Chronic welfare restrictions and adrenal gland morphology in broiler chickens Bruno R. M¨ uller,∗ Hisly Any S. Medeiros,∗ Renato S. de Sousa,† and Carla F. M. Molento∗ 1 ∗
Federal University of Paran´ a, Animal Welfare Laboratory (LABEA), Rua dos Funcion´ arios, 1540, a, Veterinary Pathology CEP80035-050, Juvevˆe, Curitiba-PR, Brazil; and † Federal University of Paran´ Laboratory, Rua dos Funcion´ arios, 1540, CEP80035-050, Juvevˆe, Curitiba-PR, Brazil
Key words: adrenal morphology, animal welfare, broiler chicken, chronic stress, cortico-medullary ratio 2015 Poultry Science 00:1–5 http://dx.doi.org/10.3382/ps/pev026 community is making efforts to meet these demands (Napolitano et al., 2007; Baptista et al., 2011; Shimmura et al., 2011; Phythian et al., 2012). Among scientifically validated indicators used to understand animal welfare, activity of the hypothalamic-pituitaryadrenal (HPA) axis is broadly assessed, and this may be considered the standard approach to the study of stress and welfare in farm animals (Morm`ede et al., 2007). The HPA axis and the sympathoadrenomedullary axis are responsible for maintaining homeostasis during stress, and the adrenal glands are essential elements common to both systems. Levels of hormones produced by the adrenal glands and gland weight are often used to assess the physiological state of animals that have endured stressful situations (H¨ otzel and Filho, 2004). Ulrich-Lai et al. (2006) reported hyperplasia of the zona fasciculata in the adrenal glands of rats exposed to chronic stress. The zona fasciculata of the mammalian gland is responsible for producing glucocorticoids, an important class of hormones released during stress, and morphological changes in this area suggest that stressful stimuli might produce important alterations in adrenal function and physiology. Avian adrenal glands are anatomically and functionally different from those in mammals. The avian adrenal parenchyma is composed of an intermingling of adrenocortical and chromaffin tissue, and is not arranged in distinct histologic regions of cortex and medulla as found in mammals (Carsia and Harvey, 2000). As a
INTRODUCTION The domestic chicken is the most common bird in the world, with the number of individuals estimated to be 19.9 billion (FAO, 2013). When considering animal welfare challenges related to farming, the domestic chicken might be considered the species with the highest number of individuals suffering the consequences of human activity, which highlights the importance of studies that aim to understand and mitigate welfare problems related to poultry production. Lameness is considered one of the most important animal welfare issues related to modern poultry production systems (Sanotra et al., 2001; Bessei, 2006; Naas et al., 2009). In addition to the possible pain directly caused by lameness, affected birds cannot hold their own weight and end up spending most of their time prostrate on the ground, which is filled with litter. Litter is defined as the combination of bedding material, excreta, feathers, wasted feed, and water. Poor handling and extended contact with this material are related to several avian health challenges (Ritz et al., 2009). The need for efficient animal welfare assessment and monitoring methods is evident, and the scientific C 2015 Poultry Science Association Inc. Received June 10, 2014. Accepted January 6, 2015. 1 Corresponding author: [email protected]
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tissues, and lymphatic tissue and blood vessels were studied. GP birds had lower BW when compared to AH birds, and when adrenal gland weight values were adjusted to BW, a greater relative adrenal weight was observed for the GP group. Adrenals from GP birds also presented a higher proportion of blood vessels when compared to AH birds. These results might indicate increased adrenal activity and evidence of the inflammatory process as a consequence of chronic stress. Results showed that gait problems caused significant adrenal gland changes, suggesting a possible role for the study of adrenal gland morphology as an indicator of chronic welfare problems in broiler chickens.
ABSTRACT Gait problems constitute an important and chronic welfare restriction for broiler chickens. The objective of the present study was to determine if adrenal gland morphology indicates chronic welfare restrictions in broiler chickens, using gait problems as the stressor. Sixty-six birds raised on a commercial unit were selected at 40 d of age and separated into groups according to gait score. One group was apparently healthy birds (AH) with gait scores of 0 to 2, and the other group had birds with gait problems (GP) that showed gait scores of 4 to 5. Birds were slaughtered and weighed, and their adrenal glands were measured and weighed; proportions of medullary and adrenocortical
¨ MULLER ET AL.
2
result, birds’ physiological responses to stressful conditions might be different from those of mammals (de Matos, 2008). A better understanding of the function of avian adrenal glands and their effects on animal welfare might be useful in guiding future strategies on poultry production. In the present study, severe gait problems were considered a source of chronic stress, and we investigated their effects on birds’ BW, the external morphology of the adrenal glands, and the ratio of adrenal cortical and medullary tissue.
MATERIAL AND METHODS
Figure 1. Checkered mask applied to histological view of adrenal glands from 40-d-old broiler chickens raised in the city of Lapa, southern Brazil; tissue identification was performed in squares A, B, C, D, and E; areas 1, 2, and 3 show cortical cells, medullary cells, and blood vessels, respectively. R (Synth , Diadema-SP, Brazil), and received an identification number. Adrenal length was defined as the distance between the cranial border and the caudal corner of the glands, and it was measured with a caliper rule. Adrenals were weighed on a precision scale, and the ratio of their relative weight to BW was also calculated. Histological sections of the adrenal glands were prepared by routine histotechnical procedure and stained by routine Mayer haematoxylin and eosin staining protocol. For each gland, 2 sections 3 μm thick were taken from the second and third quarters of the gland and for each, 3 pictures of the left, central and right areas of the histological sections were taken, using magnification of 100× on an Olympus BX40 microscope equipped with an adaptor for a Sony Cyber-shot DSC-W320 camera (Sony Corporation, Tokyo, Japan). Pictures were uploaded to a computer and received a checkered mask that determined the areas for tissue identification. Five areas were randomly chosen before reading began and were fixed for the analysis of each picture. Blind picture analysis was performed by 3 observers trained in cell type identification according to the methodology described by Bacha and Wood (1991). Observers indicated the type of tissue filling the fixed areas for analysis A, B, C, D, and E (Figure 1). A chi-square test was applied to verify interobserver coherence (P > 0.05). The observed cell types were identified as cortical and medullary cells, and lymphatic and blood vessels. Tissue tearing or deficiencies in staining were identified as artifacts. Whenever the area for tissue identification covered more than one cell type, this specific area was disconsidered, and identification was made on the next square to the right. For statistical analysis of BW and cell type proportions, the experimental units considered were individual birds. For analysis of adrenal weight and size, each gland was considered individually as an experimental unit. Results of BW and adrenal weight and size were
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This experiment was approved by the Ethics Commission for the Use of Animals of the Agrarian Sciences Sector of the Federal University of Paran´ a, Brazil, under protocol number 003/2011. For this study, a total of 66 commercial male broiler chickens were selected at the regular slaughter age of 40 d directly from a commercial broiler unit in the city of Lapa (25◦ 46 14 S; 49◦ 43 10 W), in southern Brazil. At the commercial unit, Animal density was 21 birds/m2 , and the unit was equipped with nipple drinkers, tubular feeders, wood shavings for bedding, and 6 positive pressure fans placed on the ceiling coupled with open-sided walls for thermal control. The temperature inside the commercial unit ranged from 18 to 26◦ C, and the regional mean humidity during the experiment was 85%; the humidity inside the commercial unit was not measured. Birds were randomly selected and separated into 2 groups: 33 apparently healthy (AH) birds and 33 birds with severe gait problems (GP). Selection was made at the commercial unit according to the gait score test described by Kestin et al. (1992), in which 6 categories are represented by scores ranging from 0 to 5 (0 = no impairment; 1 = detectable but unidentifiable abnormality; 2 = identifiable abnormality that has little impact on overall function; 3 = identifiable abnormality that impairs function; 4 = severe impairment of function, but the bird is still capable of walking; 5 = bird is incapable of sustained walking). Subtle changes in gait that are present in birds that score 1 or 2 are often related to morphological characteristics such as short legs and big breast volume (Morton et al., 2010); conversely, birds that score 4 or 5 have severe gait abnormalities that are considered to be consequences of pain and discomfort (Danbury et al., 2000). There is disagreement about whether birds that score 3 experience pain or if their gait is negatively affected only by physical characteristics (Corr et al., 2003); thus, birds with this score were not included in this study. Birds that scored from 0 to 2 were in the AH group, and those that scored 4 or 5 were in the GP group. After they were divided into groups, all birds were immediatelly killed in site by cervical dislocation and weighed. Adrenal glands from each bird were carefully dissected, were fixed in a 10% buffered formalin solution
WELFARE RESTRICTIONS AND BROILER ADRENAL MORPHOLOGY
3
Table 1. Mean values of BW, absolute and relative weight of adrenal glands, and length of adrenal glands from broiler chickens slaughtered at 40 d of age, in the town of Lapa, in southern Brazil. Measures
Apparently healthy (AH)
Gait problems (GP)
P-values
2.614 ± 0.030 67 ± 2 0.025 ± 0.006 7.80 ± 0.17
2.003 ± 0.038 70 ± 2 0.035 ± 0.008 8.10 ± 0.15
0.0001 0.3603 0.0001 0.2841
BW (kg) Weight of adrenals (mg) Adrenal relative weight () Adrenal length (mm)
compared by ANOVA. The number of cell types and proportions were determined for all 66 birds and were compared by the Mann-Whitney test. All statistical tests were performed using BioEstat 5.0 software (Instituto Mamirau´ a, 2007).
RESULTS As expected, gait problems affected BW (P < 0.01). The AH group presented a higher mean BW of 2.614 ± 0.030 kg when compared to the mean BW of the GP group, 2.003 ± 0.038 kg. Adrenal length did not differ significantly between groups (P = 0.28); the AH group mean was 7.80 ± 0.17 mm, and the GP group mean was 8.10 ± 0.15 mm. Adrenal gland weight was similar for both groups (P = 0.36), with observed values of 67.0 ± 2.0 and 70.0 ± 2.0 mg for the AH and GP groups, respectively. The AH group had a lower mean value of adrenal gland weight relative to BW of 0.025 ± 0.006%; the GP group mean was 0.035 ± 0.008% (P < 0.01) (Table 1). The number of cortical cells observed did not differ significantly between groups (P = 0.35; U = 413.50), with median ratios of 55.0% (40.0 to 75.0%) for the AH group and 57.0% (32.8 to 75.0%) for the GP group. Counts of medullary cells were also similar between the AH
DISCUSSION The lower BW in the GP group supports the idea that animal productivity is affected by poor welfare
A
B
GP
C
4%
60% 55.0%
2 1.75
57.0%
50%
3% b
3%
1.5
40.0%
40%
33.0%
30%
1.32
2% a
2%
1
1%
0.5
20%
10% 0%
0%
0%
0% Cortical
medullary
Blood
lymphatic
0 Cortico-medullary ratio
Figure 2. Median proportions of tissues (A and B) and median cortico-medullary ratio (C) observed in adrenal glands from broiler chickens slaughtered at 40 d of age in the town of Lapa, in southern Brazil. Medians followed by different letters represented significant differences between groups (P < 0.05).
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two groups (P = 0.12; U = 369.00), with a median proportion of 40.0% (20.0 to 56.7%) for the AH group and 33.0% (20.0 to 53.3%) for the GP group. No significant difference was found in the ratio between cortical and medullary tissues (P = 0.22; U = 394.00); the median index for the AH group was 1.32, and it was 1.75 for the GP group. Blood vessel counts were different between groups (P = 0.01; U = 301.00). The AH group had a lower proportion of blood vessels, with a median of 2.0% (0.0 to 6.7%); the GP group had a median of 3.0% (0.0 to 11.7%). Counts of other observed cells did not differ between groups. The median proportion of lymphatic tissue was 0.0% (0.0 to 5.0%) for the AH group and 0.0% (0.0 to 4.9%) for the GP group (P = 0.54; U = 437.00), and the median proportion of artifacts was 3.0% (0.0 to 8.3%) for the AH group and 3.0% (0.0 to 15.0%) for the GP group (P = 0.50; U = 433.00). Median proportions of all observed tissues and cortico-medullary ratios are shown in Figure 2.
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¨ MULLER ET AL.
Zikic et al. (2011) in broilers submitted to chronic sound stress. Yet, increased vascularization might not be specific to the adrenal glands in animals under stress, and further research would benefit from taking additional tissue samples from another organs and glands for comparison. Although lymphatic involution is known to result from increased adrenal cortex activity (Siegel, 1971; Post et al., 2003; de Matos, 2008), this was not observed in this study. Stressful stimuli clearly induce changes in adrenal morphology and may affect animal welfare (Naas et al., 2009). In addition to important changes in behavior, such as altering birds’ feeding strategies (Weeks and Nicol, 2006), stress can also cause broiler chickens to experience pain and discomfort due to severe lameness (Danbury et al., 2000). Information about avian adrenal changes in response to stress is scarce when compared to information about mammals, although it appears that adrenal glands’ main biological roles are comparable between species (Morm`ede et al., 2007). Further studies should focus on implications of avian adrenal responses to stress as they relate to poultry welfare and understanding the subtleties of how various adrenal tissues respond to chronic stress. Our results support the hypothesis that gait problems cause significant adrenal changes, suggesting a possible role for the study of adrenal gland morphology as an indicator of chronic welfare problems in broiler chickens. Birds with gait problems had lower BW, higher relative weight of adrenal glands, and higher proportion of blood vessels in the adrenal glands. Changes in adrenal morphology caused by chronic lameness in broiler chickens further indicate that this challenge is a major welfare problem that most likely causes sustained animal suffering. Advances in knowledge about the effects of chronic welfare restrictions on animal physiology enable a better understanding and encourage reflection about the practices adopted in our production systems. Our current research supports the understanding that gait problems constitute an important challenge for broiler chickens individually and also for the whole poultry industry, affecting the biological function of animals and consequently productivity. Results indicate that changes in adrenal morphology could be used as indicators of animal welfare and, if addressed in future husbandry strategies, may lead to a system that is not only more efficient but also more respectful to the animals.
ACKNOWLEDGMENTS We would like to thank the Brazilian National Council for Scientific and Technological Development for its support by means of an undergraduate scholarship during the period of the experiment. The authors also wish to thank Anderson Bonamigo, Elizabeth Santin, and Viviani Bontoni for their collaboration on this work.
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conditions (Broom, 1991). It is known that gait problems can change the time budget of many behaviors and alter feeding strategy (Weeks et al., 2000). The inability to walk and reach for food and water, especially in animals with a gait score of 5, might lead to reduced feed intake and consequent lower weight gain and may constitute a severe stressor. The absolute value of adrenal gland weight did not differ between groups. However, absolute weight is not a reliable indicator because of its high variability between conspecifics (Aire, 1980); differences between relative adrenal weights enable a better identification of morphological changes because these weights are adjusted based on each bird’s BW and thus seem particularly relevant when comparing groups with significantly different BW. The higher relative adrenal weights found in the GP group might be a result of weight loss in birds from this group, meaning that the animals reduced their BW, and gland weight remained the same. However, higher relative adrenal weights have also been reported in previous studies that relate these higher weights to increased adrenal activity in response to stress (Siegel, 1959; Harvey et al., 1984). Further investigation should focus on determining what the actual cause of increased relative adrenal weight is. No cortico-medullary ratio changes were found between groups. Changes in this ratio have been observed in water-deprived (Sharma et al., 2009) and noise-stressed broiler chickens (Zikic et al., 2011), and in heat-stressed laying hens (Aire, 1980). These studies indicate changes in adrenocortical tissue and are in agreement with Harvey et al. (1984), who state that the presence of stress-related hormones in the peripheral circulation stimulates the proliferation of adrenocortical cells. Results of the present work, however, do not show such changes. The difference in results might be due to factors influencing adrenal morphology, which are not yet clear. Age, sex, and strain seem to be important influences on the degree of responses (Siegel, 1971). Siller et al. (1975) related significant changes in corticomedullary ratio of birds occurring most markedly between 3 and 7 wk of age, when young birds can present cortical tissue prevalence of up to 90%. Also, the intensity and duration of the stressor appear to play an important role in altering adrenal morphology (Ulrich-Lai et al., 2006; Morm`ede et al., 2007). Such variability encourages new studies on the effects of stress on adrenal morphology. The methods of the present study allowed identification of important effects of stress in different tissues of the adrenal gland, not only cortical and medullary, and results showed an increased percentage of blood vessels in adrenal glands of birds in the GP group. In general, adrenal hyperemia has been scarcely described as a physiological response to stress and might have been overlooked in past studies. Edelmann (1945) indicates that the adrenal glands’ immediate response to stress is hyperemia, and an increased percentage of adrenal blood vessels has also been described by
WELFARE RESTRICTIONS AND BROILER ADRENAL MORPHOLOGY
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Aire, T. A. 1980. Morphometric study of the avian adrenal gland. J. Anat. 131:19–23. Bacha, W. J., and L. M. Wood. 1991. Atlas color de histolog´ıa veterinaria. Inter-m´edica, Buenos Aires, Argentina. Baptista, R. I. A. A., G. R. Bertani, and C. N. Barbosa. 2011. Indicadores do bem-estar em su´ınos. Ciˆencia Rural 41:1823– 1830. Bessei, W. 2006. Welfare of broilers: A review. World’s Poult. Sci. J. 62:455–466. Broom, D. 1991. Animal welfare: Concepts and measurement. J. Anim. Sci. 69:4167–4175. Carsia, R. V., and S. Harvey. 2000. Adrenals. Pages 489–537 in Sturkie’s Avian Physiology, G. C. Whittow ed. Academic Press, San Diego, USA. Corr, S. A., M. J. Gentle, C. C. McCorquodale, and D. Bennett. 2003. The effect of morphology on walking ability in the modern broiler: A gait analysis study. Anim. Welf. 12:159–171. Danbury, T., C. Weeks, J. Chambers, A. Waterman-Pearson, and S. Kestin. 2000. Self-selection of the analgesic drug carprofen by lame broiler chickens. Vet. Rec. 307–311. De Matos, R. 2008. Adrenal steroid metabolism in birds: Anatomy, physiology, and clinical considerations. Vet. Clin. North Am. 11:35–57. Edelmann, A. 1945. The significance of changes in adrenal size after periods of anoxia in rats. Exp. Biol. Med. 58:271–272. FAO. 2013. Global Livestock Production and Health Atlas. URL http://kids.fao.org/glipha/. Harvey, S., J. G. Phillips, A. Rees, and T. R. Hall. 1984. Stress and adrenal function. J. Exp. Zool. 232:633–645. H¨otzel, M. J., and L. C. P. M. Filho. 2004. Bem-estar Animal na Agricultura do S´eculo XXI. Rev. Etol. 6:3–15. Instituto Mamirau´ a. 2007. BioEstat 5.0, http://www.mamiraua. org.br/pt-br/downloads/programas/bioestat-versao-53/. Kestin, S., T. Knowles, A. Tinch, and N. Gregory. 1992. Prevalence of leg weakness in broiler chickens and its relationship with genotype. Vet. Rec. 131:190–194. Morm`ede, P., S. Andanson, B. Aup´erin, B. Beerda, D. Gu´emen´e, J. Malmkvist, X. Manteca, G. Manteuffel, P. Prunet, C. G. van Reenen, S. Richard, and I. Veissier. 2007. Exploration of the hypothalamic-pituitary-adrenal function as a tool to evaluate animal welfare. Physiol. Behav. 92:317–39. Morton, D., T. Oltenacu, C. Arnould, L. Collins, E. Le Bihan-Duval, P. Hocking, P. Sorensen, J. Hartun, L. Keeling, G. Banos, C. Berg, I. de Jong, and V. Michel. 2010. Scientific opinion on the influence of genetic parameters on the welfare and the resistance to stress of commercial broilers. Eur. Food Safety Author. (EFSA) J. 8: 1–82.
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Federal University of Paran´ a, Animal Welfare Laboratory (LABEA), Rua dos Funcion´ arios, 1540, a, Veterinary Pathology CEP80035-050, Juvevˆe, Curitiba-PR, Brazil; and † Federal University of Paran´ Laboratory, Rua dos Funcion´ arios, 1540, CEP80035-050, Juvevˆe, Curitiba-PR, Brazil
Key words: adrenal morphology, animal welfare, broiler chicken, chronic stress, cortico-medullary ratio 2015 Poultry Science 00:1–5 http://dx.doi.org/10.3382/ps/pev026 community is making efforts to meet these demands (Napolitano et al., 2007; Baptista et al., 2011; Shimmura et al., 2011; Phythian et al., 2012). Among scientifically validated indicators used to understand animal welfare, activity of the hypothalamic-pituitaryadrenal (HPA) axis is broadly assessed, and this may be considered the standard approach to the study of stress and welfare in farm animals (Morm`ede et al., 2007). The HPA axis and the sympathoadrenomedullary axis are responsible for maintaining homeostasis during stress, and the adrenal glands are essential elements common to both systems. Levels of hormones produced by the adrenal glands and gland weight are often used to assess the physiological state of animals that have endured stressful situations (H¨ otzel and Filho, 2004). Ulrich-Lai et al. (2006) reported hyperplasia of the zona fasciculata in the adrenal glands of rats exposed to chronic stress. The zona fasciculata of the mammalian gland is responsible for producing glucocorticoids, an important class of hormones released during stress, and morphological changes in this area suggest that stressful stimuli might produce important alterations in adrenal function and physiology. Avian adrenal glands are anatomically and functionally different from those in mammals. The avian adrenal parenchyma is composed of an intermingling of adrenocortical and chromaffin tissue, and is not arranged in distinct histologic regions of cortex and medulla as found in mammals (Carsia and Harvey, 2000). As a
INTRODUCTION The domestic chicken is the most common bird in the world, with the number of individuals estimated to be 19.9 billion (FAO, 2013). When considering animal welfare challenges related to farming, the domestic chicken might be considered the species with the highest number of individuals suffering the consequences of human activity, which highlights the importance of studies that aim to understand and mitigate welfare problems related to poultry production. Lameness is considered one of the most important animal welfare issues related to modern poultry production systems (Sanotra et al., 2001; Bessei, 2006; Naas et al., 2009). In addition to the possible pain directly caused by lameness, affected birds cannot hold their own weight and end up spending most of their time prostrate on the ground, which is filled with litter. Litter is defined as the combination of bedding material, excreta, feathers, wasted feed, and water. Poor handling and extended contact with this material are related to several avian health challenges (Ritz et al., 2009). The need for efficient animal welfare assessment and monitoring methods is evident, and the scientific C 2015 Poultry Science Association Inc. Received June 10, 2014. Accepted January 6, 2015. 1 Corresponding author: [email protected]
1
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 1, 2015
tissues, and lymphatic tissue and blood vessels were studied. GP birds had lower BW when compared to AH birds, and when adrenal gland weight values were adjusted to BW, a greater relative adrenal weight was observed for the GP group. Adrenals from GP birds also presented a higher proportion of blood vessels when compared to AH birds. These results might indicate increased adrenal activity and evidence of the inflammatory process as a consequence of chronic stress. Results showed that gait problems caused significant adrenal gland changes, suggesting a possible role for the study of adrenal gland morphology as an indicator of chronic welfare problems in broiler chickens.
ABSTRACT Gait problems constitute an important and chronic welfare restriction for broiler chickens. The objective of the present study was to determine if adrenal gland morphology indicates chronic welfare restrictions in broiler chickens, using gait problems as the stressor. Sixty-six birds raised on a commercial unit were selected at 40 d of age and separated into groups according to gait score. One group was apparently healthy birds (AH) with gait scores of 0 to 2, and the other group had birds with gait problems (GP) that showed gait scores of 4 to 5. Birds were slaughtered and weighed, and their adrenal glands were measured and weighed; proportions of medullary and adrenocortical
¨ MULLER ET AL.
2
result, birds’ physiological responses to stressful conditions might be different from those of mammals (de Matos, 2008). A better understanding of the function of avian adrenal glands and their effects on animal welfare might be useful in guiding future strategies on poultry production. In the present study, severe gait problems were considered a source of chronic stress, and we investigated their effects on birds’ BW, the external morphology of the adrenal glands, and the ratio of adrenal cortical and medullary tissue.
MATERIAL AND METHODS
Figure 1. Checkered mask applied to histological view of adrenal glands from 40-d-old broiler chickens raised in the city of Lapa, southern Brazil; tissue identification was performed in squares A, B, C, D, and E; areas 1, 2, and 3 show cortical cells, medullary cells, and blood vessels, respectively. R (Synth , Diadema-SP, Brazil), and received an identification number. Adrenal length was defined as the distance between the cranial border and the caudal corner of the glands, and it was measured with a caliper rule. Adrenals were weighed on a precision scale, and the ratio of their relative weight to BW was also calculated. Histological sections of the adrenal glands were prepared by routine histotechnical procedure and stained by routine Mayer haematoxylin and eosin staining protocol. For each gland, 2 sections 3 μm thick were taken from the second and third quarters of the gland and for each, 3 pictures of the left, central and right areas of the histological sections were taken, using magnification of 100× on an Olympus BX40 microscope equipped with an adaptor for a Sony Cyber-shot DSC-W320 camera (Sony Corporation, Tokyo, Japan). Pictures were uploaded to a computer and received a checkered mask that determined the areas for tissue identification. Five areas were randomly chosen before reading began and were fixed for the analysis of each picture. Blind picture analysis was performed by 3 observers trained in cell type identification according to the methodology described by Bacha and Wood (1991). Observers indicated the type of tissue filling the fixed areas for analysis A, B, C, D, and E (Figure 1). A chi-square test was applied to verify interobserver coherence (P > 0.05). The observed cell types were identified as cortical and medullary cells, and lymphatic and blood vessels. Tissue tearing or deficiencies in staining were identified as artifacts. Whenever the area for tissue identification covered more than one cell type, this specific area was disconsidered, and identification was made on the next square to the right. For statistical analysis of BW and cell type proportions, the experimental units considered were individual birds. For analysis of adrenal weight and size, each gland was considered individually as an experimental unit. Results of BW and adrenal weight and size were
Downloaded from http://ps.oxfordjournals.org/ at D H Hill Library - Acquis Dept S on March 1, 2015
This experiment was approved by the Ethics Commission for the Use of Animals of the Agrarian Sciences Sector of the Federal University of Paran´ a, Brazil, under protocol number 003/2011. For this study, a total of 66 commercial male broiler chickens were selected at the regular slaughter age of 40 d directly from a commercial broiler unit in the city of Lapa (25◦ 46 14 S; 49◦ 43 10 W), in southern Brazil. At the commercial unit, Animal density was 21 birds/m2 , and the unit was equipped with nipple drinkers, tubular feeders, wood shavings for bedding, and 6 positive pressure fans placed on the ceiling coupled with open-sided walls for thermal control. The temperature inside the commercial unit ranged from 18 to 26◦ C, and the regional mean humidity during the experiment was 85%; the humidity inside the commercial unit was not measured. Birds were randomly selected and separated into 2 groups: 33 apparently healthy (AH) birds and 33 birds with severe gait problems (GP). Selection was made at the commercial unit according to the gait score test described by Kestin et al. (1992), in which 6 categories are represented by scores ranging from 0 to 5 (0 = no impairment; 1 = detectable but unidentifiable abnormality; 2 = identifiable abnormality that has little impact on overall function; 3 = identifiable abnormality that impairs function; 4 = severe impairment of function, but the bird is still capable of walking; 5 = bird is incapable of sustained walking). Subtle changes in gait that are present in birds that score 1 or 2 are often related to morphological characteristics such as short legs and big breast volume (Morton et al., 2010); conversely, birds that score 4 or 5 have severe gait abnormalities that are considered to be consequences of pain and discomfort (Danbury et al., 2000). There is disagreement about whether birds that score 3 experience pain or if their gait is negatively affected only by physical characteristics (Corr et al., 2003); thus, birds with this score were not included in this study. Birds that scored from 0 to 2 were in the AH group, and those that scored 4 or 5 were in the GP group. After they were divided into groups, all birds were immediatelly killed in site by cervical dislocation and weighed. Adrenal glands from each bird were carefully dissected, were fixed in a 10% buffered formalin solution
WELFARE RESTRICTIONS AND BROILER ADRENAL MORPHOLOGY
3
Table 1. Mean values of BW, absolute and relative weight of adrenal glands, and length of adrenal glands from broiler chickens slaughtered at 40 d of age, in the town of Lapa, in southern Brazil. Measures
Apparently healthy (AH)
Gait problems (GP)
P-values
2.614 ± 0.030 67 ± 2 0.025 ± 0.006 7.80 ± 0.17
2.003 ± 0.038 70 ± 2 0.035 ± 0.008 8.10 ± 0.15
0.0001 0.3603 0.0001 0.2841
BW (kg) Weight of adrenals (mg) Adrenal relative weight () Adrenal length (mm)
compared by ANOVA. The number of cell types and proportions were determined for all 66 birds and were compared by the Mann-Whitney test. All statistical tests were performed using BioEstat 5.0 software (Instituto Mamirau´ a, 2007).
RESULTS As expected, gait problems affected BW (P < 0.01). The AH group presented a higher mean BW of 2.614 ± 0.030 kg when compared to the mean BW of the GP group, 2.003 ± 0.038 kg. Adrenal length did not differ significantly between groups (P = 0.28); the AH group mean was 7.80 ± 0.17 mm, and the GP group mean was 8.10 ± 0.15 mm. Adrenal gland weight was similar for both groups (P = 0.36), with observed values of 67.0 ± 2.0 and 70.0 ± 2.0 mg for the AH and GP groups, respectively. The AH group had a lower mean value of adrenal gland weight relative to BW of 0.025 ± 0.006%; the GP group mean was 0.035 ± 0.008% (P < 0.01) (Table 1). The number of cortical cells observed did not differ significantly between groups (P = 0.35; U = 413.50), with median ratios of 55.0% (40.0 to 75.0%) for the AH group and 57.0% (32.8 to 75.0%) for the GP group. Counts of medullary cells were also similar between the AH
DISCUSSION The lower BW in the GP group supports the idea that animal productivity is affected by poor welfare
A
B
GP
C
4%
60% 55.0%
2 1.75
57.0%
50%
3% b
3%
1.5
40.0%
40%
33.0%
30%
1.32
2% a
2%
1
1%
0.5
20%
10% 0%
0%
0%
0% Cortical
medullary
Blood
lymphatic
0 Cortico-medullary ratio
Figure 2. Median proportions of tissues (A and B) and median cortico-medullary ratio (C) observed in adrenal glands from broiler chickens slaughtered at 40 d of age in the town of Lapa, in southern Brazil. Medians followed by different letters represented significant differences between groups (P < 0.05).
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two groups (P = 0.12; U = 369.00), with a median proportion of 40.0% (20.0 to 56.7%) for the AH group and 33.0% (20.0 to 53.3%) for the GP group. No significant difference was found in the ratio between cortical and medullary tissues (P = 0.22; U = 394.00); the median index for the AH group was 1.32, and it was 1.75 for the GP group. Blood vessel counts were different between groups (P = 0.01; U = 301.00). The AH group had a lower proportion of blood vessels, with a median of 2.0% (0.0 to 6.7%); the GP group had a median of 3.0% (0.0 to 11.7%). Counts of other observed cells did not differ between groups. The median proportion of lymphatic tissue was 0.0% (0.0 to 5.0%) for the AH group and 0.0% (0.0 to 4.9%) for the GP group (P = 0.54; U = 437.00), and the median proportion of artifacts was 3.0% (0.0 to 8.3%) for the AH group and 3.0% (0.0 to 15.0%) for the GP group (P = 0.50; U = 433.00). Median proportions of all observed tissues and cortico-medullary ratios are shown in Figure 2.
4
¨ MULLER ET AL.
Zikic et al. (2011) in broilers submitted to chronic sound stress. Yet, increased vascularization might not be specific to the adrenal glands in animals under stress, and further research would benefit from taking additional tissue samples from another organs and glands for comparison. Although lymphatic involution is known to result from increased adrenal cortex activity (Siegel, 1971; Post et al., 2003; de Matos, 2008), this was not observed in this study. Stressful stimuli clearly induce changes in adrenal morphology and may affect animal welfare (Naas et al., 2009). In addition to important changes in behavior, such as altering birds’ feeding strategies (Weeks and Nicol, 2006), stress can also cause broiler chickens to experience pain and discomfort due to severe lameness (Danbury et al., 2000). Information about avian adrenal changes in response to stress is scarce when compared to information about mammals, although it appears that adrenal glands’ main biological roles are comparable between species (Morm`ede et al., 2007). Further studies should focus on implications of avian adrenal responses to stress as they relate to poultry welfare and understanding the subtleties of how various adrenal tissues respond to chronic stress. Our results support the hypothesis that gait problems cause significant adrenal changes, suggesting a possible role for the study of adrenal gland morphology as an indicator of chronic welfare problems in broiler chickens. Birds with gait problems had lower BW, higher relative weight of adrenal glands, and higher proportion of blood vessels in the adrenal glands. Changes in adrenal morphology caused by chronic lameness in broiler chickens further indicate that this challenge is a major welfare problem that most likely causes sustained animal suffering. Advances in knowledge about the effects of chronic welfare restrictions on animal physiology enable a better understanding and encourage reflection about the practices adopted in our production systems. Our current research supports the understanding that gait problems constitute an important challenge for broiler chickens individually and also for the whole poultry industry, affecting the biological function of animals and consequently productivity. Results indicate that changes in adrenal morphology could be used as indicators of animal welfare and, if addressed in future husbandry strategies, may lead to a system that is not only more efficient but also more respectful to the animals.
ACKNOWLEDGMENTS We would like to thank the Brazilian National Council for Scientific and Technological Development for its support by means of an undergraduate scholarship during the period of the experiment. The authors also wish to thank Anderson Bonamigo, Elizabeth Santin, and Viviani Bontoni for their collaboration on this work.
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conditions (Broom, 1991). It is known that gait problems can change the time budget of many behaviors and alter feeding strategy (Weeks et al., 2000). The inability to walk and reach for food and water, especially in animals with a gait score of 5, might lead to reduced feed intake and consequent lower weight gain and may constitute a severe stressor. The absolute value of adrenal gland weight did not differ between groups. However, absolute weight is not a reliable indicator because of its high variability between conspecifics (Aire, 1980); differences between relative adrenal weights enable a better identification of morphological changes because these weights are adjusted based on each bird’s BW and thus seem particularly relevant when comparing groups with significantly different BW. The higher relative adrenal weights found in the GP group might be a result of weight loss in birds from this group, meaning that the animals reduced their BW, and gland weight remained the same. However, higher relative adrenal weights have also been reported in previous studies that relate these higher weights to increased adrenal activity in response to stress (Siegel, 1959; Harvey et al., 1984). Further investigation should focus on determining what the actual cause of increased relative adrenal weight is. No cortico-medullary ratio changes were found between groups. Changes in this ratio have been observed in water-deprived (Sharma et al., 2009) and noise-stressed broiler chickens (Zikic et al., 2011), and in heat-stressed laying hens (Aire, 1980). These studies indicate changes in adrenocortical tissue and are in agreement with Harvey et al. (1984), who state that the presence of stress-related hormones in the peripheral circulation stimulates the proliferation of adrenocortical cells. Results of the present work, however, do not show such changes. The difference in results might be due to factors influencing adrenal morphology, which are not yet clear. Age, sex, and strain seem to be important influences on the degree of responses (Siegel, 1971). Siller et al. (1975) related significant changes in corticomedullary ratio of birds occurring most markedly between 3 and 7 wk of age, when young birds can present cortical tissue prevalence of up to 90%. Also, the intensity and duration of the stressor appear to play an important role in altering adrenal morphology (Ulrich-Lai et al., 2006; Morm`ede et al., 2007). Such variability encourages new studies on the effects of stress on adrenal morphology. The methods of the present study allowed identification of important effects of stress in different tissues of the adrenal gland, not only cortical and medullary, and results showed an increased percentage of blood vessels in adrenal glands of birds in the GP group. In general, adrenal hyperemia has been scarcely described as a physiological response to stress and might have been overlooked in past studies. Edelmann (1945) indicates that the adrenal glands’ immediate response to stress is hyperemia, and an increased percentage of adrenal blood vessels has also been described by
WELFARE RESTRICTIONS AND BROILER ADRENAL MORPHOLOGY
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