil-to-lymphocyte ratios (H/L) presented in Table 1 differed from those calculated with the presented means for heterophils and lymphocytes (0.64, 0.35, etc.) due to the effect of variance and covariance on the value of the ratio: H/L = mH/mL + 2mHs2L/mL3 − sHL/mL2, where mH and mL = the mean values of the numerator and denominator; sHL = the covariance between them; and s2L = the variance of the denominator. Mean values indicating the effect of the housing system and breed on relative asymmetry measurements are summarized in Table 2. Housing effect was significant for the relative asymmetry of leg length (P < 0.01), wattle length
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and monocytes), were counted on 1 slide of each bird (the other slide was supplementary), and the heterophilto-lymphocyte ratio was calculated. Square root transformation was used before analysis. All hens were tested for tonic immobility in a separate room on the day after the blood sampling. Tonic immobility was induced, as soon as a hen was caught, by placing the animal on its back with the head hanging in a Ushaped wooden cradle (Jones and Faure, 1981b). 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 (s) 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. Thus, tonic immobility duration ranged from 0 to 600 s. Logarithmic transformation was used before analysis. The morphological traits (measured on the same day as the blood sampling) were both right (R) and left (L) middle toe length, leg (metatarsus) length, wing (radius) length, wattle length, and leg width. Right and left values of an animal were taken during the same session. All 4 lengths and leg width were measured in millimeters using a digital calliper. Trait size was the mean of the right and left traits [(R + L)/2]. All traits showed normal frequency distributions. The fluctuating asymmetry for a trait was defined by the absolute difference between sides [⎢R − L⎥], equivalent to the mean deviation. A series of steps (Palmer, 1994; Knierim et al., 2007) was followed before identifying exhibited asymmetry as fluctuating asymmetry (normal distribution of signed right minus left differences with a mean of zero), because there are several confounding factors that complicate the analysis of asymmetry: different types of bilateral asymmetry (fluctuating asymmetry, directional asymmetry, and antisymmetry), measurement error, and relation between fluctuating asymmetry and trait size (details can be found in Campo et al., 2008). Relative fluctuating asymmetry was used for all traits [2|R − L|/(R + L)]; it had distributions that were not normal and were transformed to arc sin square root before analysis. Mean relative asymmetry was defined as the mean of the relative asymmetries of the different traits. To test the differences in heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration between treatments, a 3-way ANOVA (Sokal and Rohlf, 1981) was used with the statistical model xijkl = + Ti + Bj + TBij + rk + Trik + Brjk + TBrijk + εijkl, where xijkl = the analyzed measurement; = the overall mean; Ti = the effect of treatment (deep-litter vs. free-range housing in experiment 1 and cold stress vs. control in experiment 2); Bj = the effect of breed (j = 1 ... 6); rk = the effect of replicate (k = 1 ... 2); TBij, Trik, Brjk, and TBrijk = the interactions; and εijkl = the residual (l = 1 ... 5). Treatment and breed were considered fixed effects, and replicates were assumed to be a random
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CAMPO ET AL. Table 2. Mean relative asymmetry (×100) of various morphological traits and tonic immobility duration (s) in birds housed in deep-litter or free-range systems (n = 120) Effect Housing system Deep litter Free range SEM Breed Red-Barred Vasca Red Villafranquina Birchen Leonesa Black Menorca White-Faced Spanish White Leghorn SEM
Toe length
Leg length
Wing length
Wattle length
Leg width
Combined
Tonic immobility
3.43 3.21 0.30
1.03a 0.79b 0.07
1.31 1.29 0.11
5.38a 4.14b 0.36
4.69 3.97 0.41
3.12a 2.72b 0.12
302 236 25
2.66 3.70 4.29 3.16 3.49 2.61 0.52
0.76 1.10 0.69 0.84 1.16 0.91 0.12
1.11 1.40 1.37 1.36 1.40 1.17 0.18
5.21 3.61 5.89 5.12 4.14 4.61 0.63
3.92 4.33 4.77 4.79 4.78 3.41 0.71
2.73 2.83 3.40 3.05 2.99 2.54 0.21
342ab 398a 221bc 220bc 158c 277abc 44
Means within the same effect and column with no common superscript differ (P < 0.05).
a-c
than that of control birds (Table 4). There were significant differences among breeds in terms of lymphocyte number (P < 0.05). Lymphocyte number was significantly smaller in the Quail Castellana breed and greater in the White Leghorn population and the Buff Prat breed. Mean values indicating the effect of cold stress and breed on relative asymmetry measurements are summarized in Table 4. Cold stress effect was significant for the relative asymmetry of toe length (P < 0.001) and the combined relative asymmetry of the 5 measurements (P < 0.05). The relative asymmetry of birds with cold stress was larger than that of control birds. Cold effect was not significant for the relative asymmetry of leg length, wing length, wattle length, and leg width, although all asymmetry measurements, whether significant or not, point in the same direction, cold-stressed birds having the most asymmetrical toes, legs, wings, and wattles. Breed effect and treatment × breed interaction were not significant for any measurement.
DISCUSSION The main finding of experiment 1 was that the housing system affected heterophil-to-lymphocyte ratio and fluctuating asymmetry. Deep-litter housing significantly increased heterophil-to-lymphocyte ratio 46% in the Birchen Leonesa and 81% in the Red-Barred Vasca compared with Table 3. Mean heterophil-to-lymphocyte ratio (H:L) and heterophil number (H) in birds with or without cold stress (n = 120) H:L Breed Black Castellana Buff Prat Red-Barred Vasca Red Villafranquina Quail Castellana White Leghorn
Cold xy
0.63 0.64a,xy 0.95a,x 0.68xy 0.77xy 0.54y
H Control y
0.55 0.42b,y 0.45b,y 0.57y 0.80x 0.52y
Cold xy
36 36a,xy 46a,x 37xy 42xy 33y
Control 34y 26b,y 30b,y 34y 43x 32y
a,b Means within the same breed and trait with no common superscript differ (P < 0.05). x,y Means within the same treatment and trait with no common superscript differ (P < 0.05).
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(P < 0.05), and the combined relative asymmetry of the 5 measurements (P < 0.05). The relative asymmetry of birds housed in deep litter was larger than that of birds housed in free range. Housing effect was not significant for the relative asymmetry of toe length, wing length, and leg width, although all asymmetry measurements, whether significant or not, point in the same direction, birds housed in deep litter having the most asymmetrical toes, legs, wings, and wattles. Breed effect and treatment × breed interaction were not significant for any measurement. Housing effect approached levels of statistical significance (P = 0.05) for the tonic immobility duration (F = 3.33 with 1 and 108 df; P < 0.07), the tonic immobility duration being longer within the group of birds housed in deep litter than within the group of free-ranged birds (Table 2). There were significant differences among breeds (P < 0.001), birds from the Red Villafranquina breed having longer tonic immobility duration, with tonic immobility of birds from the White-Faced Spanish breed being shorter. Treatment × breed interaction was not significant. Cold × breed interaction was significant for heterophilto-lymphocyte ratio (P < 0.05) and heterophil number (P < 0.05), subclass means being summarized in Table 3. The effects of cold stress on heterophil-to-lymphocyte ratio and heterophil number varied from breed to breed, differences between cold-stressed and control birds being significant in 2 breeds (Red-Barred Vasca and Buff Prat). In these breeds, heterophil-to-lymphocyte ratio and heterophil number were significantly greater in cold-stressed birds and smaller in control birds. There were significant differences among breeds in terms of heterophil-to-lymphocyte ratio and heterophil number, in the cold-stressed and the control group of birds. Ratio and heterophil number were significantly greater in the Red-Barred Vasca breed and smaller in the White Leghorn population in the coldstressed group, whereas the Quail Castellana breed had significant greater heterophil-to-lymphocyte ratio and heterophil number than the other 5 breeds in the control group. Cold × breed interaction was not significant for lymphocyte number. There was a significant difference between treatments for lymphocyte number (P < 0.01), with lymphocytes of birds with cold stress being higher
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HOUSING SYSTEM, COLD STRESS, AND WELFARE Table 4. Mean lymphocyte number and relative asymmetry (×100) of various morphological traits in birds with or without cold stress (n = 120) Effect Treatment With cold stress Without cold stress SEM Breed Black Castellana Buff Prat Red-Barred Vasca Red Villafranquina Quail Castellana White Leghorn SEM
Lymphocyte number
Toe length
Leg length
Wing length
Wattle length
Leg width
Combined
58.41b 63.06a 1.13
2.60a 1.90b 0.14
0.73 0.71 0.06
1.09 0.93 0.08
2.88 2.86 0.23
3.96 3.46 0.31
2.25a 1.97b 0.09
61.30ab 64.75a 59.20ab 61.60ab 54.90b 62.70a 1.97
2.15 2.16 2.44 2.40 2.23 2.12 0.25
0.60 0.78 0.77 0.74 0.81 0.62 0.10
1.18 0.99 0.98 0.97 0.89 1.06 0.14
2.13 3.39 3.07 3.29 2.48 2.85 0.41
3.17 4.07 4.62 4.09 3.03 3.27 0.54
1.85 2.28 2.37 2.30 1.89 1.98 0.16
Means within the same effect and column with no common superscript differ (P < 0.05).
a,b
caged White Leghorn hens exposed to a cold environment had higher heterophil-to-lymphocyte ratios than those of the control hens (39.5 vs. 37.6). In relation to plasma corticosterone concentration (another stress indicator), Buckland et al. (1974) reported that the application of cold stress resulted in significant increases in plasma corticosterone levels in chicks, and Hester et al. (1996) found that caged hens exposed to a cold environment (0°C for 72 h) had higher plasma corticosterone levels than those of the control environment (39.5 vs. 37.6). In disagreement with these results, Hangalapura et al. (2004a) found a significant suppressive effect of cold stress (10°C for 7 d) on plasma corticosterone levels. The significant cold × breed interaction for heterophilto-lymphocyte ratio suggests that, depending on the genotype, the animals respond differently to different temperatures. A similar cold × breed interaction was found by Campo and Carnicer (1994) for the tonic immobility duration of several Spanish breeds of chickens including the Red-Barred Vasca, the Black Castellana, and the Buff Prat. In all 3 breeds, cold stress decreased the duration of tonic immobility, significantly in the Red-Barred Vasca breed (173 vs. 349 s), in agreement with the negative correlation between heterophil-to-lymphocyte ratio and tonic immobility duration reported by Campo and Redondo (1996, 1997) and Campo et al. (2007). Cold-stressed birds had the combined fluctuating asymmetry of morphological traits almost 15% larger than control birds without cold stress, the fluctuating asymmetry of middle toe length being more than 35% larger. This result is consistent with those found by Yalcin and Siegel (2003) in broiler embryos exposed to cooling. Relative asymmetry of wing, shank, and tibia lengths at 10 d of incubation increased in embryos exposed to cooling to 36.9°C six hours per day from 0 to 8 d of incubation, whereas the larger relative asymmetry of tibia and femur lengths at 18 d of incubation was for embryos exposed to 24 h to 21°C on d 14 of incubation, and for shank length at hatch, it was largest for embryos exposed to cooling to 36.9°C six hours per day from 10 to 18 d of incubation. Therefore, deviations from optimum environmental temperatures for chickens and embryos could contribute to
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the free-range system, hens from these breeds having significant greater heterophil number (heterophilia) and smaller lymphocyte number (lymphopenia) in the deeplitter system. In agreement with the latter result, van Loon et al. (2004) found that brown layers kept in the free-range system showed significantly higher in vitro lymphocyte proliferation than hens kept indoors in a litter system. The significant housing system × breed interaction for heterophil-to-lymphocyte ratio suggests that, depending on the genotype, the animals respond differently to different environments. Besides, deep-litter housing significantly increased the combined fluctuating asymmetry of 5 morphological traits almost 15% compared with the free-range system, the increase for the fluctuating asymmetry of 2 morphological traits (leg and wattle lengths) being almost 30%. The no significant housing effect on tonic immobility duration found in the current study at 36 wk of age agrees with the results reported by Hansen et al. (1993), who found that the duration of the tonic immobility response was unaffected by type of system in White Leghorn hens at 30 wk of age housed in cages and in aviaries, suggesting that the fearfulness is similar in both types of housing. Similarly, Weeks et al. (1993) found few differences in the behavior of broiler chickens housed in indoor and freerange environments, although it should be expected that chickens in an outdoor system performed more behavioral repertoire and suffered less from stress. As expected in experiment 2, the cold stress significantly affected heterophil-to-lymphocyte ratio and fluctuating asymmetry. Cold-stressed birds had the heterophil-to-lymphocyte ratio significantly higher, more than 50% in the Buff Prat and more than 2× in the Red-Barred Vasca, than control birds without cold stress, cold-stressed birds from these breeds having significant greater heterophil number (heterophilia); lymphocyte number was significantly smaller (lymphopenia) in cold-stressed birds from all 6 breeds. This was in agreement with the result reported by Hangalapura et al. (2004a), who found a significant enhancing effect of cold stress (10°C for 7 d) on in vitro lymphocyte proliferation. With respect to the heterophilto-lymphocyte ratio, Hester et al. (1996) also found that
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REFERENCES Buckland, R. B., K. Blagrave, and P. C. Lagne. 1974. Competitive protein-binding assay for corticoids in the peripheral plasma of the immature chicken. Poult. Sci. 53:241–245. Campo, J. L. 1991. Use of the sex-linked barring (B) gene for chick sexing on an eumelanotic columbian background. Poult. Sci. 70:1469–1473. Campo, J. L. 1998. Conservation and genetical study of Spanish chicken breeds (28). Pages 155–158 in Proc. 6th World Congr. Genet. Appl. Livest. Prod., Armidale, Australia. Univ. N. Engl., Armidale, New South Wales, Australia. Campo, J. L., and C. Carnicer. 1994. Effects of several stressors on tonic immobility reaction of chickens. Arch. Geflu¨gelkd. 88:75–78. Campo, J. L., M. G. Gil, S. G. Da´vila, and I. Mun˜oz. 2007. Genetic and phenotypic correlation between fluctuating asymmetry and two measurements of fear and stress in chickens. Appl. Anim. Behav. Sci. 102:53–64. Campo, J. L., and J. J. Jurado. 1982. Evaluation of multiple trait selection in strains of layers. Pages 869–874 in Proc. 2nd World Congr. Genet. Appl. Livest. Prod., Madrid, Spain. Editorial Garsi, Madrid, Spain. Campo, J. L., and F. Orozco. 1982. Conservation and genetical study of Spanish chicken breeds. Pages 88–93 in Proc. 2nd World Congr. Genet. Appl. Livest. Prod., Madrid, Spain. Editorial Garsi, Madrid, Spain. Campo, J. L., and F. Orozco. 1986. Genetic basis of the Melanotic Prat phenotype. Br. Poult. Sci. 27:361–367. Campo, J. L., M. T. Prieto, and S. G. Da´vila. 2008. Association between vent pecking and fluctuating asymmetry, heterophil to lymphocyte ratio, and tonic immobility duration in chickens. Appl. Anim. Behav. Sci. doi:10.1016/j.applanim. 2007.10.008. Campo, J. L., and A. Redondo. 1996. Tonic immobility reaction and heterophil to lymphocyte ratio in hens from three Spanish breeds laying pink eggshells. Poult. Sci. 75:155–159. Campo, J. L., and A. Redondo. 1997. Negative association between heterophil to lymphocyte ratio and tonic immobility reaction in hens. Pages 163–164 in Proc. 5th Eur. Symp. Poult. Welf., Wageningen, the Netherlands. Wageningen Agric. Univ., the Netherlands.
Curtis, S. E., and W. R. Stricklin. 1991. The importance of animal cognition in agricultural animal production systems: An overview. J. Anim. Sci. 69:5001–5007. Dietert, R. R., K. A. Golemboski, and R. E. Austic. 1994. Environment-immune interactions. Poult. Sci. 73:1062–1076. Gallup, G. G. 1979. Tonic immobility as a measure of fear in domestic fowl. Anim. Behav. 27:316–317. Gross, W. B., and H. S. Siegel. 1983. Evaluation of the heterophil to lymphocyte ratio as a measure of stress in chickens. Avian Dis. 27:972–979. Hangalapura, B. N., M. G. B. Nieuwland, J. Buyse, B. Kemp, and H. K. Parmentier. 2004a. Effect of duration of cold stress on plasma adrenal and thyroid hormone levels and immune responses in chicken lines divergently selected for antibody responses. Poult. Sci. 83:1644–1649. Hangalapura, B. N., M. G. B. Nieuwland, G. de Vries Reilingh, M. J. W. Heetkamp, H. van den Brand, B. Kemp, and H. K. Parmentier. 2003. Effects of cold stress on immune responses and body weight of chicken lines divergently selected for antibody responses to sheep red blood cells. Poult. Sci. 82:1692–1700. Hangalapura, B. N., M. G. B. Nieuwland, G. de Vries Reilingh, H. van den Brand, B. Kemp, and H. K. Parmentier. 2004b. Durations of cold stress modulates overall immunity of chicken lines divergently selected for antibody responses. Poult. Sci. 83:765–775. Hansen, I., B. O. Braastad, J. Storbraten, and M. Tofastrud. 1993. Differences in fearfulness indicated by tonic immobility between laying hens in aviaries and in cages. Anim. Welf. 2:105–112. Hester, P. Y., W. M. Muir, J. V. Craig, and J. L. Albright. 1996. Group selection for adaptation to multiple-hen cages: Hematology and adrenal function. Poult. Sci. 75:1295–1307. Jones, R. B., and J. M. Faure. 1981a. Tonic immobility (“righting time”) in laying hens housed in cages and pens. Appl. Anim. Ethol. 7:369–372. Jones, R. B., and J. M. Faure. 1981b. Sex and strain comparisons of tonic immobility (“righting time”) in the domestic fowl and the effects of various methods of induction. Behav. Proc. 6:47–55. Knierim, U., S. van Dongen, B. Forkman, F. A. M. Tuyttens, M. Spinka, J. L. Campo, and G. E. Weissengruber. 2007. Fluctuating asymmetry as an animal welfare indicator—A review of methodology and validity. Physiol. Behav. 92:398–421. Kujiyat, S. K., J. V. Craig, and A. D. Dayton. 1983. Duration of tonic immobility affected by housing environment in White Leghorn hens. Poult. Sci. 62:2280–2282. Lucas, A. M., and C. Jamroz. 1961. Atlas of Avian Hematology. Agriculture Monograph 25. US Dept. Agric., Washington, DC. Palmer, A. R. 1994. Fluctuating asymmetry analyses: A primer. Pages 335–364 in Developmental Instabilty: Its Origins and Evolutionary Implications. T.A. Markow, ed. Kluwer Acad., Dordrecht, the Netherlands. Parsons, P. A. 1990. Fluctuating asymmetry. An epigenetic measure of stress. Biol. Rev. Camb. Philos. Soc. 65:131–145. Snedecor, G. W., and W. G. Cochran. 1980. Statistical Methods. 7th ed. Iowa State Univ. Press, Ames. Sokal, R. R., and F. J. Rohlf. 1981. Biometry. Freeman and Co., London, UK. van Loon, D. P. R., B. Hangalapura, G. de Vries Reilingh, M. G. B. Nieuwland, B. Kemp, and H. K. Parmentier. 2004. Effect of three different housing systems on immune responses and body weight of chicken lines divergently selected for antibody responses to sheep red blood cells. Livest. Prod. Sci. 85:139– 150. Weeks, C. A., C. J. Nicol, C. M. Sherwin, and S. C. Kestin. 1993. Comparison of the behaviour of broiler chickens in indoor and free range environments. Anim. Welf. 3:179–192. Yalcin, S., and P. B. Siegel. 2003. Exposure to cold or heat during incubation on developmental stability of broiler embryos. Poult. Sci. 82:1388–1392.
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larger fluctuating asymmetry of morphological traits, because cold stress results in the use of resources that could otherwise be used for developmental control. In conclusion, results of the current study indicate that housing in deep litter with access to an outdoor area resulted in smaller heterophil-to-lymphocyte ratio and smaller fluctuating asymmetry compared with housing in deep litter without access to an outdoor area, suggesting that the housing system is associated with some measures of stress and welfare. Duration of tonic immobility was not significantly longer in the deep-litter housing, indicating that indoor vs. outdoor systems did not contribute to the fearfulness of birds. Low ambient temperatures (0 to 10°C from 24 to 36 wk of age) resulted in greater heterophilto-lymphocyte ratios and higher fluctuating asymmetry, suggesting that a cold stress seriously negatively affects the welfare of birds. The significant housing × breed and cold × breed interactions for heterophil-to-lymphocyte ratio suggest that, depending on the genotype, the animals respond differently to different environments, the RedBarred Vasca being more sensitive to the environment.
Key words: housing system, cold stress, heterophil-to-lymphocyte ratio, fluctuating asymmetry, tonic immobility 2008 Poultry Science 87:621–626 doi:10.3382/ps.2007-00466
for measuring levels of stress, welfare, and fear in chickens (Gallup, 1979; Gross and Siegel, 1983; Parsons, 1990). Studies on the effect of housing system on these criteria are very scarce and only have compared the tonic immobility duration of hens housed in cages or on litter. Adult White Leghorn hens housed in cages showed significantly longer durations of tonic immobility than those housed in floor pens (Jones and Faure, 1981a; Kujiyat et al., 1983). Hansen et al. (1993) did not find significant differences in tonic immobility duration between White Leghorn hens housed in cages and in aviaries at 30 wk of age, although hens in cages showed tonic immobility of longer duration than hens in aviaries at 70 wk of age. On the other hand, although the degree to which animals suffer when experiencing cold stress is unknown (Curtis and Stricklin, 1991), Hangalapura et al. (2004b) indicated that during the winter season, thermoregulation in homeotherms is an important energy-demanding process and may be a constraint to immune function. They concluded that the phagocytes of the immune system responded immediately to cold stress. On the contrary, Han-
INTRODUCTION Under alternative outdoor or indoor housing systems resembling more natural conditions, chickens are provided with a richer environment and perform more behavioral repertoire than in cages, all of which may enhance animal welfare. However, they are exposed to several factors including infectious and parasitic diseases, social interactions, and adverse climatic conditions (in outdoor systems) that may increase both stress and fear reactions and reduce welfare. Environmental stressors such as infectious agents, temperature, light, air quality, environmental contaminants, and diet may influence health status of poultry (Dietert et al., 1994). Heterophil-to-lymphocyte ratio, fluctuating asymmetry, and duration of tonic immobility may be used as criteria
©2008 Poultry Science Association Inc. Received November 19, 2007. Accepted January 10, 2008. 1 Corresponding author: [email protected]
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tonic immobility between hens housed in deep litter or free range. Thus, hens with access to an outdoor area were less stressed than hens without access to an outdoor area, although the fearfulness was similar in both groups of birds. In experiment 2, cocks (n = 120; 36 wk old) from 4 Spanish breeds, a synthetic breed, and the White Leghorn population, which had been housed in cages with or without a cold stress (0 to 10°C) from 24 wk of age, were used. Cold × breed interaction was significant for heterophil-to-lymphocyte ratio (P < 0.05), differences between cold-stressed and control birds being significant in 2 breeds. In these breeds (Red-Barred Vasca and Buff Prat), heterophil-to-lymphocyte ratio was significantly greater in cold-stressed birds. Cold stress effect was significant for the relative asymmetry of toe length (P < 0.001) and the combined relative asymmetry (P < 0.05), the relative asymmetry of birds with cold stress being larger than that of control birds. Thus, cold stress seriously negatively affects the welfare of cocks.
ABSTRACT The purpose of this study was to analyze the effect of housing system and cold stress on the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration of chickens. In experiment 1, hens (n = 120; 36 wk old) from 5 Spanish breeds and a White Leghorn population, which had been housed in pens with or without access to an outdoor area from 20 wk of age, were used. The effect of housing system on heterophil-to-lymphocyte ratio varied from breed to breed, differences between housing systems being significant (P < 0.05) in 2 breeds. In these breeds (Red-Barred Vasca and Birchen Leonesa), heterophil-to-lymphocyte ratio was significantly greater in hens housed in deep litter. Housing effect was significant for the relative asymmetry of leg length (P < 0.01), wattle length (P < 0.05), and the combined relative asymmetry (P < 0.05), the relative asymmetry of hens housed in deep litter being larger. There was no significant difference for the duration of
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MATERIALS AND METHODS Five Spanish breeds of chickens (Red-Barred Vasca, Red Villafranquina, Birchen Leonesa, Black Menorca, and White-Faced Spanish) and a White Leghorn population originated by crossing 3 strains selected for egg number and egg weight (Campo and Jurado, 1982) were used in experiment 1. Four Spanish breeds of chickens (Black Castellana, Buff Prat, Red-Barred Vasca, and Red Villafranquina), a synthetic breed (Quail Castellana) that originated from an F2 cross between Black Castellana and Buff Prat (Campo and Orozco, 1986; Campo, 1991), and the White Leghorn population were used in experiment 2. All these breeds are maintained at the experimental station of El Encı´n (Madrid, Spain) in a conservation program of genetic resources started in 1975 (Campo and Orozco, 1982) and have been described by Campo (1998). They are used in free-range systems for the production of white (Black Castellana), tinted (Buff Prat), brown (Red-Barred Vasca), and dark brown (Red Villafranquina) quality eggs. Black Menorca and White-Faced Spanish are 2 classical ornamental breeds, and males of the Birchen Leonesa breed are used for fly tying. Birds were reared with a density of 10 birds/m2 until 8 wk of age (sexes mixed). Artificial light was only provided
during the first week of age (23L:1D). Temperature was controlled with gas heaters (33 to 35°C at the chick level during the first week followed by a reduction of 3°C each week until to reach 18 to 20°C in the sixth week of age). Birds were moved to another all-litter house and reared with a density of 6 birds/m2 from 8 to 20 wk of age. The lighting regimen was 8L:16D. Birds were fed standard rearing diets containing 19% CP, 2,800 kcal of ME/kg, 1% Ca, and 0.5% available P until 8 wk and 15% CP, 2,700 kcal of ME/kg, 0.9% Ca, and 0.4% available P until 20 wk. Two different replicates (hatches) separated by 14 d were used in experiments 1 and 2. Hatches were made in April 12 and April 26 (experiment 1) and in June 14 and June 28 (experiment 2). A total of 120 hens was used in experiment 1 (housing system), to study the effect of the housing system on the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration at 36 wk of age. Birds were housed at 20 wk of age in pens with a raised slatted floor covering a dropping pit and straw litter on the rest of the floor; the slatted area occupied onethird of the floor. Bird density was 4 birds/m2. Birds were fed a diet containing 16% CP, 2,700 kcal of ME/kg, 3.5% Ca, and 0.5% available P. Feed and water were supplied for ad libitum consumption. The room temperature was 16 to 20°C. Feeders, drinkers, and nest boxes were in the slatted floor area. Two housing systems (deep litter and free range) without any differences in the construction were used in this experiment; birds in the free-range system had access to an outdoor area providing 4 m2 per bird. The birds were equally divided into 2 groups. Group 1 consisted of 60 birds (randomly selected), 10 birds of each population in 2 replicates of 5 birds, housed in deep litter. Group 2 consisted of 60 additional birds (randomly selected), 10 birds of each population in 2 replicates of 5 birds, housed in the free-range system. Similarly, a total of 120 cocks was used in experiment 2 (temperature effect), to study the effect of a cold stress on the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration at 36 wk of age. Birds were housed at 24 wk of age in cages; bird density was 8 birds/m2. The birds were equally divided into 2 groups. Group 1 consisted of 60 birds (randomly selected), 10 birds of each population in 2 replicates of 5 birds, kept in cages with a cold stress for 12 wk, temperature being 0 to 10°C. Group 2 consisted of 60 additional birds (randomly selected), 10 birds of each population in 2 replicates of 5 birds, kept in cages without a cold stress (control), the room temperature being 16 to 20°C. On 2 different days, birds were tested for heterophil-tolymphocyte ratio and tonic immobility duration at 36 wk of age. To obtain the heterophil-to-lymphocyte ratio, hens were carried to a separate room, and blood was collected immediately. Two drops of blood were taken from a small puncture in the comb of each bird, 1 drop being smeared on each of 2 glass slides. The smears were stained using May-Gru¨nwald and Giemsa stains (Lucas and Jamroz, 1961), approximately 2 to 4 h after methyl alcohol fixation. One hundred leukocytes, including granular (heterophils, eosinophils, and basophils) and nongranular (lymphocytes
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galapura et al. (2003, 2004a) found that antibody responses of chickens were not significantly affected by cold stress. Studies on the effect of cold stress on those criteria of stress, welfare, and fear are lacking, too. Hester et al. (1996) found that caged hens exposed to a cold environment (0°C for 72 h) had higher heterophil-to-lymphocyte ratios than those of the control environment. Yalcin and Siegel (2003) found that relative asymmetry of wing, shank, tibia, and femur lengths increased in broiler embryos exposed to cooling. Campo and Carnicer (1994) found that the effect of cold stress (1°C for 24 h) on tonic immobility duration varied from breed to breed, the difference between treatments (cold stress vs. control) being significant in a breed (Red-Barred Vasca). The purpose of the present study was to analyze the effects of housing system (litter pens with or without access to an outdoor area) and a cold stress on the heterophil-tolymphocyte ratio, the fluctuating asymmetry, and the tonic immobility duration of different breeds of chickens. It would be hypothesized that housing in litter pens with access to an outdoor area would decrease the stress and fear levels of birds, whereas a cold stress would increase them. As far as the authors know, the relationships between the outdoor or indoor housing systems and the heterophil-to-lymphocyte ratio, the fluctuating asymmetry, or the tonic immobility duration have not been studied yet, whereas the relationship between a cold stress and these criteria of stress and welfare has only been studied in 1 experiment each (Campo and Carnicer, 1994; Hester et al., 1996; Yalcin and Siegel, 2003). Duration of tonic immobility was not measured in the second experiment, because it had been previously analyzed by Campo and Carnicer (1994).
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Table 1. Mean heterophil-to-lymphocyte ratio (H:L), heterophil number (H), and lymphocyte number (L) in birds housed in deep-litter or freerange systems (n = 120) H:L Breed
Litter
Free range
Red-Barred Vasca Red Villafranquina Birchen Leonesa Black Menorca White-Faced Spanish White Leghorn
0.67a 0.68 0.54a 0.34 0.58 0.61
0.37b 0.52 0.37b 0.40 0.45 0.46
H
L
Litter
Free range
Litter
Free range
38a,x 39x 33a,x 24y 35x 36x
25b 32 25b 30 29 30
59b,y 59y 65b,y 73x 62y 61y
71a 65 73a 66 68 69
a,b Means within the same breed and trait with no common superscript differ (P < 0.05). x,y Means within the same treatment and trait with no common superscript differ (P < 0.05).
effect. Significant differences among breeds were estimated using the Student-Newman-Keuls multiple-range test (Snedecor and Cochran, 1980).
RESULTS There were no significant differences among replicates or their interactions for either experiment, and they were pooled with the residual to give a 2-way factorial model of treatment and breed effects (xijk = + Ti + Bj + TBij + εijk). Because housing system × breed interaction was significant for heterophil-to-lymphocyte ratio (P < 0.05), heterophil number (P < 0.05), and lymphocyte number (P < 0.05), subclass means are summarized in Table 1. The effects of housing system on heterophil-to-lymphocyte ratio, heterophil number, and lymphocyte number varied from breed to breed, differences between housing systems being significant in 2 breeds (Red-Barred Vasca and Birchen Leonesa). In these breeds, heterophil-to-lymphocyte ratio and heterophil number were significantly greater in hens housed in deep litter and smaller in free-ranged hens, with the opposite being true for lymphocyte number. In the deep-litter group of hens, there were no significant differences among breeds in terms of heterophil-to-lymphocyte ratio, although the Black Menorca breed had significant smaller heterophil number and greater lymphocyte number than the other 5 breeds. There were no significant differences among breeds in terms of heterophilto-lymphocyte ratio, heterophil number, and lymphocyte number in the free-range group of hens. The heterophil-to-lymphocyte ratios (H/L) presented in Table 1 differed from those calculated with the presented means for heterophils and lymphocytes (0.64, 0.35, etc.) due to the effect of variance and covariance on the value of the ratio: H/L = mH/mL + 2mHs2L/mL3 − sHL/mL2, where mH and mL = the mean values of the numerator and denominator; sHL = the covariance between them; and s2L = the variance of the denominator. Mean values indicating the effect of the housing system and breed on relative asymmetry measurements are summarized in Table 2. Housing effect was significant for the relative asymmetry of leg length (P < 0.01), wattle length
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and monocytes), were counted on 1 slide of each bird (the other slide was supplementary), and the heterophilto-lymphocyte ratio was calculated. Square root transformation was used before analysis. All hens were tested for tonic immobility in a separate room on the day after the blood sampling. Tonic immobility was induced, as soon as a hen was caught, by placing the animal on its back with the head hanging in a Ushaped wooden cradle (Jones and Faure, 1981b). 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 (s) 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. Thus, tonic immobility duration ranged from 0 to 600 s. Logarithmic transformation was used before analysis. The morphological traits (measured on the same day as the blood sampling) were both right (R) and left (L) middle toe length, leg (metatarsus) length, wing (radius) length, wattle length, and leg width. Right and left values of an animal were taken during the same session. All 4 lengths and leg width were measured in millimeters using a digital calliper. Trait size was the mean of the right and left traits [(R + L)/2]. All traits showed normal frequency distributions. The fluctuating asymmetry for a trait was defined by the absolute difference between sides [⎢R − L⎥], equivalent to the mean deviation. A series of steps (Palmer, 1994; Knierim et al., 2007) was followed before identifying exhibited asymmetry as fluctuating asymmetry (normal distribution of signed right minus left differences with a mean of zero), because there are several confounding factors that complicate the analysis of asymmetry: different types of bilateral asymmetry (fluctuating asymmetry, directional asymmetry, and antisymmetry), measurement error, and relation between fluctuating asymmetry and trait size (details can be found in Campo et al., 2008). Relative fluctuating asymmetry was used for all traits [2|R − L|/(R + L)]; it had distributions that were not normal and were transformed to arc sin square root before analysis. Mean relative asymmetry was defined as the mean of the relative asymmetries of the different traits. To test the differences in heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration between treatments, a 3-way ANOVA (Sokal and Rohlf, 1981) was used with the statistical model xijkl = + Ti + Bj + TBij + rk + Trik + Brjk + TBrijk + εijkl, where xijkl = the analyzed measurement; = the overall mean; Ti = the effect of treatment (deep-litter vs. free-range housing in experiment 1 and cold stress vs. control in experiment 2); Bj = the effect of breed (j = 1 ... 6); rk = the effect of replicate (k = 1 ... 2); TBij, Trik, Brjk, and TBrijk = the interactions; and εijkl = the residual (l = 1 ... 5). Treatment and breed were considered fixed effects, and replicates were assumed to be a random
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CAMPO ET AL. Table 2. Mean relative asymmetry (×100) of various morphological traits and tonic immobility duration (s) in birds housed in deep-litter or free-range systems (n = 120) Effect Housing system Deep litter Free range SEM Breed Red-Barred Vasca Red Villafranquina Birchen Leonesa Black Menorca White-Faced Spanish White Leghorn SEM
Toe length
Leg length
Wing length
Wattle length
Leg width
Combined
Tonic immobility
3.43 3.21 0.30
1.03a 0.79b 0.07
1.31 1.29 0.11
5.38a 4.14b 0.36
4.69 3.97 0.41
3.12a 2.72b 0.12
302 236 25
2.66 3.70 4.29 3.16 3.49 2.61 0.52
0.76 1.10 0.69 0.84 1.16 0.91 0.12
1.11 1.40 1.37 1.36 1.40 1.17 0.18
5.21 3.61 5.89 5.12 4.14 4.61 0.63
3.92 4.33 4.77 4.79 4.78 3.41 0.71
2.73 2.83 3.40 3.05 2.99 2.54 0.21
342ab 398a 221bc 220bc 158c 277abc 44
Means within the same effect and column with no common superscript differ (P < 0.05).
a-c
than that of control birds (Table 4). There were significant differences among breeds in terms of lymphocyte number (P < 0.05). Lymphocyte number was significantly smaller in the Quail Castellana breed and greater in the White Leghorn population and the Buff Prat breed. Mean values indicating the effect of cold stress and breed on relative asymmetry measurements are summarized in Table 4. Cold stress effect was significant for the relative asymmetry of toe length (P < 0.001) and the combined relative asymmetry of the 5 measurements (P < 0.05). The relative asymmetry of birds with cold stress was larger than that of control birds. Cold effect was not significant for the relative asymmetry of leg length, wing length, wattle length, and leg width, although all asymmetry measurements, whether significant or not, point in the same direction, cold-stressed birds having the most asymmetrical toes, legs, wings, and wattles. Breed effect and treatment × breed interaction were not significant for any measurement.
DISCUSSION The main finding of experiment 1 was that the housing system affected heterophil-to-lymphocyte ratio and fluctuating asymmetry. Deep-litter housing significantly increased heterophil-to-lymphocyte ratio 46% in the Birchen Leonesa and 81% in the Red-Barred Vasca compared with Table 3. Mean heterophil-to-lymphocyte ratio (H:L) and heterophil number (H) in birds with or without cold stress (n = 120) H:L Breed Black Castellana Buff Prat Red-Barred Vasca Red Villafranquina Quail Castellana White Leghorn
Cold xy
0.63 0.64a,xy 0.95a,x 0.68xy 0.77xy 0.54y
H Control y
0.55 0.42b,y 0.45b,y 0.57y 0.80x 0.52y
Cold xy
36 36a,xy 46a,x 37xy 42xy 33y
Control 34y 26b,y 30b,y 34y 43x 32y
a,b Means within the same breed and trait with no common superscript differ (P < 0.05). x,y Means within the same treatment and trait with no common superscript differ (P < 0.05).
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(P < 0.05), and the combined relative asymmetry of the 5 measurements (P < 0.05). The relative asymmetry of birds housed in deep litter was larger than that of birds housed in free range. Housing effect was not significant for the relative asymmetry of toe length, wing length, and leg width, although all asymmetry measurements, whether significant or not, point in the same direction, birds housed in deep litter having the most asymmetrical toes, legs, wings, and wattles. Breed effect and treatment × breed interaction were not significant for any measurement. Housing effect approached levels of statistical significance (P = 0.05) for the tonic immobility duration (F = 3.33 with 1 and 108 df; P < 0.07), the tonic immobility duration being longer within the group of birds housed in deep litter than within the group of free-ranged birds (Table 2). There were significant differences among breeds (P < 0.001), birds from the Red Villafranquina breed having longer tonic immobility duration, with tonic immobility of birds from the White-Faced Spanish breed being shorter. Treatment × breed interaction was not significant. Cold × breed interaction was significant for heterophilto-lymphocyte ratio (P < 0.05) and heterophil number (P < 0.05), subclass means being summarized in Table 3. The effects of cold stress on heterophil-to-lymphocyte ratio and heterophil number varied from breed to breed, differences between cold-stressed and control birds being significant in 2 breeds (Red-Barred Vasca and Buff Prat). In these breeds, heterophil-to-lymphocyte ratio and heterophil number were significantly greater in cold-stressed birds and smaller in control birds. There were significant differences among breeds in terms of heterophil-to-lymphocyte ratio and heterophil number, in the cold-stressed and the control group of birds. Ratio and heterophil number were significantly greater in the Red-Barred Vasca breed and smaller in the White Leghorn population in the coldstressed group, whereas the Quail Castellana breed had significant greater heterophil-to-lymphocyte ratio and heterophil number than the other 5 breeds in the control group. Cold × breed interaction was not significant for lymphocyte number. There was a significant difference between treatments for lymphocyte number (P < 0.01), with lymphocytes of birds with cold stress being higher
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HOUSING SYSTEM, COLD STRESS, AND WELFARE Table 4. Mean lymphocyte number and relative asymmetry (×100) of various morphological traits in birds with or without cold stress (n = 120) Effect Treatment With cold stress Without cold stress SEM Breed Black Castellana Buff Prat Red-Barred Vasca Red Villafranquina Quail Castellana White Leghorn SEM
Lymphocyte number
Toe length
Leg length
Wing length
Wattle length
Leg width
Combined
58.41b 63.06a 1.13
2.60a 1.90b 0.14
0.73 0.71 0.06
1.09 0.93 0.08
2.88 2.86 0.23
3.96 3.46 0.31
2.25a 1.97b 0.09
61.30ab 64.75a 59.20ab 61.60ab 54.90b 62.70a 1.97
2.15 2.16 2.44 2.40 2.23 2.12 0.25
0.60 0.78 0.77 0.74 0.81 0.62 0.10
1.18 0.99 0.98 0.97 0.89 1.06 0.14
2.13 3.39 3.07 3.29 2.48 2.85 0.41
3.17 4.07 4.62 4.09 3.03 3.27 0.54
1.85 2.28 2.37 2.30 1.89 1.98 0.16
Means within the same effect and column with no common superscript differ (P < 0.05).
a,b
caged White Leghorn hens exposed to a cold environment had higher heterophil-to-lymphocyte ratios than those of the control hens (39.5 vs. 37.6). In relation to plasma corticosterone concentration (another stress indicator), Buckland et al. (1974) reported that the application of cold stress resulted in significant increases in plasma corticosterone levels in chicks, and Hester et al. (1996) found that caged hens exposed to a cold environment (0°C for 72 h) had higher plasma corticosterone levels than those of the control environment (39.5 vs. 37.6). In disagreement with these results, Hangalapura et al. (2004a) found a significant suppressive effect of cold stress (10°C for 7 d) on plasma corticosterone levels. The significant cold × breed interaction for heterophilto-lymphocyte ratio suggests that, depending on the genotype, the animals respond differently to different temperatures. A similar cold × breed interaction was found by Campo and Carnicer (1994) for the tonic immobility duration of several Spanish breeds of chickens including the Red-Barred Vasca, the Black Castellana, and the Buff Prat. In all 3 breeds, cold stress decreased the duration of tonic immobility, significantly in the Red-Barred Vasca breed (173 vs. 349 s), in agreement with the negative correlation between heterophil-to-lymphocyte ratio and tonic immobility duration reported by Campo and Redondo (1996, 1997) and Campo et al. (2007). Cold-stressed birds had the combined fluctuating asymmetry of morphological traits almost 15% larger than control birds without cold stress, the fluctuating asymmetry of middle toe length being more than 35% larger. This result is consistent with those found by Yalcin and Siegel (2003) in broiler embryos exposed to cooling. Relative asymmetry of wing, shank, and tibia lengths at 10 d of incubation increased in embryos exposed to cooling to 36.9°C six hours per day from 0 to 8 d of incubation, whereas the larger relative asymmetry of tibia and femur lengths at 18 d of incubation was for embryos exposed to 24 h to 21°C on d 14 of incubation, and for shank length at hatch, it was largest for embryos exposed to cooling to 36.9°C six hours per day from 10 to 18 d of incubation. Therefore, deviations from optimum environmental temperatures for chickens and embryos could contribute to
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the free-range system, hens from these breeds having significant greater heterophil number (heterophilia) and smaller lymphocyte number (lymphopenia) in the deeplitter system. In agreement with the latter result, van Loon et al. (2004) found that brown layers kept in the free-range system showed significantly higher in vitro lymphocyte proliferation than hens kept indoors in a litter system. The significant housing system × breed interaction for heterophil-to-lymphocyte ratio suggests that, depending on the genotype, the animals respond differently to different environments. Besides, deep-litter housing significantly increased the combined fluctuating asymmetry of 5 morphological traits almost 15% compared with the free-range system, the increase for the fluctuating asymmetry of 2 morphological traits (leg and wattle lengths) being almost 30%. The no significant housing effect on tonic immobility duration found in the current study at 36 wk of age agrees with the results reported by Hansen et al. (1993), who found that the duration of the tonic immobility response was unaffected by type of system in White Leghorn hens at 30 wk of age housed in cages and in aviaries, suggesting that the fearfulness is similar in both types of housing. Similarly, Weeks et al. (1993) found few differences in the behavior of broiler chickens housed in indoor and freerange environments, although it should be expected that chickens in an outdoor system performed more behavioral repertoire and suffered less from stress. As expected in experiment 2, the cold stress significantly affected heterophil-to-lymphocyte ratio and fluctuating asymmetry. Cold-stressed birds had the heterophil-to-lymphocyte ratio significantly higher, more than 50% in the Buff Prat and more than 2× in the Red-Barred Vasca, than control birds without cold stress, cold-stressed birds from these breeds having significant greater heterophil number (heterophilia); lymphocyte number was significantly smaller (lymphopenia) in cold-stressed birds from all 6 breeds. This was in agreement with the result reported by Hangalapura et al. (2004a), who found a significant enhancing effect of cold stress (10°C for 7 d) on in vitro lymphocyte proliferation. With respect to the heterophilto-lymphocyte ratio, Hester et al. (1996) also found that
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REFERENCES Buckland, R. B., K. Blagrave, and P. C. Lagne. 1974. Competitive protein-binding assay for corticoids in the peripheral plasma of the immature chicken. Poult. Sci. 53:241–245. Campo, J. L. 1991. Use of the sex-linked barring (B) gene for chick sexing on an eumelanotic columbian background. Poult. Sci. 70:1469–1473. Campo, J. L. 1998. Conservation and genetical study of Spanish chicken breeds (28). Pages 155–158 in Proc. 6th World Congr. Genet. Appl. Livest. Prod., Armidale, Australia. Univ. N. Engl., Armidale, New South Wales, Australia. Campo, J. L., and C. Carnicer. 1994. Effects of several stressors on tonic immobility reaction of chickens. Arch. Geflu¨gelkd. 88:75–78. Campo, J. L., M. G. Gil, S. G. Da´vila, and I. Mun˜oz. 2007. Genetic and phenotypic correlation between fluctuating asymmetry and two measurements of fear and stress in chickens. Appl. Anim. Behav. Sci. 102:53–64. Campo, J. L., and J. J. Jurado. 1982. Evaluation of multiple trait selection in strains of layers. Pages 869–874 in Proc. 2nd World Congr. Genet. Appl. Livest. Prod., Madrid, Spain. Editorial Garsi, Madrid, Spain. Campo, J. L., and F. Orozco. 1982. Conservation and genetical study of Spanish chicken breeds. Pages 88–93 in Proc. 2nd World Congr. Genet. Appl. Livest. Prod., Madrid, Spain. Editorial Garsi, Madrid, Spain. Campo, J. L., and F. Orozco. 1986. Genetic basis of the Melanotic Prat phenotype. Br. Poult. Sci. 27:361–367. Campo, J. L., M. T. Prieto, and S. G. Da´vila. 2008. Association between vent pecking and fluctuating asymmetry, heterophil to lymphocyte ratio, and tonic immobility duration in chickens. Appl. Anim. Behav. Sci. doi:10.1016/j.applanim. 2007.10.008. Campo, J. L., and A. Redondo. 1996. Tonic immobility reaction and heterophil to lymphocyte ratio in hens from three Spanish breeds laying pink eggshells. Poult. Sci. 75:155–159. Campo, J. L., and A. Redondo. 1997. Negative association between heterophil to lymphocyte ratio and tonic immobility reaction in hens. Pages 163–164 in Proc. 5th Eur. Symp. Poult. Welf., Wageningen, the Netherlands. Wageningen Agric. Univ., the Netherlands.
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larger fluctuating asymmetry of morphological traits, because cold stress results in the use of resources that could otherwise be used for developmental control. In conclusion, results of the current study indicate that housing in deep litter with access to an outdoor area resulted in smaller heterophil-to-lymphocyte ratio and smaller fluctuating asymmetry compared with housing in deep litter without access to an outdoor area, suggesting that the housing system is associated with some measures of stress and welfare. Duration of tonic immobility was not significantly longer in the deep-litter housing, indicating that indoor vs. outdoor systems did not contribute to the fearfulness of birds. Low ambient temperatures (0 to 10°C from 24 to 36 wk of age) resulted in greater heterophilto-lymphocyte ratios and higher fluctuating asymmetry, suggesting that a cold stress seriously negatively affects the welfare of birds. The significant housing × breed and cold × breed interactions for heterophil-to-lymphocyte ratio suggest that, depending on the genotype, the animals respond differently to different environments, the RedBarred Vasca being more sensitive to the environment.