Poult. Sci.
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Poult Sci 2008. 87:1540-1543. doi:10.3382/ps.2008-00067
© 2008 Poultry Science Association
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GENETICS

Effect of Bad Collocation of Wing Tag on Feather Amelanosis, Heterophil-to-Lymphocyte Ratio, Fluctuating Asymmetry, and Tonic Immobility Duration in White-Faced Black Spanish Hens

J. L. Campo1 and S. G. Dávila

Departamento de Mejora Genética Animal, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Apartado 8.111, 28080 Madrid, Spain

1 Corresponding author: jlcampo{at}inia.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
The purpose of the present study was to analyze the effects of bad collocation of the wing tag on feather amelanosis, the heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration at 140 d of age in hens from the White-Faced Black Spanish breed. A total of 52 females were used. There was a significant difference (P < 0.05) for the heterophil-to-lymphocyte ratio and the tonic immobility duration between groups of females with bad or good collocation of the wing tag, with the ratio being higher and the duration being longer in the former group. Females with bad collocation of the wing tag had significant heterophilia and lymphopenia (P < 0.05). There was a significant difference (P < 0.05) in the fluctuating asymmetry of the middle and hind toe lengths, the combined asymmetry of the 4 toes, the fluctuating asymmetry of the earlobe area, and the combined asymmetry of toe and leg lengths and earlobe and wattle areas, with the asymmetry of birds with bad collocation of the wing tag being larger than that of birds with good collocation of the wing tag. Results indicate that bad collocation of the wing tag negatively affects measures of stress, such as the heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration.

Key Words: wing tag bad collocation • feather amelanosis • heterophil-to-lymphocyte ratio • fluctuating asymmetry • tonic immobility


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Wing-tagged chicks from a conservation program of Spanish chicken breeds sometimes develop a callus around the tag as a consequence of a bad collocation by the experimenter. In the White-Faced Black Spanish breed, some of the birds with a callus also showed spontaneous amelanosis in the feathers around the wing tag at 56 d of age (moving to the second rearing house). This was not a temporary phenomenon, and birds showed amelanosis at 140 d of age (moving to the laying house) and at 252 d of age (moving to the breeding house). All of these spontaneous amelanotic birds were females. Birds from other Spanish breeds (females and males) and White-Faced Black Spanish males sometimes develop a callus as a consequence of bad collocation of the wing tag, but they never show feather amelanosis around the tag. The White-Faced Black Spanish breed carries the extended black allele and is the only one in the world that has enlarged earlobes, with the white earlobes covering the whole face. It is interesting to note that this trait is not seen in young birds but develops as the birds mature, and the white pigment is made up of purine bases (Smyth, 1990).

Because the immune system has been shown to be involved in the etiology of the Smyth chicken line, a model for autoimmune amelanosis and human vitiligo (see Smyth, 1989 for a review), it was considered that the amelanosis of the aforementioned birds might have an autoimmune component that was the result of the wing tag insult and the subsequent inflammatory reaction, with the melanocytes being destroyed and eliminated by the immune system. Erf and Smyth (1996) found that chickens from the Smyth line exhibiting feather amelanosis had significantly higher proportions of heterophils and lower proportions of lymphocytes than control birds. Maxwell and Robertson (1998) suggested that avian leukocytes will respond to problems associated with stress and trauma. A possible association between vitiligo and scleroderma was discussed by Bonifati et al. (2006).

The purpose of the present study was to analyze the effect of bad collocation of the wing tag on feather amelanosis and the heterophil-to-lymphocyte ratio, a reliable indicator of stress in poultry (Gross and Siegel, 1983), in hens of the White-Faced Black Spanish breed of chickens. Additionally, the effects of bad collocation of the wing tag on fluctuating asymmetry, a measure that has been reported to reflect chronic stress and welfare status (Parsons, 1990), and duration of tonic immobility, a traditional measure of fearfulness in poultry (Gallup, 1979), were analyzed.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 REFERENCES
 
The White-Faced Black Spanish breed of chickens is maintained at the experimental station of El Encín (Madrid, Spain) in a conservation program of genetic resources begun in 1975 (Campo and Orozco, 1982). It is a classical ornamental breed internationally known by poultry fanciers, which was admitted to the American Standard of Perfection in 1874. Its plumage is extended black, and birds have a white face and large earlobes meeting in front and extending below the wattles.

Chicks were wing tagged at 1 d of age (zip size-3; National Band and Tag Co., Newport, KY). All tagging was done by the same individual. After the individual had inserted the band through the wing with the thumb and forefinger, another individual closed the band until the eyelet was inserted into the hole, and sealed the eyelet with an applicator. Chicks were reared with a density of 10 birds/m2 until 56 d of age (sexes mixed). Artificial light was provided only 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 reaching 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 56 to 140 d of age; the flock size was 200. 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 56 d of age, and 15% CP, 2,700 kcal of ME/kg, 0.9% Ca, and 0.4% available P until 140 d of age.

Four replicates (hatches) separated by 14 d were used in the experiment. A total of 52 pullets were used to study the effect of bad collocation of the wing tag on the heterophil-to-lymphocyte ratio, fluctuating asymmetry, and tonic immobility duration at 140 d of age. Group 1 (bad collocation of the wing tag) consisted of the 26 females (9, 5, 6, and 6 in each replicate) that exhibited the normal extended black coloration of the breed but showed a callus and white feathers around the wing tag. The mean percentage of these females was 3.25% (4.5, 2.5, 3, and 3% in each replicate). Group 2 (good collocation of the wing tag) consisted of 26 additional females (randomly selected from the 191, 195, 194, and 194 remaining females) that did not show any signs of callus and feather amelanosis and that had normal extended black coloration in all the feathers. The number of sampled birds was equal to that of group 1 (9, 5, 6, and 6 in each replicate). Hens from both groups were phenotypically similar, with the white feathers of hens with feather amelanosis being apparent only when the wing was stretched.

On 2 different days, birds were tested for the heterophil-to-lymphocyte ratio (the first day) and tonic immobility duration (the second day) at 140 d of age. To obtain the heterophil-to-lymphocyte ratio, birds 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, and one drop was smeared on each of 2 glass slides. The smears were stained with May-Grünwald 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 and monocytes), were counted on one slide of each bird (the other slide was supplementary), and the heterophil-to-lymphocyte ratio was calculated. All counts were made by the same person (S. G. Dávila).

All birds were tested for tonic immobility in a separate room on the day after the blood sampling. Tonic immobility was induced, as soon as a bird was caught, by placing the bird on its back with the head hanging in a U-shaped wooden cradle (Jones and Faure, 1981). The bird was restrained for 10 s. The observer sat in full view of the bird, approximately 1 m away, and fixed his eyes on the bird to give 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, duration of tonic immobility ranged from 0 to 600 s.

The measured morphological traits (on the same day as the blood sampling) were both right (R) and left (L) outer, middle, inner, and hind toe, and leg (metatarsus) lengths, and ear-lobe and wattle areas. Right and left values of a bird were taken during the same session. All 7 lengths and ear-lobe and wattle widths were measured in millimeters by using a digital caliper; ear-lobe and wattle areas were calculated by multiplying lengths by widths. Trait length 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 several confounding factors complicate the analysis of asymmetry: different types of bilateral asymmetry (fluctuating asymmetry, directional asymmetry, and antisymmetry), measurement error, and the relationship between fluctuating asymmetry and trait size (details can be found in Campo et al., 2005, 2007). Relative fluctuating asymmetry was used for all traits [2|R – L|/(R + L)]. Combined 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 groups, a 2-way ANOVA (Sokal and Rohlf, 1981) was used with the statistical model xijk = µ + Gi + rj + Grij + {varepsilon}ijk, where xijk was the analyzed measurement, µ was the overall mean, Gi was the effect of group (females with bad or good collocation of the wing tag), rj was the effect of replicate (j = 1...4), Grij was the interaction, and {varepsilon}ijk was the residual (the number of birds in the individual subclasses was unequal, with k ranging from 5 to 9). Group was considered a fixed effect and replicates were assumed to be a random effect. Square root (heterophil-to-lymphocyte ratio), logarithmic (tonic immobility duration), or arcsine square root (relative fluctuating asymmetry) transformations were used before analysis, but indicated mean values are not transformed.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
REFERENCES
 
Why females with bad collocation of the wing tag from the White-Faced Black Spanish breed showed feather amelanosis, whereas males did not, posed an interesting question. Feather amelanosis might be controlled hormonally (estrogens vs. androgens) and be sex-limited, with androgenic hormones being protective. Although sex differences in the incidence of the Smyth line amelanosis were inconsistent (Smyth et al., 1981) when a significant difference was present, females were associated with the increased incidence (Smyth, 1989). In addition, it has been reported that the incidence of amelanosis in the Smyth line chicks was significantly lower in birds receiving testosterone (Smyth, 1989). On the other hand, genetic differences among breeds were important to the occurrence of feather amelanosis as a consequence of bad collocation of the wing tag. Although the extended black plumage seemed to be necessary, other Spanish breeds with this plumage color (Black Castellana and Black Menorca) never showed feather amelanosis. Enlarged white earlobes, which are exclusive of the White-Faced Black Spanish breed, might be the key trait. Black melanin appears to be associated with expression of the Smyth line amelanosis, although the role of the extended black gene is not clear, and both black and red melanins appear to be susceptible to amelanosis (Smyth et al., 1981).

Replicates and replicate x group interaction were not significant for any measurement, and they were pooled with the residual to give a one-way model of the group effect (xij = µ + Gi + {varepsilon}ij). There was a significant difference between groups for the heterophil-to-lymphocyte ratio (P < 0.05), with the ratio of birds with bad collocation of the wing tag being higher than that of birds with good collocation of the wing tag (Table 1Go). This fact indicated that the former group is more stressful than the latter one. In birds with bad collocation of the wing tag, there was a significant (P < 0.05) increase in heterophil number (heterophilia) and a significant (P < 0.05) decrease in lymphocytes (lymphopenia), with the increase of heterophils being a response to the stressful effect of the bad collocation of the wing tag. These findings are in agreement with those reported by Maxwell and Robertson (1998), who suggested a response of the avian leukocytes to problems associated with trauma. They also agreed with those reported by Erf and Smyth (1996), who indicated that birds from the Smyth line with spontaneous feather amelanosis had a significantly greater number of heterophils and a smaller number of lymphocytes than Brown line control birds. The association between the wing tag-induced callus and feather amelanosis is consistent with the simultaneous occurrence of epidermis amelanosis in humans (vitiligo) and scleroderma, which was analyzed by Bonifati et al. (2006).


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  		Table 1. Mean heterophil-to-lymphocyte ratio, heterophil number, lymphocyte number, and tonic immobility duration (s) in groups of females from the White-Faced Black Spanish breed with bad or good collocation of the wing tag (n = 52)
 
The heterophil-to-lymphocyte ratios presented in Table 1Go differed from those calculated with the presented means for heterophils and lymphocytes (0.63 and 0.50) because of the effects of variance and covariance on the value of the ratio: H/L = mH/mL + 2mHs L2/mL3 – sHL/mL2 with mH and mL being the mean values of the numerator and denominator, sHL being the covariance between them, and sL2 being the variance of the denominator.

There was a significant (P < 0.05) group effect on tonic immobility duration (Table 1Go). Birds with bad collocation of the wing tag had a longer tonic immobility duration than birds with good collocation of the wing tag, indicating that a bad collocation affected the fearfulness of birds.

Mean values indicating the effect of the group on relative asymmetry measurements are summarized in Tables 2Go and 3Go. Group effect was significant (P < 0.05) for the relative asymmetry of middle and hind toe lengths, the combined relative asymmetry of the 4 toes, the ear-lobe area, and the combined relative asymmetry of leg length, ear-lobe and wattle areas, and toes lengths. The relative asymmetry of birds with bad collocation of the wing tag was larger than that of birds with good collocation of the wing tag, as a consequence of the chronic stress produced by the wing tag insult.


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  		Table 2. Mean relative asymmetry (x 100) in outer, middle, inner, and hind toe lengths, and combined toes in groups of females from the White-Faced Black Spanish breed with bad or good collocation of the wing tag (n = 52)
 

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  		Table 3. Mean relative asymmetry (x 100) of leg length, ear-lobe area, wattle area, and combined toes, leg, ear-lobe, and wattle in groups of females from the White-Faced Black Spanish breed with bad or good collocation of the wing tag (n = 52)
 
In conclusion, results of the current study indicate that bad collocation of the wing tag in hens from the WhiteFaced Black Spanish breed of chickens was associated with feather amelanosis around the wing tag, a greater heterophil-to-lymphocyte ratio, greater fluctuating asymmetry, and longer tonic immobility duration, suggesting that bad collocation of the wing tag negatively affects some measures of stress.

Received for publication February 11, 2008. Accepted for publication April 3, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 REFERENCES
 
Bonifati, C., G. Impara, A. Morrone, A. Pietrangeli, and M. Carducci. 2006. Simultaneous occurrence of linear scleroderma and homolateral segmental vitiligo. J. Eur. Acad. Dermatol. Venereol. 20:63–65.[CrossRef][Web of Science][Medline]

Campo, J. L., M. G. Gil, S. G. Dávila, and I. Muñoz. 2005. Estimation of heritability for fluctuating asymmetry in chickens by restricted maximum likelihood. Effects of age and sex. Poult. Sci. 84:1689–1697.

Campo, J. L., M. G. Gil, S. G. Dávila, and I. Muñoz. 2007. Effect of lighting stress on fluctuating asymmetry, heterophil-to-lymphocyte ratio, and tonic immobility duration in eleven breeds of chickens. Poult. Sci. 86:37–45.[Abstract/Free Full Text]

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. Livestock Prod., Madrid, Spain. Editorial Garsi, Madrid, Spain.

Erf, G. F., and J. R. Smyth. 1996. Alterations in blood leukocyte populations in Smyth line chickens with autoimmune vitiligo. Poult. Sci. 75:351–356.[Web of Science][Medline]

Gallup, G. G. 1979. Tonic immobility as a measure of fear in domestic fowl. Anim. Behav. 27:316–317.[CrossRef][Web of Science]

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.[CrossRef][Web of Science][Medline]

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.[CrossRef][Web of Science]

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.[CrossRef][Medline]

Lucas, A. M., and C. Jamroz. 1961. Atlas of Avian Hematology. Agric. Monograph 25. USDA, Washington, DC.

Maxwell, M. H., and G. W. Robertson. 1998. The avian heterophil leukocyte: A review. World’s Poult. Sci. J. 54:155–178.[CrossRef][Web of Science]

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 Academic, Dordrecht, the Netherlands.

Parsons, P. A. 1990. Fluctuating asymmetry. An epigenetic measure of stress. Biol. Rev. Camb. Philos. Soc. 65:131–145.[Medline]

Smyth, J. R. 1989. The Smyth chicken: A model for autoimmune amelanosis. Crit. Rev. Poult. Biol. 2:1–19.

Smyth, J. R. 1990. Genetics of plumage, skin and eye pigmentation in chickens. Pages 109–167 in Poultry Breeding and Genetics. R. D. Crawford, ed. Elsevier, Amsterdam, the Netherlands.

Smyth, J. R., R. E. Boissy, and K. V. Fite. 1981. The DAM chicken: A model for spontaneous postnatal cutaneous and ocular amelanosis. J. Hered. 72:150–156.[Medline]

Sokal, R. R., and F. J. Rohlf. F.J. 1981. Biometry. Freeman and Co., London, UK.





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