GENERAL
AND
COMPARATIVE
ENDOCRINOLOGY
ffect of Stressing
35, 153-159 (1978)
Factors on Corticosterme of Laying Hens G.BEUVINGAND
Spelderholt
G.M.
Levels in t
AVONDER
Institute for Poultry Research, Ministry of Agriculture Beekbergerz, The Netherlands
and Fisheries,
Accepted January 18, 1978 The injection of ACTH into young or old laying hens and roosters caused an increase in plasma corticosterone within a few minutes. The increase was more rapid in younger than in older birds, but in the older birds the increase lasted longer. The effect of various stressor agents on the corticosterone content in plasma of laying hens is described. Immobilization by hand or by crating gave higher concentrations than thirst, hunger, or heat. Adaptation to repeated handling could not be shown.
Although interest in stress has considerably increased during the last few years, particularly in relation to animal welfare (Freeman, 19X), it is still difficult to measure stress with physiological parameters. This may be partly due to the fact that, in the literature, stress is often badly defined and can have several meanings. To assess the physiologica aspects of stress, a choice must be made between parameters which are by themselves not identical to stress. The daily rhythm in corticosterone level and the influence of egg laying on this level were reported in an earlier study using the hen (Beuving and Vender, 1977). In this study the influence of ACTH injections and of several stressful factors on plasma corticosterone levels is described. MATEFWILS
AND METHODS
General. The animals used were White Leghorn laying hens from an experimental line of the Institute. All birds (2.5-40 weeks old except for two groups of birds in the ACTH experiment) were housed individually in cages both before and during the experiments. The daily light period in all experiments lasted from 06.00 to 20.00 hr. Cannulation of the wing artery was performed at least 38 hr before the first blood samples were taken. The technique of cannulation has been described elsewhere (Beuving and Vender, 1977). The rate of blood removal through the cannula is
normally 0.5 mUmin but can be greater if a shorter cannula is used. A total of 2 to 3 ml of blood was removed in all the experiments. Corticosterone was determined by competitive IXOtein binding in all individual blood samples (Beuving and Vender, 1977). ACTH (porcine) was obtained from Mann Research Laboratories (New York, New York). ACTH injecfions. In this experiment three grraps of six chickens were used: I26-week-old laying hens (1.6 to 2.1 kg), l26-week-old roosters from the same line (2.3 to 2.7 kg), and 36-week-old laying hens (1.5 to I .9 kg). The birds were cannulated in both the right and the left wing arteries. Porcine ACTH 1.2 IU/kg, dissolved ia 0.9% NaCl, was injected into one cannula at Time 0 at least 2 hr after egg laying TV avoid the influence of ovttlation and egg Iaying ore the hormone levels. Blood samples were taken in each group at 3& & l2& 172 .min, and so on as indicated in Fig. 1. During blood sampling the birds were able to move freely irk their cages. It took abom 3 mix-~ ?Q remove the blood sample. Handlitzg of the birds. In these experiments? cannuiated birds of 33 to 37 weeks old were used. Again, the treatments were begun at leas1 2 hr after egg laying. Reference samples were taken just before the start of the experiment. At Time 0, the bird was taken from the cage, put onto a table, and !mmobikzed by hand. In the first experiment, chickens were held for 7+ min !Fig. 2). The time needed to take the blood samples was 30 sec. In the following experiment. blood samples were taken after repeated handling (dux&io~: of blood sampling 24 min). The cannulated birds were taken from the cage at 08.30, 10.30. 12.30 and 14.X
154
BEUVING
AND
hr for 5 successive days and held on a table for 5 min. At 08.35 and at 12.35 hr, blood samples were taken after the 5-min immobilization period. In a third experiment, cannulated birds were held by hand on a table for I hr, after which bIood was removed, a process requiring 1 min. &r&g. Fifteen laying hens (34 weeks old) were placed into a crate (82.5 x 63.5 x 28 cm) for 7 hr without food and water. In each crate 2 of the 15 hens were cannulated. The hens were transferred from the cages (f = 0) to the crate and blood samples were taken within 24 min. Heuf stress. The temperature in the room containing the cannulated birds (31 weeks old) in their usual cages was increased over a I-hr period from 23 to 37 (08.30-09.30 hr). The relative humidity was 25%. Deprivation offood and water. Food was withheld from 40-week-old chickens for 5 days and water was withheld for 24 days. Blood samples were taken as
20
40
VONDER stated in Table 2. Food and water were taken away on the first day at 20.00 hr. Water was given again after the blood samples at 08.00 hr had been taken.
RESULTS
AND DISCUSSION
The injection of porcine ACTH increased the corticosterone concentration considerably within a few minutes in all groups of chickens (Fig. 1). The response was greater in the younger than in the older hens (Fig. l), but in the older birds the increase lasted longer. Therefore, the same average maximum (about 50 r&ml) was reached in all three groups. Although the moment when the hormone level began to increase cannot be determined precisely, it may be
60
,
,
80
100
120
MINUTES
FIG. I. Changes in plasma corticosterone concentrations (@ml) after the intraarterial injection (t = 0) of ACTH in three groups of chickens: 36week-old laying hens (C-O, N = 6), 126week-old laying hens (CO, N = 6) and 126-week-old roosters (v-v, N = 6). The ? SE of the arithmetic mean (@ml) varied from 0.7 (t = 0 min) to 7.4 (r = 24 min) for the young laying hens, from 0.9 (t = 0 min) to 9.4 (r = 3+ mm) for the old laying hens, and from 0.6 (t = 0 min) to 3.2 (t = 464 min) for the roosters.
§TRESS AND PLASMA
CORTICOSTERONE
assumed to be less than 2 min after ACTH administration At 34 min after injection, the values were significantly different from the values at Time 0 in all three groups (P < 0.01). In a previous experiment (Beuving and Vender, 1977), blood samples were taken every 20 min during egg laying. After oviposition the values returned to the “normal” level for that time of day. Weiss and Brand (1974) found maxima of 2.5 rig/ml corticosterone 40 min after an ACTH injection into laying hens, while Culbert and Wells (1975) found peak levels of SO rig/ml corticosterone 30 min after the intramuscu-
2
K-d HENS
155
lar injection of ACTH (~yna~tb~~~. ferences from our values may be caused by the differences in the m hormone assay and also by diff~~e~~es in type and age of the birds. Also, the amounts of ACTH injected varied considerably: l-2 II-J/kg in our experiments, 4 (Weiss and Brand, 1974), an (Culbert and Wells, 1975). The influerme of is shown in Fig. 2. cause the varian the mean from chickens was heterogeneous, the in values were transformed using
4
6
8
MINUTES
FIG. 2. The influence of immobilization by hand for 7+ min on pIasma corticosterone. The points are averages of values from 14 cannulated chickens. The Time 0 sample was taken just before the chickens were moved from their cages. The vertical lines represent ? SE of the arithmetic mean.
156
BEUVING
AND
logarithms in all statistical operations. Using Student’s t test on such data, we found a significant increase in corticosterone (P < 0.05) after only 45 set of immobilization. Culbert and Wells (1975) found no influences of immobilization but their initial values were too high to find small differences such as we found. Etches (1976) did not find an effect of handling on the corticosterone level, but he took the bIood samples within I min. During immobilization the corticosterone concentration initially increased rapidly, but after 3 to 4 min the steepness of the rise gradually decreased. It was observed that some birds hardly reacted to the immobilization while others showed a very strong response, so that the concentrations ranged between 2 and greater than 20 rig/ml 7+ min after immobilization. In this particular experiment, blood samples were taken close to each other and this may have caused a hemorrhagic shock, thereby having an important influence on the hormone concentrations, especially in the last part of the curve. The influence of prolonged (1 hr) im-
FIG. vertical
3. Corticosterone lines represent
VONDER
mobilization by hand is shown in Fig. 3. Following a large increase during the first 5 min, a slower increase continued during the entire hour of immobilization. Duncan’s multiple range test revealed significant increases in all groups (P < 0.01) above that at Time 0. The concentrations in this experiment were somewhat lower than in the preceding one, perhaps because hemorrhagic shock was less marked. The influence of repeated handling (four times a day during 5 days) is seen in Table I. A significant increase in corticosterone could be shown after the first period of handling (p < 0.05). If all values taken after handling are pooled, the resultant mean shows a significant increase compared with the values at Time 0 (P < 0.01). No significant decrease could be shown during this experiment. Adaptation to the influence of repeated handling was not apparent. Strangely enough, the samples taken in the afternoon had slightly higher hormone values than those taken in the morning. From the results on the daily rhythm (Beuving and Vender, 1977) the reverse situation was expected. Possibly, in the afternoon
concentration in plasma of 1 I laying k SE of the arithmetic mean.
hens
during
prolonged
immobilization.
The
STRESS AND PLASMA
CQRTICQSTERONE TABLE
15-T
1
INFLUENCE OF REPEATED HANDLING ON 5 §UC~EWVE DAYS IN NINE CANNULATED LAYING HISS”
THE
First day Day time Arithmetic mean of corticosterone conconcentration (rig/ml) *SE of arithmetic mean Retransformed values of the geometric mean
IN HENS
Second day
Third day
Fourth day
Fifth day
08.00
08.35
12.35
08.35
12.35
08.35
12.35
08.35
12.35
08.35
12.35
2.3
6.1
6.5
4.0
4.3
4.2
4.8
4.3
4.8
4.0
5.1
0.9
1.2
1.5
0.9
0.8
0.8
0.8
0.7
1.2
0.7
i~2
I.9
5.3
3.6
3.0
3.6
3.5
4.1
4.0
2.9
3.4
3.9
u The hens were immobilized by hand for 5 min at 08.30, 10.30. 12.30, and 14.30 hr. Blood samples were taken ?wice a day at 08.35 and 12.35 hr; on the first day a reference value was determined at 08.00 hr”
the birds are more sensitive to stimuli than 2). The combined values of the second and in the morning. the third day, compared with the combined Another method of immobilization was to values of the fifth and the sixth day pen 1.5birds in a crate (Fig. 4). Application significantly (P < 0.01). No effect o of Duncan’s test showed a significant in- tion could be shown because the level. r-ecrease after crating (I’ < 0.01). No sig- turned to normal after water was giv nificant differences could be shown during the fourth day, but adaptation may crating. In tins experiment, too, no adapta- occurred (Table 2). At the end of the tion to immobilization was found. period, all hens had stopped laymg. Withholding water for 2$ days and food The effect of heat stress (1 week at 37’) is for 5 days is normally used commercially to shown in Table 3. The hormone vahres force a moult. Deprivation of water had a were elevated on the first day but gr istinct effect on the hormone level (Table fell to a lower level during the ex 12
10 I
HO”RS
FIG. 4. The effect of penning 15 laying hens into a crate, Two animals in each crate were camulated. The points are the averages of the values of 10 chickens. The vertical lines represent 2 SE of the arithmetic meax
1%
BEUVING
AND
VONDER
TABLE THE
INFLUENCE
2
OF WITHHOLDING
FOOD
Third W Day time 07.45 Arithmetic mean of corticosterone concentration (@ml) 3.1 ?SE of arithmetic mean 0.36 Retransformed values of the geometric mean 2.9
AND
WATERY
Fourth dw
Fifth dv
Sixth dw
07.45
15.15
07.45
15.15
07.45
15.15
07.45
15.15
07.45
t-5.15
3.8 0.49
4.5 0.52
4.4 0.55
3.2 0.72
5.5 1.04
3.1 0.64
2.7 0.74
2.6 0.44
2.8 0.68
2.3 0.40
3.6
4.3
4.1
2.4
4.7
2.6
1.8
2.3
2.1
2.1
Q Water was withheld for 2 4 days and food was withheld for 5 days (N = IO). Water and food were removed on the first day at 20.00 hr; water was given again on the fourth day at 08.00 hr. TABLE THE
INFLUENCE
OF HEAT
STRESS
3
(37’)
ON IO CHICKENS
First dw Day time Arithmetic corticosterone centration
FOR 7 DAYP
Second W
OS.00 09.15 10.00 Il.30
Third W
13.15 15.30 09.00 15.30 09.00 15.30
09.00
09.00
mean of con-
(@ml) ? SE of arithmetic mean Retransformed values of the geometric mean
3.1 0.63
5.3 1.00
4.9 0.96
5.0 0.80
3.6 0.68
2.6 0.64
4.0 0.58
3.3 0.55
4.4 0.79
3.8 0.41
3.3 0.55
3.2 0.65
2.6
3.5
3.3
3.8
2.0
1.8
3.7
2.5
3.5
3.5
2.8
2.8
a Heating started on the first day at 08.30 hr after the first samples had been taken.
period. However, a Student’s t test showed no significant differences between the hormone values on successive days. Although deprivation of food and water as well as exposure to heat should supposedly be heavy stressors, the response of the adrenal gland does not support this supposition. When the values for the plasma corticosterone content in the various experiments are compared, it is clear that immobilization, whether for a long or short periods by hand or in a crate, caused a higher corticosterone content in the plasma of laying hens than heat, hunger, thirst, or while egg laying (Beuving and Vender, 1977). Although the response to the stress of immobilization for 7 min or to ACTH injection varied greatly, the individual response pattern was not inconsistent. On the other
hand, the response to immobilization by crating for 7 hours or by handling for I hr showed a more inconsistent pattern. The corticosterone content in plasma after particular stimuli may be a good criterion for identifying stressed birds. It has been used as such by Edens and Siegel (1975) for chickens injected with ACTH and by Brown (I 967) for turkeys exposed to cold stress. However, the present data show that it must be ascertained that the response to the stressor is not inconsistent. ACKNOWLEDGMENTS We wish to thank Dr. R. N. van Lookeren Campagne of the University of Groningen for his commerits on both the style and the content. We are also greatly indebted to Miss Riet Verbeek and Mr. F. van K est eren for their skilfnl technical assistance and to Mr. I’. J. W. van Schagen, who carried out the statis-
r& an&=.
STRESS
AND
PLASMA
CORTICOSTERQNE
REFERENCES
h:oi.
65, 363-376.
Edens. F. W., and Siegel. H. S. (1975). Adrenal re-
HEN§
159
sponses in high and low ACTS response lures of chickens during acute heat stress. &TT. Co!q. Endocrinol.
Beuving, G., and Vender, G. M. A. (1977). Daily rhythm of corticosterone in laying hens and the influence of egg laymg. J. Rep?&. FUR. 51, 169-173, Browti, K. I, (1967). Environmentahy imposed stress. f/7 “Environmental Control in Poultry Production” (T. C. Carter. ed.), pp. IOI-t 13. Oliver and Boyd, Edinburgh. Cuibert, J., and Wells, J. W. (197s). Aspects of adrenal function in the domestic fowl. J. Endo-
K’l
125~ 64-73.
Etches, R. J. (1976). A radioimmunoassay for corticosterone and its apphcation to the measurement of stress in pouhry. 5Zero& ZgY X3-773. Freeman, B. M. (1976). $tress and the domestic fowl: A physiological reappraisal. World’,? PM/F. Sci~ J. 3z9 249-256. Weiss, .J., and Brand, J. H. (1974). Untersuchungen tiber die NNR-Funktion bei landw~rts~~aftlic~en Nutztieren mit HiKe der Cortisol- und Corticosteronbestimmung nach dem Prinzip der konkurrierenden Eiweissbindungsanatyse. Z. r/ef. Med. .4& 225-242.
AND
COMPARATIVE
ENDOCRINOLOGY
ffect of Stressing
35, 153-159 (1978)
Factors on Corticosterme of Laying Hens G.BEUVINGAND
Spelderholt
G.M.
Levels in t
AVONDER
Institute for Poultry Research, Ministry of Agriculture Beekbergerz, The Netherlands
and Fisheries,
Accepted January 18, 1978 The injection of ACTH into young or old laying hens and roosters caused an increase in plasma corticosterone within a few minutes. The increase was more rapid in younger than in older birds, but in the older birds the increase lasted longer. The effect of various stressor agents on the corticosterone content in plasma of laying hens is described. Immobilization by hand or by crating gave higher concentrations than thirst, hunger, or heat. Adaptation to repeated handling could not be shown.
Although interest in stress has considerably increased during the last few years, particularly in relation to animal welfare (Freeman, 19X), it is still difficult to measure stress with physiological parameters. This may be partly due to the fact that, in the literature, stress is often badly defined and can have several meanings. To assess the physiologica aspects of stress, a choice must be made between parameters which are by themselves not identical to stress. The daily rhythm in corticosterone level and the influence of egg laying on this level were reported in an earlier study using the hen (Beuving and Vender, 1977). In this study the influence of ACTH injections and of several stressful factors on plasma corticosterone levels is described. MATEFWILS
AND METHODS
General. The animals used were White Leghorn laying hens from an experimental line of the Institute. All birds (2.5-40 weeks old except for two groups of birds in the ACTH experiment) were housed individually in cages both before and during the experiments. The daily light period in all experiments lasted from 06.00 to 20.00 hr. Cannulation of the wing artery was performed at least 38 hr before the first blood samples were taken. The technique of cannulation has been described elsewhere (Beuving and Vender, 1977). The rate of blood removal through the cannula is
normally 0.5 mUmin but can be greater if a shorter cannula is used. A total of 2 to 3 ml of blood was removed in all the experiments. Corticosterone was determined by competitive IXOtein binding in all individual blood samples (Beuving and Vender, 1977). ACTH (porcine) was obtained from Mann Research Laboratories (New York, New York). ACTH injecfions. In this experiment three grraps of six chickens were used: I26-week-old laying hens (1.6 to 2.1 kg), l26-week-old roosters from the same line (2.3 to 2.7 kg), and 36-week-old laying hens (1.5 to I .9 kg). The birds were cannulated in both the right and the left wing arteries. Porcine ACTH 1.2 IU/kg, dissolved ia 0.9% NaCl, was injected into one cannula at Time 0 at least 2 hr after egg laying TV avoid the influence of ovttlation and egg Iaying ore the hormone levels. Blood samples were taken in each group at 3& & l2& 172 .min, and so on as indicated in Fig. 1. During blood sampling the birds were able to move freely irk their cages. It took abom 3 mix-~ ?Q remove the blood sample. Handlitzg of the birds. In these experiments? cannuiated birds of 33 to 37 weeks old were used. Again, the treatments were begun at leas1 2 hr after egg laying. Reference samples were taken just before the start of the experiment. At Time 0, the bird was taken from the cage, put onto a table, and !mmobikzed by hand. In the first experiment, chickens were held for 7+ min !Fig. 2). The time needed to take the blood samples was 30 sec. In the following experiment. blood samples were taken after repeated handling (dux&io~: of blood sampling 24 min). The cannulated birds were taken from the cage at 08.30, 10.30. 12.30 and 14.X
154
BEUVING
AND
hr for 5 successive days and held on a table for 5 min. At 08.35 and at 12.35 hr, blood samples were taken after the 5-min immobilization period. In a third experiment, cannulated birds were held by hand on a table for I hr, after which bIood was removed, a process requiring 1 min. &r&g. Fifteen laying hens (34 weeks old) were placed into a crate (82.5 x 63.5 x 28 cm) for 7 hr without food and water. In each crate 2 of the 15 hens were cannulated. The hens were transferred from the cages (f = 0) to the crate and blood samples were taken within 24 min. Heuf stress. The temperature in the room containing the cannulated birds (31 weeks old) in their usual cages was increased over a I-hr period from 23 to 37 (08.30-09.30 hr). The relative humidity was 25%. Deprivation offood and water. Food was withheld from 40-week-old chickens for 5 days and water was withheld for 24 days. Blood samples were taken as
20
40
VONDER stated in Table 2. Food and water were taken away on the first day at 20.00 hr. Water was given again after the blood samples at 08.00 hr had been taken.
RESULTS
AND DISCUSSION
The injection of porcine ACTH increased the corticosterone concentration considerably within a few minutes in all groups of chickens (Fig. 1). The response was greater in the younger than in the older hens (Fig. l), but in the older birds the increase lasted longer. Therefore, the same average maximum (about 50 r&ml) was reached in all three groups. Although the moment when the hormone level began to increase cannot be determined precisely, it may be
60
,
,
80
100
120
MINUTES
FIG. I. Changes in plasma corticosterone concentrations (@ml) after the intraarterial injection (t = 0) of ACTH in three groups of chickens: 36week-old laying hens (C-O, N = 6), 126week-old laying hens (CO, N = 6) and 126-week-old roosters (v-v, N = 6). The ? SE of the arithmetic mean (@ml) varied from 0.7 (t = 0 min) to 7.4 (r = 24 min) for the young laying hens, from 0.9 (t = 0 min) to 9.4 (r = 3+ mm) for the old laying hens, and from 0.6 (t = 0 min) to 3.2 (t = 464 min) for the roosters.
§TRESS AND PLASMA
CORTICOSTERONE
assumed to be less than 2 min after ACTH administration At 34 min after injection, the values were significantly different from the values at Time 0 in all three groups (P < 0.01). In a previous experiment (Beuving and Vender, 1977), blood samples were taken every 20 min during egg laying. After oviposition the values returned to the “normal” level for that time of day. Weiss and Brand (1974) found maxima of 2.5 rig/ml corticosterone 40 min after an ACTH injection into laying hens, while Culbert and Wells (1975) found peak levels of SO rig/ml corticosterone 30 min after the intramuscu-
2
K-d HENS
155
lar injection of ACTH (~yna~tb~~~. ferences from our values may be caused by the differences in the m hormone assay and also by diff~~e~~es in type and age of the birds. Also, the amounts of ACTH injected varied considerably: l-2 II-J/kg in our experiments, 4 (Weiss and Brand, 1974), an (Culbert and Wells, 1975). The influerme of is shown in Fig. 2. cause the varian the mean from chickens was heterogeneous, the in values were transformed using
4
6
8
MINUTES
FIG. 2. The influence of immobilization by hand for 7+ min on pIasma corticosterone. The points are averages of values from 14 cannulated chickens. The Time 0 sample was taken just before the chickens were moved from their cages. The vertical lines represent ? SE of the arithmetic mean.
156
BEUVING
AND
logarithms in all statistical operations. Using Student’s t test on such data, we found a significant increase in corticosterone (P < 0.05) after only 45 set of immobilization. Culbert and Wells (1975) found no influences of immobilization but their initial values were too high to find small differences such as we found. Etches (1976) did not find an effect of handling on the corticosterone level, but he took the bIood samples within I min. During immobilization the corticosterone concentration initially increased rapidly, but after 3 to 4 min the steepness of the rise gradually decreased. It was observed that some birds hardly reacted to the immobilization while others showed a very strong response, so that the concentrations ranged between 2 and greater than 20 rig/ml 7+ min after immobilization. In this particular experiment, blood samples were taken close to each other and this may have caused a hemorrhagic shock, thereby having an important influence on the hormone concentrations, especially in the last part of the curve. The influence of prolonged (1 hr) im-
FIG. vertical
3. Corticosterone lines represent
VONDER
mobilization by hand is shown in Fig. 3. Following a large increase during the first 5 min, a slower increase continued during the entire hour of immobilization. Duncan’s multiple range test revealed significant increases in all groups (P < 0.01) above that at Time 0. The concentrations in this experiment were somewhat lower than in the preceding one, perhaps because hemorrhagic shock was less marked. The influence of repeated handling (four times a day during 5 days) is seen in Table I. A significant increase in corticosterone could be shown after the first period of handling (p < 0.05). If all values taken after handling are pooled, the resultant mean shows a significant increase compared with the values at Time 0 (P < 0.01). No significant decrease could be shown during this experiment. Adaptation to the influence of repeated handling was not apparent. Strangely enough, the samples taken in the afternoon had slightly higher hormone values than those taken in the morning. From the results on the daily rhythm (Beuving and Vender, 1977) the reverse situation was expected. Possibly, in the afternoon
concentration in plasma of 1 I laying k SE of the arithmetic mean.
hens
during
prolonged
immobilization.
The
STRESS AND PLASMA
CQRTICQSTERONE TABLE
15-T
1
INFLUENCE OF REPEATED HANDLING ON 5 §UC~EWVE DAYS IN NINE CANNULATED LAYING HISS”
THE
First day Day time Arithmetic mean of corticosterone conconcentration (rig/ml) *SE of arithmetic mean Retransformed values of the geometric mean
IN HENS
Second day
Third day
Fourth day
Fifth day
08.00
08.35
12.35
08.35
12.35
08.35
12.35
08.35
12.35
08.35
12.35
2.3
6.1
6.5
4.0
4.3
4.2
4.8
4.3
4.8
4.0
5.1
0.9
1.2
1.5
0.9
0.8
0.8
0.8
0.7
1.2
0.7
i~2
I.9
5.3
3.6
3.0
3.6
3.5
4.1
4.0
2.9
3.4
3.9
u The hens were immobilized by hand for 5 min at 08.30, 10.30. 12.30, and 14.30 hr. Blood samples were taken ?wice a day at 08.35 and 12.35 hr; on the first day a reference value was determined at 08.00 hr”
the birds are more sensitive to stimuli than 2). The combined values of the second and in the morning. the third day, compared with the combined Another method of immobilization was to values of the fifth and the sixth day pen 1.5birds in a crate (Fig. 4). Application significantly (P < 0.01). No effect o of Duncan’s test showed a significant in- tion could be shown because the level. r-ecrease after crating (I’ < 0.01). No sig- turned to normal after water was giv nificant differences could be shown during the fourth day, but adaptation may crating. In tins experiment, too, no adapta- occurred (Table 2). At the end of the tion to immobilization was found. period, all hens had stopped laymg. Withholding water for 2$ days and food The effect of heat stress (1 week at 37’) is for 5 days is normally used commercially to shown in Table 3. The hormone vahres force a moult. Deprivation of water had a were elevated on the first day but gr istinct effect on the hormone level (Table fell to a lower level during the ex 12
10 I
HO”RS
FIG. 4. The effect of penning 15 laying hens into a crate, Two animals in each crate were camulated. The points are the averages of the values of 10 chickens. The vertical lines represent 2 SE of the arithmetic meax
1%
BEUVING
AND
VONDER
TABLE THE
INFLUENCE
2
OF WITHHOLDING
FOOD
Third W Day time 07.45 Arithmetic mean of corticosterone concentration (@ml) 3.1 ?SE of arithmetic mean 0.36 Retransformed values of the geometric mean 2.9
AND
WATERY
Fourth dw
Fifth dv
Sixth dw
07.45
15.15
07.45
15.15
07.45
15.15
07.45
15.15
07.45
t-5.15
3.8 0.49
4.5 0.52
4.4 0.55
3.2 0.72
5.5 1.04
3.1 0.64
2.7 0.74
2.6 0.44
2.8 0.68
2.3 0.40
3.6
4.3
4.1
2.4
4.7
2.6
1.8
2.3
2.1
2.1
Q Water was withheld for 2 4 days and food was withheld for 5 days (N = IO). Water and food were removed on the first day at 20.00 hr; water was given again on the fourth day at 08.00 hr. TABLE THE
INFLUENCE
OF HEAT
STRESS
3
(37’)
ON IO CHICKENS
First dw Day time Arithmetic corticosterone centration
FOR 7 DAYP
Second W
OS.00 09.15 10.00 Il.30
Third W
13.15 15.30 09.00 15.30 09.00 15.30
09.00
09.00
mean of con-
(@ml) ? SE of arithmetic mean Retransformed values of the geometric mean
3.1 0.63
5.3 1.00
4.9 0.96
5.0 0.80
3.6 0.68
2.6 0.64
4.0 0.58
3.3 0.55
4.4 0.79
3.8 0.41
3.3 0.55
3.2 0.65
2.6
3.5
3.3
3.8
2.0
1.8
3.7
2.5
3.5
3.5
2.8
2.8
a Heating started on the first day at 08.30 hr after the first samples had been taken.
period. However, a Student’s t test showed no significant differences between the hormone values on successive days. Although deprivation of food and water as well as exposure to heat should supposedly be heavy stressors, the response of the adrenal gland does not support this supposition. When the values for the plasma corticosterone content in the various experiments are compared, it is clear that immobilization, whether for a long or short periods by hand or in a crate, caused a higher corticosterone content in the plasma of laying hens than heat, hunger, thirst, or while egg laying (Beuving and Vender, 1977). Although the response to the stress of immobilization for 7 min or to ACTH injection varied greatly, the individual response pattern was not inconsistent. On the other
hand, the response to immobilization by crating for 7 hours or by handling for I hr showed a more inconsistent pattern. The corticosterone content in plasma after particular stimuli may be a good criterion for identifying stressed birds. It has been used as such by Edens and Siegel (1975) for chickens injected with ACTH and by Brown (I 967) for turkeys exposed to cold stress. However, the present data show that it must be ascertained that the response to the stressor is not inconsistent. ACKNOWLEDGMENTS We wish to thank Dr. R. N. van Lookeren Campagne of the University of Groningen for his commerits on both the style and the content. We are also greatly indebted to Miss Riet Verbeek and Mr. F. van K est eren for their skilfnl technical assistance and to Mr. I’. J. W. van Schagen, who carried out the statis-
r& an&=.
STRESS
AND
PLASMA
CORTICOSTERQNE
REFERENCES
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Edens. F. W., and Siegel. H. S. (1975). Adrenal re-
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159
sponses in high and low ACTS response lures of chickens during acute heat stress. &TT. Co!q. Endocrinol.
Beuving, G., and Vender, G. M. A. (1977). Daily rhythm of corticosterone in laying hens and the influence of egg laymg. J. Rep?&. FUR. 51, 169-173, Browti, K. I, (1967). Environmentahy imposed stress. f/7 “Environmental Control in Poultry Production” (T. C. Carter. ed.), pp. IOI-t 13. Oliver and Boyd, Edinburgh. Cuibert, J., and Wells, J. W. (197s). Aspects of adrenal function in the domestic fowl. J. Endo-
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Etches, R. J. (1976). A radioimmunoassay for corticosterone and its apphcation to the measurement of stress in pouhry. 5Zero& ZgY X3-773. Freeman, B. M. (1976). $tress and the domestic fowl: A physiological reappraisal. World’,? PM/F. Sci~ J. 3z9 249-256. Weiss, .J., and Brand, J. H. (1974). Untersuchungen tiber die NNR-Funktion bei landw~rts~~aftlic~en Nutztieren mit HiKe der Cortisol- und Corticosteronbestimmung nach dem Prinzip der konkurrierenden Eiweissbindungsanatyse. Z. r/ef. Med. .4& 225-242.
