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British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effects of blood sampling on plasma concentrations of corticosterone and glucose in laying hens caged in groups a
a
a
H. Lagadic , J. M. Faure , A. D. Mills & J. B. Williams
a
a
Station de Recherches Avicoles , Institut National de la Recherche Agronomique, Centre de Tours , Nouzilly, Monnaie, 37380, France Published online: 08 Nov 2007.
To cite this article: H. Lagadic , J. M. Faure , A. D. Mills & J. B. Williams (1990) Effects of blood sampling on plasma concentrations of corticosterone and glucose in laying hens caged in groups, British Poultry Science, 31:4, 823-829, DOI: 10.1080/00071669008417313 To link to this article: http://dx.doi.org/10.1080/00071669008417313
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
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British Poultry Science (1990) 31: 823-829
EFFECTS OF BLOOD SAMPLING ON PLASMA CONCENTRATIONS OF CORTICOSTERONE AND GLUCOSE IN LAYING HENS CAGED IN GROUPS H. LAGADIC, J. M. FAURE, A. D. MILLS 1 AND J. B. WILLIAMS Station de Recherches Avicoles, Institut National de la Recherche Agronomique—Centre de Tours, Nouzilly, 37380, Monnaie, France
Downloaded by [University of Calgary] at 15:00 07 April 2015
Received for publication 24th January 1989
Abstract 1. The effects of taking a blood sample from one bird in a caged group on plasma concentrations of corticosterone and glucose in birds from its own group and birds from other groups were investigated. 2. Two blood sampling protocols were used: successive (all birds within a group were sampled one immediately after another) and alternative (birds from different groups were sampled one after another until all birds in all groups had been sampled). 3. Neither sampling protocol nor between or within group sampling rank was related to plasma concentrations of corticosterone and glucose. 4. The time taken to remove a blood sample (generally more than 45 s but less than 2 min) did not influence circulating corticosterone and glucose. 5. In individual birds plasma concentrations of corticosterone and glucose were poorly correlated with one another. 6. It is concluded that it is possible to take blood samples from a bird, kept in a group, without affecting plasma concentrations of corticosterone and glucose in other birds from that group or in birds from other groups in other cages. INTRODUCTION
Although some controversy exists concerning the relationship between physiological measures of stress and distress (Ewbank, 1985; Rushen, 1987), concern for the welfare of domestic animals kept under intensive husbandry conditions has led to the need to develop objective measures of stress which can be used under 'farm conditions' (Ewbank, 1981). At present, changes in certain of the physiological variables associated with Selye's (1937) 'General 1
To whom correspondence and reprint requests should be addressed. 823
Downloaded by [University of Calgary] at 15:00 07 April 2015
824
H. LAGADIC£rAL.
Adaptation Syndrome' and Cannon's (1935) "flight-fight reaction" are the most widely used non-behavioural measures of stress (see Wiepkema and van Adrichem (1987) for references). With battery-caged domestic fowls, such studies have mainly been carried out on animals kept in individual cages, whereas under commercial conditions these birds are usually kept in groups of 4 or 5. Furthermore, because the measurements require birds being caught and restrained for blood sampling, the sampling of one animal may disturb not only the animal itself but also the other members of the group and thereby artificially distort the results. Two commonly used physiological indices of stress in domestic fowl are plasma concentrations of corticosterone and glucose, both of which increase after exposure to a stressor (Freeman, 1976; Saleh and Jaksch, 1977). If, as suggested above, taking a blood sample from one bird influences other birds then these variables should increase with both the time taken to remove a blood sample and within and between group sampling order. The present study had three aims: to determine if the procedures of catching and handling of one bird from a caged group influenced plasma corticosteone and plasma glucose concentrations in other birds, to investigate the influence of the time taken to remove a blood sample from a bird on these variables and to determine if a relationship existed between plasma corticosterone and plasma glucose concentration.
MATERIALS AND METHODS
The animals were 144 ISA-Brown layer hens. From 20 weeks of age to the end of the experiment the animals were housed in groups of 4 in battery cages. Within these cages the space allowance per animal was 0-045 m2. All the birds in a given group were individually identifiable by means of coloured wing badges. All the cages were located in the same poultry house. At the time of the experiment the animals were 58 weeks of age. Food and water were available ad libitum. The lighting regimen in the poultry house was 15L:9D. Approximately one week before the start of the experiment, the feathers covering the wing veins were plucked from each bird to facilitate blood sampling. Blood samples (5 ml) were taken on each day of two periods of three days. These two periods of three days were separated by an interval of 4 days. All samples were taken between 15-00 and 16-00 h (that is, 4 to 5 h before the end of the photoperiod), because circulating corticosterone concentrations are at their lowest at this time of day (Beuving and Vonder, 1977) and changes in this variable are most readily detectable at this time of day (Hill, 1983; Koelkebeck et al., 1986). On each day of sampling three groups of 4 birds were sampled according to one of each of the following sample protocols. Alternative. Birds from each of the three groups were sampled alternatively (bird 1 from group 1, then bird 1 from group 2, bird 1 from group 3, bird 2 from group 1, bird 2 from group 2 and so forth through to bird 4 from group 3). Successive. The 4 birds within a group were sampled one immediately after
Downloaded by [University of Calgary] at 15:00 07 April 2015
BLOOD SAMPLING AND STRESS IN HENS
825
another and the three groups were sampled successively (birds 1 to 4 from group 1 then birds 1 to 4 from group 2 and so forth). The order in which the two sampling protocols were carried out was reversed on successive sampling days. Over the 6 d of the experiment each protocol was replicated 18 times. For each sample from each bird the following information was noted; the time at which the bird was taken from its cage, the time at which blood sampling was commenced and the time at which blood sampling finished. Blood samples were taken from each bird on one occasion and under one sampling protocol only. The interval between samples from individual birds was greater under the alternative protocol than under the successive protocol simply because of the time taken to move between cages and to open the cage doors. No attempt was made to standardise the intervals between blood samplings under the two protocols because one of the aims of the experiment was to compare the two protocols under practical conditions. All blood samples were taken using heparinised syringes. Immediately after collection, the samples were transferred to pre-numbered tubes and temporarily stored on ice. To prepare and assay the blood samples they were centrifuged at 2000 g for 10 min at 4°C and the plasma was divided into two fractions and stored at — 20°C until subsequently analysed. One 400 /d fraction was used to measure plasma glucose with an autoanalyser (Beckman Glucose Analyser 2, Beckman Instruments Inc, Galway, Ireland). The other fraction was assayed for corticosterone using the radioimmunoassay described by Etches (1976). The sensitivity, precision and inter-assay variation association of this last assay are as follows: sensitivity; ED50 (median dose), 40-60 ±1-8 pg; precision (P), coefficient of variation, 10-00>P> 10-32 for the range 17 to 323 pg; inter-assay variation, 13-18%. Each sample from each animal in each group was classed according to each of the two following sampling conditions: within group sampling rank within a sampling protocol (1 to 4), and between groups sampling rank (on a given day), within a sampling protocol (1 to 3). All results were analysed using the non-parametric analysis of variance by ranks described by Meddis (1984). Where, on the basis of the literature (see Introduction and Results), a priori hypotheses could be put forward to describe the effects of the various sampling conditions on plasma corticosterone and plasma glucose concentrations, specific alternative hypotheses tests (Meddis, 1984) were initially used to analyse all results. In these tests, where the results obtained should be consistent with a given hypothesis, the significance of differences between the various sampling conditions are expressed in terms of 'z' and its associated probability. If no a priori hypothesis could be formulated or the probability level obtained in the specific alternative hypothesis test was non-significant (i.e. P>0-05), then a nonspecific alternative hypotheses test (Meddis, 1984) was used to test the significance of differences between the various sampling conditions. In these tests the significance of differences is expressed in terms of 'FT and its associated probability. In those analyses where correlation coefficients were calculated the values given are Spearman rank correlation coefficients.
826
H. LAGADIC ETAL.
RESULTS
Table 1 shows the effect of within group sampling rank on plasma concentrations of corticosterone and glucose under the alternative and successive sampling protocols. Under neither protocol was the effect of sampling rank significant. TABLE 1
Effect of within group sampling rank on plasma concentrations of corticosterone and glucose under two sampling protocols (alternative and successive) in groups of laying hens. Values are means (±SD)
Downloaded by [University of Calgary] at 15:00 07 April 2015
Corticosterone (ng/ml) Sampling protocol Within groups sampling rank 1 2 3 4 Non-parametric analysis of variance Specific test Nonspecific test
Alternative 1-53 1-23 1-66 1-65
(±0-62) (±0-46) (±0-62) (±0-68)
z = l-37 (P=0-09) H=5-59 (P=0-13)
Successive 1-40 1-47 1-58 1-48
(±0-59) (±0-29) (±0-57) (±0-57)
z=0-59 (P=0-28) H=0-98 (P>0-80)
Glucose (mg/ml) Alternative
2-27 2-26 2-32 2-32
(±0-13) (±0-13) (±0.12) (±0-16)
z = 0-95 (P=0-17) //=l-46 (P>0-50)
Successive 2-24 2-26 2-25 2-27
(±0-12) (±0-14) (±0-16) (±0-13)
z = 0-81 (P=0-21) H=l-36
Table 2 shows the effect of between groups sampling rank on plasma concentrations of corticosterone and glucose under the alternative and successive sampling protocols. The effect of sampling rank was not significant under either protocol. TABLE 2
Effect of group sampling rank on plasma concentrations of corticosterone and glucose under two sampling protocols (alternative and successive) in groups of laying hens. Values are means (± SD)
Corticosterone (ng/ml) Sampling protocol Group sampling rank 1 2
3 Non-parametric analysis of variance Specific test Nonspecific test
Alternative
1-60 1-48
(±0-61) (±0-44)
1-49
(±0-77)
z= -M7 (P>0-50) H=l-40 (P>0-30)
Successive
1-57 1-45 1-43
(±0-75) (±0-40) (±0-28)
z=-0-14 (P>0-50) H=0-03 (P>0-98)
Glucose (mg/ml) Alternative
2-29 2-27 2-32
(±0-12) (±0-12) (±0-16)
z = 0-41 (P=0-34) //=l-32 (P>0-50)
Successive
2-25 2-22 2-29
(±0-12) (±0-14) (±0-15)
z = 0-56 (P=0-29) //=3-62 (P=0-16)
No a priori hypothesis could be put forward to explain possible influences
Downloaded by [University of Calgary] at 15:00 07 April 2015
BLOOD SAMPLING AND STRESS IN HENS
827
of type of sampling protocol on circulating corticosterone and glucose. Therefore, only nonspecific analyses were performed. Sampling protocol type had no significant effect on plasma corticosterone (alternative protocol, 1-48 ±0-51 ng/ml; successive protocol, 1-52 ±0-62 ng/ml; H= 0-001; P>0-95). However, plasma glucose concentrations were higher under the alternative protocol than the successive protocol and this difference approached significance at the 5% level (alternative protocol, 2#29±0-14 mg/ml; successive protocol, 2-26 ±0-14 mg/ml; H= 2-96; DF=l; P= 0-08). Under the alternative sampling protocol plasma concentrations of corticosterone and glucose were significantly correlated with one another (Spearman's p=0-24, z=l*96, P
British Poultry Science Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cbps20
Effects of blood sampling on plasma concentrations of corticosterone and glucose in laying hens caged in groups a
a
a
H. Lagadic , J. M. Faure , A. D. Mills & J. B. Williams
a
a
Station de Recherches Avicoles , Institut National de la Recherche Agronomique, Centre de Tours , Nouzilly, Monnaie, 37380, France Published online: 08 Nov 2007.
To cite this article: H. Lagadic , J. M. Faure , A. D. Mills & J. B. Williams (1990) Effects of blood sampling on plasma concentrations of corticosterone and glucose in laying hens caged in groups, British Poultry Science, 31:4, 823-829, DOI: 10.1080/00071669008417313 To link to this article: http://dx.doi.org/10.1080/00071669008417313
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden.
Downloaded by [University of Calgary] at 15:00 07 April 2015
Terms & Conditions of access and use can be found at http://www.tandfonline.com/ page/terms-and-conditions
British Poultry Science (1990) 31: 823-829
EFFECTS OF BLOOD SAMPLING ON PLASMA CONCENTRATIONS OF CORTICOSTERONE AND GLUCOSE IN LAYING HENS CAGED IN GROUPS H. LAGADIC, J. M. FAURE, A. D. MILLS 1 AND J. B. WILLIAMS Station de Recherches Avicoles, Institut National de la Recherche Agronomique—Centre de Tours, Nouzilly, 37380, Monnaie, France
Downloaded by [University of Calgary] at 15:00 07 April 2015
Received for publication 24th January 1989
Abstract 1. The effects of taking a blood sample from one bird in a caged group on plasma concentrations of corticosterone and glucose in birds from its own group and birds from other groups were investigated. 2. Two blood sampling protocols were used: successive (all birds within a group were sampled one immediately after another) and alternative (birds from different groups were sampled one after another until all birds in all groups had been sampled). 3. Neither sampling protocol nor between or within group sampling rank was related to plasma concentrations of corticosterone and glucose. 4. The time taken to remove a blood sample (generally more than 45 s but less than 2 min) did not influence circulating corticosterone and glucose. 5. In individual birds plasma concentrations of corticosterone and glucose were poorly correlated with one another. 6. It is concluded that it is possible to take blood samples from a bird, kept in a group, without affecting plasma concentrations of corticosterone and glucose in other birds from that group or in birds from other groups in other cages. INTRODUCTION
Although some controversy exists concerning the relationship between physiological measures of stress and distress (Ewbank, 1985; Rushen, 1987), concern for the welfare of domestic animals kept under intensive husbandry conditions has led to the need to develop objective measures of stress which can be used under 'farm conditions' (Ewbank, 1981). At present, changes in certain of the physiological variables associated with Selye's (1937) 'General 1
To whom correspondence and reprint requests should be addressed. 823
Downloaded by [University of Calgary] at 15:00 07 April 2015
824
H. LAGADIC£rAL.
Adaptation Syndrome' and Cannon's (1935) "flight-fight reaction" are the most widely used non-behavioural measures of stress (see Wiepkema and van Adrichem (1987) for references). With battery-caged domestic fowls, such studies have mainly been carried out on animals kept in individual cages, whereas under commercial conditions these birds are usually kept in groups of 4 or 5. Furthermore, because the measurements require birds being caught and restrained for blood sampling, the sampling of one animal may disturb not only the animal itself but also the other members of the group and thereby artificially distort the results. Two commonly used physiological indices of stress in domestic fowl are plasma concentrations of corticosterone and glucose, both of which increase after exposure to a stressor (Freeman, 1976; Saleh and Jaksch, 1977). If, as suggested above, taking a blood sample from one bird influences other birds then these variables should increase with both the time taken to remove a blood sample and within and between group sampling order. The present study had three aims: to determine if the procedures of catching and handling of one bird from a caged group influenced plasma corticosteone and plasma glucose concentrations in other birds, to investigate the influence of the time taken to remove a blood sample from a bird on these variables and to determine if a relationship existed between plasma corticosterone and plasma glucose concentration.
MATERIALS AND METHODS
The animals were 144 ISA-Brown layer hens. From 20 weeks of age to the end of the experiment the animals were housed in groups of 4 in battery cages. Within these cages the space allowance per animal was 0-045 m2. All the birds in a given group were individually identifiable by means of coloured wing badges. All the cages were located in the same poultry house. At the time of the experiment the animals were 58 weeks of age. Food and water were available ad libitum. The lighting regimen in the poultry house was 15L:9D. Approximately one week before the start of the experiment, the feathers covering the wing veins were plucked from each bird to facilitate blood sampling. Blood samples (5 ml) were taken on each day of two periods of three days. These two periods of three days were separated by an interval of 4 days. All samples were taken between 15-00 and 16-00 h (that is, 4 to 5 h before the end of the photoperiod), because circulating corticosterone concentrations are at their lowest at this time of day (Beuving and Vonder, 1977) and changes in this variable are most readily detectable at this time of day (Hill, 1983; Koelkebeck et al., 1986). On each day of sampling three groups of 4 birds were sampled according to one of each of the following sample protocols. Alternative. Birds from each of the three groups were sampled alternatively (bird 1 from group 1, then bird 1 from group 2, bird 1 from group 3, bird 2 from group 1, bird 2 from group 2 and so forth through to bird 4 from group 3). Successive. The 4 birds within a group were sampled one immediately after
Downloaded by [University of Calgary] at 15:00 07 April 2015
BLOOD SAMPLING AND STRESS IN HENS
825
another and the three groups were sampled successively (birds 1 to 4 from group 1 then birds 1 to 4 from group 2 and so forth). The order in which the two sampling protocols were carried out was reversed on successive sampling days. Over the 6 d of the experiment each protocol was replicated 18 times. For each sample from each bird the following information was noted; the time at which the bird was taken from its cage, the time at which blood sampling was commenced and the time at which blood sampling finished. Blood samples were taken from each bird on one occasion and under one sampling protocol only. The interval between samples from individual birds was greater under the alternative protocol than under the successive protocol simply because of the time taken to move between cages and to open the cage doors. No attempt was made to standardise the intervals between blood samplings under the two protocols because one of the aims of the experiment was to compare the two protocols under practical conditions. All blood samples were taken using heparinised syringes. Immediately after collection, the samples were transferred to pre-numbered tubes and temporarily stored on ice. To prepare and assay the blood samples they were centrifuged at 2000 g for 10 min at 4°C and the plasma was divided into two fractions and stored at — 20°C until subsequently analysed. One 400 /d fraction was used to measure plasma glucose with an autoanalyser (Beckman Glucose Analyser 2, Beckman Instruments Inc, Galway, Ireland). The other fraction was assayed for corticosterone using the radioimmunoassay described by Etches (1976). The sensitivity, precision and inter-assay variation association of this last assay are as follows: sensitivity; ED50 (median dose), 40-60 ±1-8 pg; precision (P), coefficient of variation, 10-00>P> 10-32 for the range 17 to 323 pg; inter-assay variation, 13-18%. Each sample from each animal in each group was classed according to each of the two following sampling conditions: within group sampling rank within a sampling protocol (1 to 4), and between groups sampling rank (on a given day), within a sampling protocol (1 to 3). All results were analysed using the non-parametric analysis of variance by ranks described by Meddis (1984). Where, on the basis of the literature (see Introduction and Results), a priori hypotheses could be put forward to describe the effects of the various sampling conditions on plasma corticosterone and plasma glucose concentrations, specific alternative hypotheses tests (Meddis, 1984) were initially used to analyse all results. In these tests, where the results obtained should be consistent with a given hypothesis, the significance of differences between the various sampling conditions are expressed in terms of 'z' and its associated probability. If no a priori hypothesis could be formulated or the probability level obtained in the specific alternative hypothesis test was non-significant (i.e. P>0-05), then a nonspecific alternative hypotheses test (Meddis, 1984) was used to test the significance of differences between the various sampling conditions. In these tests the significance of differences is expressed in terms of 'FT and its associated probability. In those analyses where correlation coefficients were calculated the values given are Spearman rank correlation coefficients.
826
H. LAGADIC ETAL.
RESULTS
Table 1 shows the effect of within group sampling rank on plasma concentrations of corticosterone and glucose under the alternative and successive sampling protocols. Under neither protocol was the effect of sampling rank significant. TABLE 1
Effect of within group sampling rank on plasma concentrations of corticosterone and glucose under two sampling protocols (alternative and successive) in groups of laying hens. Values are means (±SD)
Downloaded by [University of Calgary] at 15:00 07 April 2015
Corticosterone (ng/ml) Sampling protocol Within groups sampling rank 1 2 3 4 Non-parametric analysis of variance Specific test Nonspecific test
Alternative 1-53 1-23 1-66 1-65
(±0-62) (±0-46) (±0-62) (±0-68)
z = l-37 (P=0-09) H=5-59 (P=0-13)
Successive 1-40 1-47 1-58 1-48
(±0-59) (±0-29) (±0-57) (±0-57)
z=0-59 (P=0-28) H=0-98 (P>0-80)
Glucose (mg/ml) Alternative
2-27 2-26 2-32 2-32
(±0-13) (±0-13) (±0.12) (±0-16)
z = 0-95 (P=0-17) //=l-46 (P>0-50)
Successive 2-24 2-26 2-25 2-27
(±0-12) (±0-14) (±0-16) (±0-13)
z = 0-81 (P=0-21) H=l-36
Table 2 shows the effect of between groups sampling rank on plasma concentrations of corticosterone and glucose under the alternative and successive sampling protocols. The effect of sampling rank was not significant under either protocol. TABLE 2
Effect of group sampling rank on plasma concentrations of corticosterone and glucose under two sampling protocols (alternative and successive) in groups of laying hens. Values are means (± SD)
Corticosterone (ng/ml) Sampling protocol Group sampling rank 1 2
3 Non-parametric analysis of variance Specific test Nonspecific test
Alternative
1-60 1-48
(±0-61) (±0-44)
1-49
(±0-77)
z= -M7 (P>0-50) H=l-40 (P>0-30)
Successive
1-57 1-45 1-43
(±0-75) (±0-40) (±0-28)
z=-0-14 (P>0-50) H=0-03 (P>0-98)
Glucose (mg/ml) Alternative
2-29 2-27 2-32
(±0-12) (±0-12) (±0-16)
z = 0-41 (P=0-34) //=l-32 (P>0-50)
Successive
2-25 2-22 2-29
(±0-12) (±0-14) (±0-15)
z = 0-56 (P=0-29) //=3-62 (P=0-16)
No a priori hypothesis could be put forward to explain possible influences
Downloaded by [University of Calgary] at 15:00 07 April 2015
BLOOD SAMPLING AND STRESS IN HENS
827
of type of sampling protocol on circulating corticosterone and glucose. Therefore, only nonspecific analyses were performed. Sampling protocol type had no significant effect on plasma corticosterone (alternative protocol, 1-48 ±0-51 ng/ml; successive protocol, 1-52 ±0-62 ng/ml; H= 0-001; P>0-95). However, plasma glucose concentrations were higher under the alternative protocol than the successive protocol and this difference approached significance at the 5% level (alternative protocol, 2#29±0-14 mg/ml; successive protocol, 2-26 ±0-14 mg/ml; H= 2-96; DF=l; P= 0-08). Under the alternative sampling protocol plasma concentrations of corticosterone and glucose were significantly correlated with one another (Spearman's p=0-24, z=l*96, P
