INTRODUCTION. S. G. Dávila, 1 J. L. Campo, M. G. Gil, M. T. Prieto, and O. Torres

Effects of auditory and physical enrichment on 3 measurements of fear and stress (tonic immobility duration, heterophil to lymphocyte ratio, and fluctuating asymmetry) in several breeds of layer chicks S. G. Dávila, 1 J. L. Campo, M. G. Gil, M. T. Prieto, and O. Torres Departamento de Mejora Genética Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, 28080 Madrid, Spain ABSTRACT The purpose of this study was to analyze the effect of auditory enrichment (by means of classical music) or physical enrichment (by means of hanging colored string bunches and barley grains on the floor) on tonic immobility duration, heterophil to lymphocyte ratio, and fluctuating asymmetry (FA) in chicks of several layer breeds. In experiment 1, 192 chicks from 8 Spanish breeds and 1 White Leghorn population were reared in cages with or without music auditory enrichment until 8 wk of age. The effect of music auditory enrichment was significant for heterophil to lymphocyte ratio (P < 0.05). The ratios were higher in chicks reared without music than in those reared with music, suggesting that auditory enrichment from classical music reduces stress in chicks. There were significant differences in morphological trait measurements (relative asymmetry of wing length, leg width, and combined asymmetry; P < 0.05), being greater in chicks reared without music. This result suggests that FA is a good indicator for stress level in chicks, given that it follows INTRODUCTION Environmental enrichment for domestic animals is receiving growing attention. It can be defined as an improvement in the biological functioning of captive (domestic, zoo, or laboratory) animals, resulting from modifications to their environment (Newberry, 1995). More concisely, it is a procedure designed to increase environmental complexity (Jones, 1996). A variety of environmental modifications have been used as environmental enrichment methods in domestic animals, including physical and auditory enrichment. the same trend as that found for heterophil to lymphocyte ratio. There was a significant treatment by breed interaction (P < 0.05) for tonic immobility duration, indicating no consistent effect by auditory enrichment on tonic immobility duration across breeds. In experiment 2, 180 chicks from 3 Spanish breeds were housed in pens with or without physical enrichment (colored plastic string bunches and barley grains on the floor) until 6 wk of age. The effect of physical enrichment on tonic immobility duration, heterophil to lymphocyte ratio, and FA was not significant, indicating no effect on fear and stress in layer chicks. In conclusion, auditory enrichment by means of classical music is a reliable method for reducing stress levels in several breeds of layer chicks. However, music auditory enrichment was not effective in reducing fearfulness in any of the layer breeds. Physical enrichment by means of colored plastic string bunches and floor barley grains does not appear to be an effective method for reducing stress and fear in layer chicks. Key words: auditory enrichment, physical enrichment, tonic immobility duration, heterophil to lymphocyte ratio, fluctuating asymmetry 2011 Poultry Science Association Inc. Received May 9, 2011. Accepted July 23, 2011. 1 Corresponding author: sgdavila@inia.es 2011 Poultry Science 90 :2459 2466 doi:10.3382/ps.2011-01595 Physical enrichment has positive effects on the behavior of poultry (Braastad, 1990; Gvaryahu et al., 1994; Jones and Carmichael, 1998; Sherwin et al., 1999; Jones et al., 2000; McAdie et al., 2005; Dixon et al., 2010) and pigs (Fraser et al., 1991; Trickett et al., 2009), and improves welfare, productivity, and quality of chickens (Jones et al., 1980; Edmond et al., 2005; Leone and Estévez, 2008). Reports that music is good for chickens even appear in commercial journals (Anonymous, 1994). However, auditory enrichment does have some previously contradictory results in domestic animals. Uetake et al. (1997) found positive effects from auditory enrichment on behavior in dairy cows, whereas Cloutier et al. (2000) observed negative effects on stress in pigs, and Christensen and Knight (1975) found no significant effect on growing performance in meat-type chickens. 2459

2460 Dávila et al. The relationship between environmental enrichment and tonic immobility duration (a traditional measure of fearfulness in poultry; Gallup, 1979) has been analyzed in a few previous studies. Jones et al. (1991) and Jones and Waddington (1992) found a reduction in tonic immobility duration through physical enrichment (variety of colored objects) in Japanese quails and brown layers, respectively. Campo et al. (2005) found that auditory enrichment through classical music increased tonic immobility duration in adult laying hens housed in floor pens. Gvaryahu et al. (1989), studying meat broilers, tested the combined effects of auditory enrichment by means of classical music and physical enrichment by means of hanging red gloves. They reported that treated chickens had shorter tonic immobility durations than control chickens at 6 wk, although there was no significant difference at 7 wk. Nicol (1992), studying fear responses in broilers as affected by auditory enrichment through classical music and physical enrichment through colored plastic rings, milk bottle tops, and grass, demonstrated that duration of tonic immobility was shorter in enriched birds than in nonenriched birds. Using other fear indicators, Jones (1982) reported that physical enrichment significantly reduced fear levels in brown layers, suggesting that environmental enrichment may have a wide-ranging effect on fear; however, Miller and Mench (2005) observed that physical enrichment had no significant effect on fear in Japanese quails. The relationship between environmental enrichment and heterophil to lymphocyte ratio (a reliable indicator of stress in poultry; Gross and Siegel, 1983) has been studied less thoroughly. Ladd et al. (1992) observed that regular exposure to country music reduced this ratio in white layers, but found the ratio to be similar in the control group and in birds that listened to classical music. Martrenchar et al. (2001) found no significant effects from physical enrichment by means of metal objects (iron sheets and metal chains) and straw in the heterophil to lymphocyte ratio in turkeys. Also, Campo et al. (2005) found that auditory enrichment through classical music did not affect the heterophil to lymphocyte ratio in laying hens. The objective of the present study was to analyze the effect of auditory enrichment by means of classical music or physical enrichment by means of hanging colored string bunches and administering floor barley grains on tonic immobility duration, heterophil to lymphocyte ratio, and fluctuating asymmetry (FA) in several breeds of layer chicks. Fluctuating asymmetry, defined as the small and random deviations from perfect symmetry, caused by environmental and genetic stress during development (van Valen, 1962), has been reported to reflect chronic stress and welfare status (Parsons, 1990; Moller and Swaddle, 1997). Our current study consisted of 2 experiments. Experiment 1 assessed the effect of auditory enrichment and experiment 2 assessed the effect of physical enrichment on layer chicks. We hypothesized that chicks reared under environmental enrichment conditions would show a shorter duration of tonic immobility, decreased heterophil to lymphocyte ratio, and less FA in bilateral traits compared with control chicks. Relative to the existing literature, ours is the first study to analyze the effect of environmental enrichment on FA, at the same time complementing previous experiments in the evaluation of the effect of environmental enrichment on tonic immobility duration and heterophil to lymphocyte ratio. MATERIALS AND METHODS General Procedure In the current study, layer chicks from 8 Spanish breeds: Black-Barred Andaluza, Black-Breasted Red Andaluza, Blue Andaluza, Black Castellana, Buff Prat, Red-Barred Vasca, Red Villafranquina, and Quail Castellana (a synthetic breed originating from an F 2 cross between Black Castellana and Buff Prat; Campo and Orozco, 1986; Campo, 1991), along with 1 White Leghorn population (created by crossing 3 strains selected for egg number and weight; Campo and Jurado, 1982) were assessed. The Andaluza and Castellana breeds are white-shell egg layers, the Prat breed is a tinted-shell egg layer, and the Vasca and Villafranquina breeds are brown- and dark brown-shell egg layers. These breeds are maintained at the experimental station of El Encín (Madrid, Spain) in a conservation program of genetic resources that started in 1975 (Campo and Orozco, 1982) and have been described by Campo (1998). All of the chicks were reared under standard temperatures that were controlled by electric or gas heaters (experiments 1 and 2, respectively; 33 35 C at chick level for wk 1, followed by a reduction of 3 C/wk until the temperature reached 18 20 C at 6 wk of age). Artificial light was only provided during the first week (23L:1D). Chicks were fed standard rearing diets containing 19% CP, 2,800 kcal of ME/kg, 1% Ca, and 0.5% available P. Feed and water were supplied for ad libitum consumption. To measure the duration of tonic immobility, chicks were caught randomly and carried in an upright position to a separate room. A few seconds after the chick was caught, tonic immobility was induced by placing the bird on its back with its head hanging in a U- shaped wooden cradle (Jones and Faure, 1981). The chick was restrained for 10 s, during which time the observer sat in full view of the chick (approximately 1 m away) with his eyes fixed on the chick, providing fear-inducing properties of eye contact. If the chick remained immobile for 10 s after the experimenter removed his hands, a stopwatch was initiated in order to record the latency duration (s) until the chick righted itself. If the chick righted itself in less than 10 s, then it was considered that tonic immobility was not induced and the restraint procedure was repeated (3 times maximum). If the chick did not show a righting response over the 10-min test period, a maximum score of 600 s was assigned for the latency duration. Thus, tonic

AUDITORY AND PHYSICAL ENRICHMENT IN LAYER BREEDS immobility duration ranged from 0 to 600 s, and was logarithmically transformed before analysis. The heterophil to lymphocyte ratio was obtained the day following the test for tonic immobility. To measure the ratio, chicks were caught randomly and carried to a separate room where 2 drops of blood were immediately collected from a small puncture in the comb of each chick; 1 drop was smeared on each of 2 glass slides. The smears were stained (using May-Grünwald and Giemsa stains; Lucas and Jamroz, 1961) approximately 2 to 4 h after methyl-alcohol fixation. One hundred leukocytes, including granular (heterophil, eosinophil, and basophil) and nongranular leukocytes (lymphocyte and monocyte), were counted from 1 slide of each chick (the other slide was supplementary and not analyzed), and the heterophil to lymphocyte ratio was calculated. The ratios were square root transformed before analysis. The chick morphological trait measurements were taken after the blood sampling. These measurements consisted of right (R) and left (L) middle toe length (from the metatarsus to the nail), leg (metatarsus) length (from the hock joint to the middle toe), wing (radius) length (from the humerus joint to the carpus), and leg width (in the hock joint). Both R and L values from a given bird were taken during the same session. The 3 length measurements and the leg width measurement were recorded in millimeters using a digital caliper. The trait size was recorded as the mean of the right and left traits [(R + L)/2]. All of the traits showed a normal frequency distribution. The FA of a trait was defined by the absolute differences between sides [ R L ]. A series of steps (Palmer, 1994; Knierim et al., 2007) was followed before identifying the exhibited asymmetry as FA (normal distribution of signed R L differences with a mean of zero) because several confounding factors can complicate the analysis of asymmetry (details can be found in the preliminary study by Campo et al., 2008). First, the presence of antisymmetry (nonnormal distribution with a mean of zero) was determined by inspecting the distribution of (R L). Departures from normality were assessed using kurtosis measures; antisymmetry is characterized by bimodal (or broad-peaked) distributions and tends to be platykurtic (more intermediate values than the normal distribution). Second, FA and measurement error are normally distributed over a mean of zero. Thus, it is essential to show that the variance in asymmetry observed between individuals is greater than the variance due to measurement error; FA is often small and sometimes of the same magnitude as measurement error. The effect of measurement error was calculated from a subsample of 20 randomly selected birds and measured 3 times on 3 days. These repeated measurements were analyzed using a 2-way ANOVA (Leamy, 1984), with the side (R, L) as a fixed factor and bird as a random factor (1 and 19 df). Their interaction was 19 df and the measurement error was 80 df. A significant variation between sides indicates directional asymmetry (normal distribution with a nonzero mean), whereas a significant interaction indicates significant FA (in the absence of antisymmetry). In the preliminary study by Campo et al. (2008), toe, leg, and wing lengths were considered suitable for the study of FA based on the presence of a high FA to measurement error and the absence of both directional asymmetry and antisymmetry. Leg width exhibited a significant level of FA and directional asymmetry; it was included in the study by subtracting the mean of (R L), a measure of directional asymmetry, from each value of FA to eliminate directional asymmetry (van Valen, 1962). Finally, the product-moment correlation between FA and trait value was used in determining their independence of each other. If a positive relationship was found between the mean value and the asymmetry of a trait, this effect was removed by dividing the absolute asymmetry score by the trait mean, defined as the relative FA: [2 R L / (R + L)]. Relative FA was used for all traits; it had distributions that were not normal and were arcsin square root transformed before analysis. As an alternative to the analysis of FA based on the 4 single morphological traits, combined relative asymmetry was considered (defined as the mean of the relative asymmetries of the different traits). The analysis based on the combined FA may be a more reliable indicator of stress (Leung et al., 2000). All measurements were made by S. G. Dávila. Auditory Enrichment Layer chicks from 8 different breeds (Black-Barred Andaluza, Black-Breasted Red Andaluza, Blue Andaluza, Black Castellana, Buff Prat, Quail Castellana, Red Villafranquina, and White Leghorn) were used in experiment 1. They were reared in cages from d 1 to 8 wk of age at a density of 4 birds/m 2. Cages had 2 drinkers and 2 feeders on the outside. In total, the 192 chicks from 3 different replicates, which performed at 3 separate times (64 chicks in each replicate), were used to study the effect of auditory enrichment on tonic immobility duration, heterophil to lymphocyte ratio, and FA at 8 wk of age. There were 12 cages (8 chicks/cage, 1 chick from each breed) in each treatment (4 cages/ replicate). Two auditory treatments were used. Treatment 1 (auditory enrichment) consisted of 96 chicks (32 in each replicate) reared with classical music (Mozart s string quartets) between 0900 and 1400 h from d 1 to 8 wk of age for 3 d/wk; the sound level was a maximum of 75 db (ranging from 70 75 db from the background noises plus music). Treatment 2 (control) consisted of 96 additional chicks (32 in each replicate) reared without music; the sound level was a maximum of 65 db (ranging from 60 65 db from the chicks and fan noises). Physical Enrichment 2461 Layer chicks from 3 breeds (Black Castellana, Buff Prat, and Red-Barred Vasca) were used in experiment

2462 Dávila et al. 2. The chicks from all 3 breeds were reared together in an all-litter floor pen from d 1 to 6 wk of age at a density of 10 birds/m 2. In total, the 180 chicks from 3 different replicates, which performed at 3 separate times (60 chicks in each replicate), were sampled to study the effect of physical enrichment on tonic immobility duration, heterophil to lymphocyte ratio, and FA at 6 wk of age. There were 3 pens in each treatment (1 pen/ replicate). Two physical treatments were used. Treatment 1 (physical enrichment) consisted of 90 randomly selected chicks (30 in each replicate, 10 chicks from each breed) reared with plastic string bunches (red, yellow, green, and blue) hanging at the chick s level (1 string bunch/50 chicks) and barley grains on the pen floor from d 1 to 6 wk of age. The enrichment items were not changed; barley grains were given daily. The chicks interacted with the enrichment items throughout the experiment. Jones et al. (2002) indicated that plastic string bunches are particularly attractive for both chicks and adults, and can retain a bird s interest over a lengthy period. Treatment 2 (control) consisted of 90 additional randomly selected chicks (30 in each replicate, 10 chicks from each breed) reared without colored string bunches or barley grains. Statistical Analysis A 3-way ANOVA (Sokal and Rohlf, 1981) was used with the statistical model: x ijkl = m + T i + B j + TB ij + r k + Tr ik + Br jk + TBr ijk + e ijkl, where x ijkl is the analyzed measurement, m is the overall mean, T i is the effect of the treatment (auditory or physical enrichment vs. control in experiments 1 and 2, respectively), B j is the effect of breed (j = 1 to 8 and j = 1 to 3 in experiments 1 and 2, respectively), r k is the effect of replicate (k = 1 to 3), TB ij, Tr ik, Br jk, and TBr ijk are the interactions, and e ijkl is the residual (k = 1 to 4 and k = 1 to 10 in experiments 1 and 2, respectively). Treatment and breed were considered fixed effects and replicates were considered to be a random effect. When there were no significant differences among replicates or their interactions, they were pooled with the residual to give a 2-way factorial model of treatment and breed effects (x ijk = m + T i + B j + TB ij + e ijk ). Significant differences among breeds were estimated using the Student-Newman-Keuls multiple-range test (Snedecor and Cochran, 1980). The SAS statistical package, GLM procedure, was used for data analysis (SAS Institute, Cary, NC). RESULTS Table 1. Mean heterophil to lymphocyte ratio in layer chicks reared with or without auditory enrichment by means of classical music (experiment 1) Item Heterophil: lymphocyte Auditory enrichment Yes 0.32 b No 0.37 a Black-Barred Andaluza 0.29 ab Black-Breasted Red Andaluza 0.41 ab Blue Andaluza 0.33 ab Black Castellana 0.44 a Buff Prat 0.25 ab Quail Castellana 0.43 a Red Villafranquina 0.21 b White Leghorn 0.43 a Error mean square 0.06 a,b Means within the same effect with no common letters differ (P < 0.05). There was no significant interaction found between auditory enrichment and layer breed (F = 1.09; P = 0.37) for the heterophil to lymphocyte ratio (experiment 1). Mean values indicating the effects of auditory enrichment and breed are summarized in Table 1. The effect of auditory enrichment by means of classical music was significant (F = 4.08; P = 0.03), with heterophil to lymphocyte values being higher in chicks reared without music than in those reared with music. There were significant differences among breeds (F = 3.49; P = 0.002) with respect to heterophil to lymphocyte ratio, which was greater in the Black Castellana, Quail Castellana, and White Leghorn breeds and smaller in the Red Villafranquina breed. The effect of auditory enrichment on tonic immobility duration varied from breed to breed (significant treatment by breed interaction; F = 2.14; P = 0.04; Table 2). Difference between treatments was significant in the Black Castellana breed, with the tonic immobility duration being longer within the group of control chicks than within the group of chicks reared with music. There was no significant interaction between auditory enrichment and breed for relative FA of toe length (F = 1.49; P = 0.18), wing length (F = 1.05; P = 0.40), Table 2. Mean duration of tonic immobility (s) in layer chicks reared with or without auditory enrichment by means of classical music (experiment 1) Item Yes Auditory enrichment Black-Barred Andaluza 397 215 Black-Breasted Red Andaluza 133 196 Black Castellana 141 b 330 a Blue Andaluza 150 228 Buff Prat 148 266 Quail Castellana 150 256 Red Villafranquina 245 215 White Leghorn 359 242 Error mean square 34,392 a,b Means within the same breed with no common letters differ (P < 0.05). No

AUDITORY AND PHYSICAL ENRICHMENT IN LAYER BREEDS 2463 Table 3. Mean relative asymmetry (values 100) of morphological traits in layer chicks reared with or without auditory enrichment by means of classical music (experiment 1) 1 Item Toe length Wing length Leg length Leg width Combined 2 Auditory enrichment Yes 3.13 1.55 b 1.64 6.64 b 3.24 b No 3.84 2.27 a 1.92 10.13 a 4.54 a Black-Barred Andaluza 3.33 1.99 2.16 8.07 3.89 Black-Breasted Red Andaluza 3.78 2.10 1.62 7.29 3.70 Blue Andaluza 3.98 1.94 1.78 7.78 3.87 Black Castellana 3.65 1.64 1.41 10.56 4.32 Buff Prat 3.32 1.64 1.94 7.12 3.50 Quail Castellana 4.08 2.33 1.30 6.80 3.63 Red Villafranquina 2.73 1.88 1.67 10.22 4.13 White Leghorn 3.01 1.67 2.31 8.28 3.82 Error mean square 0.10 0.04 0.02 0.85 0.08 a,b Means within the same effect and column with no common letters differ (P < 0.05). 1 Relative asymmetry is fluctuating asymmetry [ Right (R) Left (L) ] divided by the trait mean [(R + L)/2] :2 R L /(R + L). 2 Combined = toe length, leg length, wing length, and leg width. leg length (F = 1.34; P = 0.24), leg width (F = 0.27; P = 0.96), or combined relative asymmetry (F = 0.32; P = 0.94). The effect of music on relative FA is shown in Table 3. There were significant differences in relative asymmetry of wing length (F = 5.87; P = 0.02) and leg width (F = 6.42; P = 0.01), being greater in the chicks reared without music. The combined relative asymmetry of the 4 bilateral traits (toe length, leg length, wing length, and leg width) was also greater in the control chicks (F = 10.09; P = 0.002). There were no significant differences for relative asymmetry of toe length (F = 2.71; P = 0.10) or leg length (F = 0.74; P = 0.39). There were no significant differences among breeds for relative asymmetry of toe length (F = 0.39; P = 0.91), wing length (F = 0.52; P = 0.82), leg length (F = 1.71; P = 0.11), leg width (F = 0.44; P = 0.88), or combined relative asymmetry (F = 0.05; P = 0.99). In experiment 2, there was no significant interaction between physical enrichment and breed in the heterophil to lymphocyte ratio (F = 0.70; P = 0.50) or in the tonic immobility duration (F = 0.70; P = 0.50). The effect of physical enrichment on heterophil to lymphocyte ratio or tonic immobility duration was not significant (F = 0.90; P = 0.34, and F = 0.44; P = 0.51, respectively; Table 4). There were no significant differences among breeds in the heterophil to lymphocyte ratio (F = 2.94; P = 0.06) or in the tonic immobility duration (F = 0.69; P = 0.51). There was no significant interaction between physical enrichment and breed in relative FA of toe length (F = 0.08; P = 0.92), wing length (F = 0.26; P = 0.77), leg length (F = 0.85; P = 0.43), leg width (F = 1.42; P = 0.25), or combined relative asymmetry (F = 1.14; P = 0.32). The effect of physical enrichment on relative FA is shown in Table 5. There were no significant differences in the relative asymmetry of toe length (F = 2.71; P = 0.10), wing length (F = 0.21; P = 0.65), leg length (F = 0.01; P = 0.92) or leg width (F = 0.17; P = 0.68). The combined relative asymmetry was also not significant (F = 0.79; P = 0.37). There were no significant differences among breeds in the relative asymmetry of toe length (F = 0.26; P = 0.77), wing length (F = 1.10; P = 0.34), leg length (F = 0.32; P = 0.73), leg width (F = 1.64; P = 0.20) or combined relative asymmetry (F = 0.93; P = 0.40). DISCUSSION The significant difference in heterophil to lymphocyte ratio found between layer chicks reared with auditory enrichment and control layer chicks reared without enrichment is in agreement with the expected change, suggesting that environmental enrichment by means of classical music reduces stress in chicks. The heterophil to lymphocyte ratio was 0.32 for the chicks with auditory enrichment. This indicates a moderate effect from music, bearing in mind that heterophil to lymphocyte ratios of 0.2, 0.5, and 0.8 are characteristic of low, optimal, and high degrees of stress, respectively (Gross and Siegel, 1993). Control chicks had a heterophil to lymphocyte ratio that was 15% higher than chicks reared with classical music, showing significant heterophilia, but no significant lymphopenia. These results were consistent in all breeds. Ladd et al. (1992) and Campo et al. (2005) did not observe a significant effect from Table 4. Mean heterophil to lymphocyte ratio and tonic immobility duration in layer chicks reared with or without physical enrichment by means of colored string bunches and barley grains (experiment 2) Item Heterophil: lymphocyte Tonic immobility (s) Physical enrichment Yes 0.40 281 No 0.42 318 Black Castellana 0.46 315 Buff Prat 0.39 308 Red-Barred Vasca 0.39 275 Error mean square 0.06 41,235

2464 Dávila et al. Table 5. Mean relative asymmetry (values 100) of morphological traits in layer chicks reared with or without physical enrichment by means of colored string bunches and barley grains (experiment 2) 1 Item Toe length Wing length Leg length Leg width Combined 2 Physical enrichment Yes 3.71 2.60 2.21 5.53 3.51 No 2.80 2.85 2.28 5.29 3.30 Black Castellana 2.83 2.02 2.31 5.99 3.29 Buff Prat 3.76 2.28 2.14 4.73 3.23 Red-Barred Vasca 3.16 3.88 2.27 5.50 3.70 Error mean square 0.15 0.48 0.02 0.17 0.05 1 Relative asymmetry is fluctuating asymmetry [ Right (R) Left (L) ] divided by the trait mean [(R + L)/2] :2 R L /(R + L). 2 Combined = toe length, leg length, wing length, and leg width. auditory enrichment by means of music on heterophil to lymphocyte ratio in adult laying hens, which is in agreement with the better expected effects of early environmental enrichment in comparison with effects in adult birds. The results observed in this study indicate a significant effect from auditory enrichment on the FA of wing length and leg width. Control chicks showed significantly higher relative asymmetry of wing length (46%) and leg width (52%) compared with the chicks reared with classical music. The combined relative asymmetry of the 4 bilateral traits was also significantly higher in control chicks (40%) than in chicks reared with music. This result suggests that FA is a good indicator of the stress level in chicks, given that it follows the same trend as the heterophil to lymphocyte ratio, which is considered to be the best indicator of stress in chickens. In agreement with these facts, Jones (1996) strongly recommended auditory enrichment through music. There was no consistent effect from auditory enrichment on tonic immobility duration (significant treatment by breed interaction), with the different breeds being affected differently by the environmental enrichment with classical music. According to the expectations, auditory enrichment reduced tonic immobility duration in 5 of the 8 breeds of layers studied, although only significantly in the Black Castellana breed. Control chicks in the Black Castellana breed had durations of tonic immobility that were more than 2 times longer than in chicks reared with classical music. In disagreement with this result, Campo et al. (2005) found a significant effect from auditory enrichment through music on tonic immobility duration in 36-wk-old hens from 2 Spanish breeds (Black-Breasted Red Andaluza and Birchen Leonesa) housed in floor pens. This discrepancy might be because of the age of the birds, the type of housing, and the breed, although Reed et al. (1993) indicated that early enrichment during rearing showed fear-reducing effects that persisted into adulthood in brown layers. Contrary to the expectations, chicks reared with physical enrichment by means of colored string bunches and barley grains did not show shorter durations of tonic immobility, decreased heterophil to lymphocyte ratio, or less FA compared with control chicks. These stress indicators might signal the absence of a stressful environment, but do not signal the presence of an enriched environment. In agreement with our results, Martrenchar et al. (2001) did not find a significant effect on heterophil to lymphocyte ratio from physical enrichment by means of metal objects and straw in young turkeys, and likewise, Miller and Mench (2005) did not find significant effects from physical enrichment in Japanese quail using the emergence, novel food, and flight distance fear tests. A significantly shorter tonic immobility duration in birds housed with physical enrichment was found by Jones et al. (1991) and Jones and Waddington (1992) in quails and brown layers, respectively. However, in their experiments the colored objects were changed at intervals of 3 d in order to avoid habituation in the birds. Periodic changes of the stimulus objects do not seem necessary, although the enrichment effect might be enhanced if such changes are made (Jones, 1982). Jones and Carmichael (1999) did not find evidence of habituation in experiments assessing chicks reared in a physically enriched environment by means of string bunches. Using the open field and the emergence fear tests, Jones (1982) suggested that early environmental enrichment decreases fearfulness in fear-inducing situations in brown layers. Obviously, the effect from physical enrichment can be different depending on the type of enrichment used, which in our case was the color of the string bunch. Jones et al. (2000) suggested that white or yellow strings may be preferred to green, blue, or red ones, or combinations of 2 or more colors. Gvaryahu et al. (1989) and Nicol (1992) found a shorter duration of tonic immobility at 6 wk of age in meat chickens reared with both types of environmental enrichment (auditory and physical) simultaneously. These findings disagree with the lack of significance found in our 2 experiments on layers, which considered auditory and physical enrichment separately. However, Gvaryahu et al. (1989) did not find significant effects from auditory plus physical enrichment at 7 wk of age, which is in agreement with our results obtained at 6 to 8 wk of age in layers. Campo and Carnicer (1993) observed significant variation in tonic immobility duration associated with age in both males and females until 8 wk of age. Environmental enrichment is one of

AUDITORY AND PHYSICAL ENRICHMENT IN LAYER BREEDS the suggested methods for reducing fear levels in chickens (Jones, 1996), and it is also starting to be applied to commercial animals for the improvement of welfare, along with free-range outdoor access and the prohibition of cages. These resultswere not corroborated by the results from both experiments in the current study. In conclusion, auditory enrichment by means of classical music resulted in an improvement for the chicks, whereas physical enrichment by means of colored plastic string bunches and barley grains did not. Auditory enrichment can be considered a reliable method for reducing the stress level, heterophil to lymphocyte ratio, and FA indicators in layer chicks, although it is not an effective method for reducing fearfulness. Physical enrichment did not significantly affect tonic immobility duration, heterophil to lymphocyte ratio, or FA, and should not be considered an effective method for reducing stress or fear levels in chicks. REFERENCES Anonymous. 1994. Music good for chickens. World Poult. 10:7. Braastad, B. O. 1990. Effects on behavior and plumage of a keystimuli floor and a perch in triple cages for laying hens. Appl. Anim. Behav. Sci. 27:127 139. 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. Pages 155 158 in Proc. 6th World Congr. Genet. Appl. Livestock Prod., Armidale, Australia. Univ. New England, Armidale, New South Wales, Australia. Campo, J. L., and C. Carnicer. 1993. Realized heritability of tonic immobility in White Leghorn hens: A replicated single-generation test. Poult. Sci. 72:2193 2199. Campo, J. L., M. G. Gil, and S. G. Dávila. 2005. Effects of specific noise and music stimuli on stress and fear levels of laying hens of several breeds. Appl. Anim. Behav. Sci. 91:75 84. 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. Ed. 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. Ed. 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. Dávila. 2008. Association between vent pecking and fluctuating asymmetry, heterophil to lymphocyte ratio, and tonic immobility duration in chickens. Appl. Anim. Behav. Sci. 113:87 97. Christensen, A. C., and A. D. Knight. 1975. Observations on the effects of music exposure to growing performance of meat-type chicks. Poult. Sci. 54:619 621. Cloutier, S., D. M. Weary, and D. Fraser. 2000. Can ambient sound reduce distress in piglets during weaning and restraint? J. Appl. Anim. Welf. Sci. 3:107 116. Dixon, L. M., I. J. Duncan, and G. J. Mason. 2010. The effects of four types of enrichment on feather-pecking behavior in laying hens housed in barren environments. Anim. Welf. 19:429 435. Edmond, A., L. A. King, S. E. Solomon, and M. M. Bain. 2005. Effect of environmental enrichment during the rearing phase on subsequent eggshell quality in broiler breeders. Br. Poult. Sci. 46:182 189. Fraser, D., P. A. Phillips, B. K. Thompson, and T. Tennessen. 1991. Effect of straw on the behavior of growing pigs. Appl. Anim. Behav. Sci. 30:307 318. 2465 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. Gross, W. B., and P. B. Siegel. 1993. General principles of stress and welfare. Pages 21 34 in Livestock, Handling and Transport. T. Grandin, ed. CABI, Wallingford, UK. Gvaryahu, G., E. Ararat, E. Asaf, M. Lev, J. I. Weller, B. Robinzon, and N. Snapir. 1994. An enrichment object that reduces aggressiveness and mortality in caged laying hens. Physiol. Behav. 55:313 316. Gvaryahu, G., D. L. Cunningham, and A. Van Tienhoven. 1989. Filial imprinting, environmental enrichment, and music application. Effects on behavior and performance of meat-strain chicks. Poult. Sci. 68:211 217. Jones, R. B. 1982. Effects of early environmental enrichment upon open-field behavior and timidity in the domestic chick. Dev. Psychobiol. 15:105 111. Jones, R. B. 1996. Fear and adaptability in poultry: Insights, implications, and imperatives. World s Poult. Sci. J. 52:131 174. Jones, R. B., and N. L. Carmichael. 1998. Pecking at string by individually caged, adult laying hens: Color preferences and their stability. Appl. Anim. Behav. Sci. 60:11 23. Jones, R. B., and N. L. Carmichael. 1999. Responses of domestic chicks to selected pecking devices presented for varying durations. Appl. Anim. Behav. Sci. 64:125 140. Jones, R. B., N. L. Carmichael, and E. Rayner. 2000. Pecking preferences and predispositions in domestic chicks: Implications for the development of environmental enrichment devices. Appl. Anim. Behav. Sci. 69:291 312. Jones, R. B., and J. M. Faure. 1981. Sex and strain comparisons of tonic immobility ( righting time ) in the domestic fowl and the effects of various methods of induction. Behav. Processes 6:47 55. Jones, R. B., S. Harvey, B. O. Hughes, and A. Chadwick. 1980. Growth and the plasma concentrations of growth hormone and prolactin in chicks: Effects of environmental enrichment, sex, and strain. Br. Poult. Sci. 21:457 462. Jones, R. B., T. M. McAdie, C. McCorquodale, and L. J. Keeling. 2002. Pecking at other birds and at string enrichment devices by adult laying hens. Br. Poult. Sci. 43:337 343. Jones, R. B., A. D. Mills, and J. M. Faure. 1991. Genetic and experiential manipulation of fear-related behavior in Japanese-quail chicks (Coturnix coturnix japonica). J. Comp. Psychol. 105:15 24. Jones, R. B., and D. Waddington. 1992. Modification of fear in domestic chicks, Gallus gallus domesticus, via regular handling and early environmental enrichment. Anim. Behav. 43:1021 1033. 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. Ladd, J. K., J. L. Albright, A. M. Beck, and B. T. Ladd. 1992. Behavioral and physiological studies on the effect of music on animals. J. Anim. Sci. 70:170. Leamy, L. 1984. Morphometric studies in inbred and hybrid house mice. V. Directional and fluctuating asymmetry. Am. Nat. 123:579 593. Leone, E. H., and I. Estévez. 2008. Economic and welfare benefits of environmental enrichment for broiler breeders. Poult. Sci. 87:14 21. Leung, B., M. R. Forbes, and D. Houle. 2000. Fluctuating asymmetry as a bioindicator of stress: Comparing efficacy of analysis involving multiple traits. Am. Nat. 155:101 115. Lucas, A. M., and C. Jamroz. 1961. Atlas of Avian Hematology. Agriculture Monograph 25. USDA, Washington, DC. Martrenchar, A., D. Huonnic, and J. P. Cotte. 2001. Influence of environmental enrichment on injurious pecking and perching behaviour in young turkeys. Br. Poult. Sci. 42:161 170. McAdie, T. M., L. J. Keeling, H. J. Blokhuis, and R. B. Jones. 2005. Reduction in feather pecking and improvement of feather condition with the presentation of a string device to chickens. Appl. Anim. Behav. Sci. 93:67 80.

2466 Dávila et al. Miller, K. A., and J. A. Mench. 2005. The differential effects of four types of environmental enrichment on the activity budgets, fearfulness, and social proximity preference of Japanese quail. Appl. Anim. Behav. Sci. 95:169 187. Moller, A. P., and J. P. Swaddle. 1997. Asymmetry, Developmental Stability, and Evolution. Oxford University Press, Oxford, UK. Newberry, R. C. 1995. Environmental enrichment: Increasing the biological relevance of captive environments. Appl. Anim. Behav. Sci. 44:229 243. Nicol, C. J. 1992. Effects of environmental enrichment and gentle handling on behavior and fear responses of transported broilers. Appl. Anim. Behav. Sci. 33:367 380. Palmer, A. R. 1994. Fluctuating asymmetry analyses: A primer. Pages 335 364 in Developmental Instability: Its Origins and Evolutionary Implications. T. A. Markow, ed. Kluwer Academic, Dordrecht, the Netherlands. Parsons, P. A. 1990. Fluctuating asymmetry. An epigenetic measure of stress. Biol. Rev. Camb. Philos. Soc. 65:131 145. Reed, H. J., L. J. Wilkins, S. D. Austin, and N. G. Gregory. 1993. The effect of environmental enrichment during rearing on fear reactions and depopulation trauma in adult caged hens. Appl. Anim. Behav. Sci. 36:39 46. Sherwin, C. M., P. D. Lewis, and G. C. Perry. 1999. Effects of environmental enrichment, fluorescent and intermittent lighting, on injurious pecking amongst male turkey poults. Br. Poult. Sci. 40:592 598. Snedecor, G. W., and W. G. Cochran. 1980. Statistical Methods. 7th ed. Iowa State University Press, Ames. Sokal, R. R., and F. J. Rohlf. 1981. Biometry. Freeman and Co., London, UK. Trickett, S. L., J. H. Guy, and S. A. Edwards. 2009. The role of novelty in environmental enrichment for the weaned pig. Appl. Anim. Behav. Sci. 116:45 51. Uetake, K., J. F. Hurnik, and L. Johnson. 1997. Effect of music on voluntary approach of dairy cows to an automatic milking system. Appl. Anim. Behav. Sci. 53:175 182. van Valen, L. 1962. A study of fluctuating asymmetry. Evolution 16:125 142.