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Changes in the adrenal steroidogenic responsiveness of the mallard duck (Anas platyrhynchos) during early post-natal development
Camp. Biochem. Physioi. Vol. 92A, No. 3, pp. 403-403, Printed in Great Britain
1989
0300-9629189 $3.00 + 0.00 1989 Pergamon Press plc
CHANGES IN THE ADRENAL STEROIDOGEN~C RESPONSIVENESS OF THE MALLARD DUCK (ANAS PLATYRHYNCHOS) DURING EARLY POST-NATAL DEVELOPMENT Department
W. N. HOLMES, J. L. REWNDO and JAMES CRCJNSHAW of Biological Sciences, University of California. Santa Barbara, CA 93106, USA. Telephone: (805) 961-3511 (Received 26 July 1988)
AWract-1. Plasma ~n~nt~tions of corticosteronc (B), aldosterone (Aldo) and d~xy~r~sterone (DGC) were measured in mallard d~klin~ imm~iately before and after exposure to acute immobi~~tion stress. 2. Except for transient declines in B and DGC between the 4th and 14th days after hatching, the resting concentration of each harmone did not change significantly during post-natal development. 3. The stress-induced in Aldo was maximal at hatching while maximal increases in B and DGC did not occur until one day later. 4. Thereafter the magnitude of the stress-induced increases in the concentrations of all of the hormones decreased steadily and on the 21st and 28th days after hatching only B increased sibilantly in response to stress.
INTRODUCTION
The pattern of post-natal growth in the duck is such that the rate of increase in body weight is pro~rtionately greater than that of the adrenal glands. As a result, a sharp decline in the weight of the adrenal glands relative to the body weight occurs during the first few weeks of post-natal life. Later, this rate of decline lessens and at about six months of age the relative weight of the glands stabilize at a value ranging from onequarter to one-third of that seen in the neonate (Holmes and Kelly, 1976; Holmes and Cronshaw, 1980). As the adrenal gIands grow, however, the rate of corticosterone synthesis per unit mass of tissue decreases, and as a result a fairly steady resting plasma concentration of the hormone is maintained throughout the first six-month period of post-natal development (Holmes and Kelly, 1976). In the mature mallard duck, three corticosteroids are found in the plasma, namely corticosterone, aldosterone and 1I-deoxycorticosterone (Redondo ef al., 1988). The steroidogenic properties of the adrenal gland at hatching and during early post-natal development, however, have not been described for this species. The purpose of this study, therefore, was to determine the relative abundances of these corticosteroids in plasma at the time of hatching and to assess, in terms of stress-induced changes the corticosteroid concentrations, in plasma corticotropic responsiveness of the adrenal steroidogenic tissue in ducklings during the first month of post-natal development. MATERIALS AND
METHODS
Animals Fertilized mallard duck eggs were purchased from a commercial breeder whistling Wings. Hanover, IL) and
incubated at 37.4’C and 85% relative humidity. Newly hatched ducklings were maintained for the first 2 days 37.4”C and thereafter at 27”C, and throughout their postnatal development they were exposed to a daily photoperiod of 12hr light (08:~~:~hr) and 12hr dark. Food (Gamebird Developer, Ralston-Purina) and drinking water (tap water) were available udlibirum, and fresh supplies were provided each day at 08:OOhr. Prorocol A blood sample was taken by heart puncture within 1 min of removing a bird from its cage. The bird was then gently restrained by wrapping it in muslin gauze, and a second blood sample was taken by heart puncture 30min later. Each bird was then weighed and the recorded body weight was corrected for the mass of blood removed during sampling. The birds were considered to be unstressed when the first blood sample was taken, and at the time when the second blood sample was taken they were considered to have experienced mild stress in response to the earlier blood sampling procedure and the ensuing 30-min period of restraint. The first blood sample was taken from the ducklings that were 4 days old and older at II :00 hr, but experiments on the newly hatched and one-day-old ducklings were started at the time when they had fully emerged from the shell and 24 hr later, respectively. Each blood sample was collected in a heparinized syringe and represented approximately 1% (v/w) of the body weight. Since it was not possible to obtain a sufficiently large sample of blood from newly hatched and very young ducklings, samples taken from several birds were pooled. These pooled samples were centrifuged for 5 min at 123OOg and 5°C and stored at -20°C. To ensure that the ducklings used in these experiments wem achy disturbed, th :y were binge and handled by only one person (JLR) from the day of hatching until they were 28 days old. A group of five male and five female birds derived from the same stuck were maintained until they were 6 months old. At this time, each bird was treated in exactly the same way as the ducklings. Because of their size, however, it was possible to take two Sml blood samples from each bird. 403
W. N.
404
HOLMESet
Sample purificarion Plasma samples were thawed at room temperature and a 2.0ml aliquot of each sample was transferred to a new scintillation vial containing 8000 dpm of chromatographitally pure [i ,2-‘HI-corticosterone, (1,Z3H]-aldosterone and [I ,2-3H]-deoxycorticosterone, (Amersham Corporation, Arlington Heights, IL). The samples were allowed to equilibrate for I hr at room temperature before extraction with 10 volumes cold anhydrous ethyl ether. The ether phase was separated, dried under nitrogen, redissolved in 0.5 ml benzene:cyclohexane:methanol: water (25: 12.5 :4 : 1) and purified by gel filtration chromatography using Sephadex LH-20 and the same solvent system. The fractions containing labelled corticosterone, aldosterone and deoxycorticosterone were collected and pooled separately, evaporated by dryness under nitrogen and redissolved in 1.Oml of buffer containing 0.1 M tricine, 0.18 M NaCl and 0.015 M NaN, at pH 8, The radioactivity contained in 0.2 ml of this extract was used to determine the recovery of each steroid and the remainder of sample was stored at -20°C. Hormone analysis The concentrations of corticosterone, aldosterone and deoxycorticosterone were measured by radioimmune assay (Nishida er al., 1976; Mayes et al., 1970; Arnold and James, 1971). Corticosterone antiserum was produced in rabbits immunized with corticosterone--2~-h~isuccinyl thyroglobulin (ICN Biomedicals Inc., Costa Mesa, CA). Sensitivity for this assay was 15 pg corticosterone per assay tube, intra-assay variation was 5.0% and interassay variation was 12.8%. Aldosterone antiserum was raised in sheep with an aldosterone-3-oxine-bovine serum albumin conjugate (Arnel Products Co.. New York, NY); sensitivity was IO pg of aldosterone per assay tube and intra- and interassay variation was 3.0% and 10.3%. respectively. Deoxycorticosterone antiserum was produced in rabbits using a deoxycorticosterone-3-oxine-bovine serum albumin conjugate (Endocrine Sciences, Tarzana, CA); sensitivity was 4 pg deoxycorticosterone per assay tube and intra- and interassay variation was 8.3% and 6.8%, respectively. Statistics Ali values are expressed as means + standard error of the mean (SEM) and were compared by the Student r-test (Sokal and Rohlf, 1969). Regressions were fitted by the method of least squares.
RESULTS Body weights The mean body weight of the ducklings upon emergence from the egg was 40.3 _C0.89 g (N = 70); this value represented about 70% of the mean weight of the fertilized eggs at the start of incubation. Immediately after hatching, the body weights of the ducklings started to decline and after 24 hr the mean body weight was significantly less than at hatching (22.1 It 1.4 g, N = 40, P -C0.01). This decline in body weight continued for at least the next 3 days and on the 4th day the mean weight was still significantly lower than it was at hatching (32.0 k 0.9 g, N = 30, P c 0.01). After the 4th day, however, the ducklings started to increase in weight and this continued in a linear fashion until the end of the experimental period on the 28th day after hatching; the average daily rate of increase during this interval was 12.1 f 0.38 g (r = 0.97, P < 0.01 with 77 degrees of freedom) and the mean body weight of the ducklings on the 28th
al.
day after hatching was 237.8 + 10.7g (N = 5). Between the 28th and 180th day after hatching the body weights of the birds continued to increase at an average rate of approximately 5 g per day, and at 180 days the mean body weights of the males and the females were 1052f 35 (N = 5) and 973+45g (n = 5), respectively. Plasma corticosteroid concentrations Before exposure to stress (Tabfe 1). During the first 4 days after hatching the plasma concentration of corticosterone remained constant and during this period the mean value was 11.2 + 1.2 ng per ml (N = 17). By the end of the first week, however, the concentration of corticosterone had declined significantly and it remained at this low level until at least the 14th day after hatching. Thereafter, the concentration increased, and at 3 and 4 weeks of age it was not significantty different from the concentration recorded at hatching. The plasma concentration of aldosterone increased almost two-fold during the first 24 hr after hatching and it did not vary significantly during the ensuing 4 weeks. The pattern of post-natal change in plasma deoxycorticosterone concentration was qualitatively similar to that of corticosterone; a significantly lower concentration compared with that measured on the 1st day was recorded on the 4th day after hatching (P < 0.01 with 8 degrees of freedom), and significantly lower concentrations persisted until at least the 14th day after hatching, The mean concentrations of corticosterone, aldosterone and deoxycorticosterone recorded in the group of five mature unstressed males (180 days old) did not differ significantly from the corresponding values recorded for the group of five unstressed females of the same age (Table 2). Neither the mean concentration of each hormone recorded in either the males or the females, nor the combined mean values recorded for both the males and the females, differed significantly from the corresponding values measured in the 4-week-old ducklings (cf. Tables 1 and 2). After exposure to stress (Table 1). The plasma concentrations of all three hormones in al1 of the age groups up to and including the tCday_old ducklings increased significantly during the 30 min period of restraint. Exposure to stress also caused the corticosterone concentration, but not the aldosterone and deoxycorticosterone concentrations, to increase significantly in the 21- and 28-day-old ducklings. In the mature male and female birds, however, significant stress-induced increases in both corticosterone and aidosterone concentrations were recorded, but a significant stress-induced increase in the plasma deoxycorticosterone concentration was recorded only in the mature males (cf. Tables 1 and 2). In general, the stress-inducible increases in plasma corticosteroid concentrations were maximal at or soon after hatching, and for each hormone the mag nitude of the increase declined steadily during the ensuing weeks of post-natal development (Fig. 1). During the first 24 hr after hatching the stressinduced change in the plasma corticosterone concentration increased significantly (P < 0.05 with 10 degrees of freedom) by approximately one-fifth.
Adrenal Table
steroidogenic
responsiveness
405
I. The plasma corticosteroid concentrations (mean It SEM) in mallard ducklings exoosure to a 30-min wriod of gentle immobilization stress Corticosteroid
--___-
concentration
Corticosterone Age (days)
f5.1)
Prestress
-- stress
II.7 k2.3
58.4tt I: 1.7
11.6NS If-I.8
66.8tt 23.5
10.3NS f1.7
fng/ml plasma)
Aldosterone Prestress -~______ 0.234 i 0.020
___-
~oxycorticosterone Prestress
Stress
I .742tt k 0.270
0.472 + 0.093
I s05tt k 0.069
0.460’ f 0.074
I .704tt *0.140
0.412NS * 0.034
f0.117
&.6W t2.9
0.362NS io.064
1.soztt to.137
0.298NS & 0.028
I .349tt f0.082
5.8’ * 1.7
46.3tt +4.2
0.427NS +0.111
1.464tt kO.070
0.236. *0.043
zhO.124
6.8’ +0.4
32.0tt k4.0
0.423* +0.056
0.978t 20.137
0.200. f 0.033
0.645t +0.107
9.3NS k2.6
25.1t k5.l
0.454’ + 0.068
0.728ND &0.168
0.33lNS kO.141
OS98ND io.200
10.3NS + 2.9
29.7tt c3.5
0.403* +0.138
0.580ND &O.lOb
0.404NS &0.078
0.63lND +0.152
2.064tt
1.164tt
Plasma samples taken from newly hatched ducklings (age = 0 days) and at I, 4, 7, 14, 21 and 28 days after hatching, The first number in parentheses indicates the number of pooled plasma samples analysed to determine the mean concentration, and the second number represents number of birds that contributed to each pooled blood sample. lP -c 0.05 and NS = not significant with respect to the corresponding value in newly hatched ducklings (age = day 0). tiP -c 0.01, tP < 0.05 and ND = not significantly different from the corresponding value recorded prior to exposure to stress.
Between the 1st and the 21st days after hatching, however, the magnitude of the inducible increase declined at an average daily rate of 1.64 + 0.33 ngiml (r = 0.73, P < 0.01 with 21 degrees of freedom). On the 28th day after hatching the inducible increase in plasma corticosterone concentration was the same as that observed on the 21st day and similar to that seen in the male and female adult birds; the mean inducible increases measured in the 21-, 28- and 180-day-old birds (19.0 i 2.2 ng per ml plasma, IV = 19) being only one-third of the maximum inducible response measured on the 1st day after hatching. A maximal stress-inducible increase in plasma aldosterone concentration occurred in the newly hatched ducklings, and this degree of responsiveness was sustained until at least the 4th day after hatching (Fig. 1). Between hatching and the 4th day of postnatal development, the mean inducible increase was 1.410 + 0.125 ng/ml plasma (N = 17). After the 4th day the stress-inducible increase in plasma aldosterone concentration started to decline at a mean rate of 0.05 k 0.008 nglmliday (r = 0.79, P < 0.01 with 21 degrees of freedom) and on the 21st and the 28th days after hatching no significant increase was detectable (Fig. 1). During the course of maturation, however, the stress-inducible increase in plasma aldosterone concentration was partially restored, and at 180 days of age, mean increases of 0.927 + 0.173 ng per ml plasma (N = 10) were recorded; this value was significantly higher than that recorded in the 28-day-old ducklings (P < 0.01 with 13 degrees of freedom) and represented about 60% of the value recorded at hatching. The pattern of change in stress-inducible increases in deoxycorticosterone concentrations roughly followed that of corticosterone (Fig. 1). The inducible
increase was maximal on the 1st day after hatching (1.652 f 0.099 ng per ml plasma, N = 5) and declined thereafter at 0.047 f 0.007 ng/mI/day (I = 0.79, P < 0.01 with 27 degrees of freedom) until no significant inducible response was detectable on the 21st and the 28th days. In the mature birds, however, a small but significant inducible increase in plasma deoxycorticosterone concentration was detectable in the male, but not the female, birds (Table 2).
DISCUSSION
Regular daily patterns of increase and decrease in resting plasma corticosterone concentrations have been observed in several species of birds (Boissin and Assenmacher, 1968; Chan and Phillips, 1973; ElHalawani ef at., 1973; Gorsline and Holmes, 1981). Indeed, in the duck, such a pattern of change is already established at the time of hatching (Weiss et al., 1977), and thereafter one or more zeitgebers, such as the daily photoperiod, the diurnal cycle of activity and the feeding regimen, sustain and entrain this cyclicity. There is also reason to believe that the highest plasma concentrations of corticosterone that occur during the diurnal cycle reflect rates of hormone synthesis that may be close to maximal for the adrenal steroidogenic tissue (El-Halawani et al., 1973). Thus, if a bird is exposed to a particular stressor when the resting level is maximal, the stressinduced increase in cortocosterone concentration may be minimal whereas the response may be maximal during the nadir. It is for these reasons, therefore, that the birds used in the present experiments were maintained on a standardized feeding and watering regime, were exposed to a constant daily photoperiod and were subjected to stress at a time close to the
W. N. HOLMESet al.
406
Table 2. The plasma corticosteroid concentrations (mean It SEM) in 160.day old mature male and female mallard ducks immediately before and after their exposure to a 30 min period of gentle immobilization stress
Males (5)
Corticosterone _--__ Stress ___^_~ 8.1 30.2tt 22.5 k3.8
Females (5)
9.8 f 3.0
Combined (W
8.8 f2.0
Prestress
Aldosterone _---_--Prestress stress
Deoxycorticosterone Prestress
stress
0.239 kO.042
1.341tt i0.273
0.431 *0.107
0.9w rtO.132
27.2tt + 2.6
0.265 kO.042
0.931tt io.131
0.520 fO.115
0.755ND kO.144
28.9tt f2.4
0.250 kO.037
1.177t.t *0.191
0.472 +0.080
0.837t rto.099
ttP< 0.01, fP x 0.05and ND = not significant wilh respect to the corresponding value recorded before exposure to stress
estimated nadir that occurs at about middle of the light phase (Gorsline and Holmes, 1981). The rapid and profound increases in the plasma corticosteroid concentrations that occurred when the newly hatched ducklings were subjected to stress indicate that the mechanisms which regulate the synthesis and release of these hormones are already well-established at the end of embryogenesis. This is in marked contrast to the domestic chicken which shows no stress-induced rise in plasma corticosterone
Corticosterone 50
40 30 20 IO fi u
0
1
4
7
14
21
28
180
Aldosterone 21
4
0
-a
E
2
1
4
7
14
21
28
180
Deoxycorticosterone 21
0
1
4
7
14
21
28
180
Age in days after hatching
Fig. 1. Stressor-induced increases in the plasma corticosteroid concentrations (mean k SEM) in mallard ducklings during the first month of post-natal development, and in mature male and female birds (combined values) derived from the same stock and maintained in the laboratory for 180 days prior to experimentation.
concentration until about one week after hatching (Wise and Frye, 1973). The adrenal steroidogenic tissue in the neonate chicken, however, responds readily to exogenous ACTH (Wise and Frye, 1973). Thus, as in mammals, the failure of the neonate chicken to respond to stress may reflect an immature functional status of the hypothalamo-pituitary axis rather than steroidogenic incompetence (Jailer, 1950; Levine et al., 1967; Milkovic and Milkovic, 1959, 1963). In this respect, it is interesting to note that the incubation period of the chicken is only 21 days while that of the mallard duck is 26 days. Thus, the chicken develops the capacity to respond maximally to a stressor at a time during post-natal development that corresponds to almost exactly to the developmental age of the neonate mallard duck. There is evidence to suggest that both the sympatho-m~ullary and the hypothalam~pituitary-adrenal axes are SimuitaneousIy stimulated when birds are exposed to any one of a variety of stressors (Freeman, 1983; Harvey et al., 1984; Rees et al., 1984). Thus, increases in the circulating concentrations of catecholamines, as well as corticosteroids, characterize short-term responses to stress (Brown and Nestor, 1973; El-Halawani et al, 1973; Buckland et al., 1974; Howard, 1974; Davison, 1975; Edens and Siegel, 1975; Zachariasen and Newcomer, 1975; Jurani et al., 1978). Furthermore, it has been suggested that stress-induced increases in plasma catecholamine concentrations may act to augment the stress-induced adrenal steroidogenesis. For example, intravenously administered epineph~ne, norepinephrine, P-adrenergic agonists and a-adrenergic antagonists have each been shown to potentiate the effects of both endogenous and exogenous ACTH while an a-adrenergic agonist had the opposite effect (Freeman and Manning, 1979; Rees et al., 1985). The manner in which catecholamines may act to cause increases in plasma corticosteroid concentrations, however, is not clear. One explanation may be that catecholamines increase blood flow through the adrenal glands (Harrison and Hoey, 1960) and thereby facilitate the diffusion of hormone from the steroidogenic cells. Such an effect would certainly be consistent with the very rapid increase in plasma corticosterone that occurs when birds are subjected to stress (Beuving and Vonder, 1978; Harvey et al., 1980). Another possibility is that potentiation of the hypothalamo-hypophyseal axis may occur through stimulation of /I-adrenergic receptors located in either the pituitary, as in mammals, (Tilders et ai., 1980) or the adrenal gland, as has been suggested for
Adrenal steroidogenic responsiveness
the domestic chicken (Rees et al., 1985). Studies in vitro have also suggested that direct stimulation of /3-adrenergic receptors present in cultured bovine adrenal cells may stimulate aldosterone release (McKenna et al., 1980; De Lean et al., 1984). The interpretation of these results, however, does not acknowledge the possibility that the release of aldosterone occurred following adrenergic stimulation of an intracellular renin-angiotensin system associated with the cultured cells (Ryan, 1967; Inagami et al., 1984). Also, we have been unable to identify any effect of epinephrine and nor-epinephrine on corticosteroid hormone release from superfused adrenal tissue taken from neonate mallard ducklings (Al-Ghawas, Holmes and Cronshaw, unpublished data) and Benyamina et al. (1987) have reported a failure to detect any stimulatory effect of these compounds on superfused frog adrenal tissue (unpublished data, in Benyamina et al., 1987). Finally, catecholamine-induced increases in plasma corticosterone concentrations have been attributed to a direct aminergic action on the hypothalamohypophyseal axis; in mammals fl-receptors located in the pituitary are believed to mediate this action (Axelrod and Reisine, 1984) but in birds there is some evidence for their presence in the adrenal gland (Rees et al., 1985). Clearly, the control mechanisms that regulate the stress-induced changes in adrenal steroidogenic activity are complex and have still to be defined unequivocally. For the bird, however, the experimental evidence does not seem to favor a hypothesis based on a direct stimulation of adrenal steroid hormone synthesis by catecholamines released as part of the stress reflex.
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1989
0300-9629189 $3.00 + 0.00 1989 Pergamon Press plc
CHANGES IN THE ADRENAL STEROIDOGEN~C RESPONSIVENESS OF THE MALLARD DUCK (ANAS PLATYRHYNCHOS) DURING EARLY POST-NATAL DEVELOPMENT Department
W. N. HOLMES, J. L. REWNDO and JAMES CRCJNSHAW of Biological Sciences, University of California. Santa Barbara, CA 93106, USA. Telephone: (805) 961-3511 (Received 26 July 1988)
AWract-1. Plasma ~n~nt~tions of corticosteronc (B), aldosterone (Aldo) and d~xy~r~sterone (DGC) were measured in mallard d~klin~ imm~iately before and after exposure to acute immobi~~tion stress. 2. Except for transient declines in B and DGC between the 4th and 14th days after hatching, the resting concentration of each harmone did not change significantly during post-natal development. 3. The stress-induced in Aldo was maximal at hatching while maximal increases in B and DGC did not occur until one day later. 4. Thereafter the magnitude of the stress-induced increases in the concentrations of all of the hormones decreased steadily and on the 21st and 28th days after hatching only B increased sibilantly in response to stress.
INTRODUCTION
The pattern of post-natal growth in the duck is such that the rate of increase in body weight is pro~rtionately greater than that of the adrenal glands. As a result, a sharp decline in the weight of the adrenal glands relative to the body weight occurs during the first few weeks of post-natal life. Later, this rate of decline lessens and at about six months of age the relative weight of the glands stabilize at a value ranging from onequarter to one-third of that seen in the neonate (Holmes and Kelly, 1976; Holmes and Cronshaw, 1980). As the adrenal gIands grow, however, the rate of corticosterone synthesis per unit mass of tissue decreases, and as a result a fairly steady resting plasma concentration of the hormone is maintained throughout the first six-month period of post-natal development (Holmes and Kelly, 1976). In the mature mallard duck, three corticosteroids are found in the plasma, namely corticosterone, aldosterone and 1I-deoxycorticosterone (Redondo ef al., 1988). The steroidogenic properties of the adrenal gland at hatching and during early post-natal development, however, have not been described for this species. The purpose of this study, therefore, was to determine the relative abundances of these corticosteroids in plasma at the time of hatching and to assess, in terms of stress-induced changes the corticosteroid concentrations, in plasma corticotropic responsiveness of the adrenal steroidogenic tissue in ducklings during the first month of post-natal development. MATERIALS AND
METHODS
Animals Fertilized mallard duck eggs were purchased from a commercial breeder whistling Wings. Hanover, IL) and
incubated at 37.4’C and 85% relative humidity. Newly hatched ducklings were maintained for the first 2 days 37.4”C and thereafter at 27”C, and throughout their postnatal development they were exposed to a daily photoperiod of 12hr light (08:~~:~hr) and 12hr dark. Food (Gamebird Developer, Ralston-Purina) and drinking water (tap water) were available udlibirum, and fresh supplies were provided each day at 08:OOhr. Prorocol A blood sample was taken by heart puncture within 1 min of removing a bird from its cage. The bird was then gently restrained by wrapping it in muslin gauze, and a second blood sample was taken by heart puncture 30min later. Each bird was then weighed and the recorded body weight was corrected for the mass of blood removed during sampling. The birds were considered to be unstressed when the first blood sample was taken, and at the time when the second blood sample was taken they were considered to have experienced mild stress in response to the earlier blood sampling procedure and the ensuing 30-min period of restraint. The first blood sample was taken from the ducklings that were 4 days old and older at II :00 hr, but experiments on the newly hatched and one-day-old ducklings were started at the time when they had fully emerged from the shell and 24 hr later, respectively. Each blood sample was collected in a heparinized syringe and represented approximately 1% (v/w) of the body weight. Since it was not possible to obtain a sufficiently large sample of blood from newly hatched and very young ducklings, samples taken from several birds were pooled. These pooled samples were centrifuged for 5 min at 123OOg and 5°C and stored at -20°C. To ensure that the ducklings used in these experiments wem achy disturbed, th :y were binge and handled by only one person (JLR) from the day of hatching until they were 28 days old. A group of five male and five female birds derived from the same stuck were maintained until they were 6 months old. At this time, each bird was treated in exactly the same way as the ducklings. Because of their size, however, it was possible to take two Sml blood samples from each bird. 403
W. N.
404
HOLMESet
Sample purificarion Plasma samples were thawed at room temperature and a 2.0ml aliquot of each sample was transferred to a new scintillation vial containing 8000 dpm of chromatographitally pure [i ,2-‘HI-corticosterone, (1,Z3H]-aldosterone and [I ,2-3H]-deoxycorticosterone, (Amersham Corporation, Arlington Heights, IL). The samples were allowed to equilibrate for I hr at room temperature before extraction with 10 volumes cold anhydrous ethyl ether. The ether phase was separated, dried under nitrogen, redissolved in 0.5 ml benzene:cyclohexane:methanol: water (25: 12.5 :4 : 1) and purified by gel filtration chromatography using Sephadex LH-20 and the same solvent system. The fractions containing labelled corticosterone, aldosterone and deoxycorticosterone were collected and pooled separately, evaporated by dryness under nitrogen and redissolved in 1.Oml of buffer containing 0.1 M tricine, 0.18 M NaCl and 0.015 M NaN, at pH 8, The radioactivity contained in 0.2 ml of this extract was used to determine the recovery of each steroid and the remainder of sample was stored at -20°C. Hormone analysis The concentrations of corticosterone, aldosterone and deoxycorticosterone were measured by radioimmune assay (Nishida er al., 1976; Mayes et al., 1970; Arnold and James, 1971). Corticosterone antiserum was produced in rabbits immunized with corticosterone--2~-h~isuccinyl thyroglobulin (ICN Biomedicals Inc., Costa Mesa, CA). Sensitivity for this assay was 15 pg corticosterone per assay tube, intra-assay variation was 5.0% and interassay variation was 12.8%. Aldosterone antiserum was raised in sheep with an aldosterone-3-oxine-bovine serum albumin conjugate (Arnel Products Co.. New York, NY); sensitivity was IO pg of aldosterone per assay tube and intra- and interassay variation was 3.0% and 10.3%. respectively. Deoxycorticosterone antiserum was produced in rabbits using a deoxycorticosterone-3-oxine-bovine serum albumin conjugate (Endocrine Sciences, Tarzana, CA); sensitivity was 4 pg deoxycorticosterone per assay tube and intra- and interassay variation was 8.3% and 6.8%, respectively. Statistics Ali values are expressed as means + standard error of the mean (SEM) and were compared by the Student r-test (Sokal and Rohlf, 1969). Regressions were fitted by the method of least squares.
RESULTS Body weights The mean body weight of the ducklings upon emergence from the egg was 40.3 _C0.89 g (N = 70); this value represented about 70% of the mean weight of the fertilized eggs at the start of incubation. Immediately after hatching, the body weights of the ducklings started to decline and after 24 hr the mean body weight was significantly less than at hatching (22.1 It 1.4 g, N = 40, P -C0.01). This decline in body weight continued for at least the next 3 days and on the 4th day the mean weight was still significantly lower than it was at hatching (32.0 k 0.9 g, N = 30, P c 0.01). After the 4th day, however, the ducklings started to increase in weight and this continued in a linear fashion until the end of the experimental period on the 28th day after hatching; the average daily rate of increase during this interval was 12.1 f 0.38 g (r = 0.97, P < 0.01 with 77 degrees of freedom) and the mean body weight of the ducklings on the 28th
al.
day after hatching was 237.8 + 10.7g (N = 5). Between the 28th and 180th day after hatching the body weights of the birds continued to increase at an average rate of approximately 5 g per day, and at 180 days the mean body weights of the males and the females were 1052f 35 (N = 5) and 973+45g (n = 5), respectively. Plasma corticosteroid concentrations Before exposure to stress (Tabfe 1). During the first 4 days after hatching the plasma concentration of corticosterone remained constant and during this period the mean value was 11.2 + 1.2 ng per ml (N = 17). By the end of the first week, however, the concentration of corticosterone had declined significantly and it remained at this low level until at least the 14th day after hatching. Thereafter, the concentration increased, and at 3 and 4 weeks of age it was not significantty different from the concentration recorded at hatching. The plasma concentration of aldosterone increased almost two-fold during the first 24 hr after hatching and it did not vary significantly during the ensuing 4 weeks. The pattern of post-natal change in plasma deoxycorticosterone concentration was qualitatively similar to that of corticosterone; a significantly lower concentration compared with that measured on the 1st day was recorded on the 4th day after hatching (P < 0.01 with 8 degrees of freedom), and significantly lower concentrations persisted until at least the 14th day after hatching, The mean concentrations of corticosterone, aldosterone and deoxycorticosterone recorded in the group of five mature unstressed males (180 days old) did not differ significantly from the corresponding values recorded for the group of five unstressed females of the same age (Table 2). Neither the mean concentration of each hormone recorded in either the males or the females, nor the combined mean values recorded for both the males and the females, differed significantly from the corresponding values measured in the 4-week-old ducklings (cf. Tables 1 and 2). After exposure to stress (Table 1). The plasma concentrations of all three hormones in al1 of the age groups up to and including the tCday_old ducklings increased significantly during the 30 min period of restraint. Exposure to stress also caused the corticosterone concentration, but not the aldosterone and deoxycorticosterone concentrations, to increase significantly in the 21- and 28-day-old ducklings. In the mature male and female birds, however, significant stress-induced increases in both corticosterone and aidosterone concentrations were recorded, but a significant stress-induced increase in the plasma deoxycorticosterone concentration was recorded only in the mature males (cf. Tables 1 and 2). In general, the stress-inducible increases in plasma corticosteroid concentrations were maximal at or soon after hatching, and for each hormone the mag nitude of the increase declined steadily during the ensuing weeks of post-natal development (Fig. 1). During the first 24 hr after hatching the stressinduced change in the plasma corticosterone concentration increased significantly (P < 0.05 with 10 degrees of freedom) by approximately one-fifth.
Adrenal Table
steroidogenic
responsiveness
405
I. The plasma corticosteroid concentrations (mean It SEM) in mallard ducklings exoosure to a 30-min wriod of gentle immobilization stress Corticosteroid
--___-
concentration
Corticosterone Age (days)
f5.1)
Prestress
-- stress
II.7 k2.3
58.4tt I: 1.7
11.6NS If-I.8
66.8tt 23.5
10.3NS f1.7
fng/ml plasma)
Aldosterone Prestress -~______ 0.234 i 0.020
___-
~oxycorticosterone Prestress
Stress
I .742tt k 0.270
0.472 + 0.093
I s05tt k 0.069
0.460’ f 0.074
I .704tt *0.140
0.412NS * 0.034
f0.117
&.6W t2.9
0.362NS io.064
1.soztt to.137
0.298NS & 0.028
I .349tt f0.082
5.8’ * 1.7
46.3tt +4.2
0.427NS +0.111
1.464tt kO.070
0.236. *0.043
zhO.124
6.8’ +0.4
32.0tt k4.0
0.423* +0.056
0.978t 20.137
0.200. f 0.033
0.645t +0.107
9.3NS k2.6
25.1t k5.l
0.454’ + 0.068
0.728ND &0.168
0.33lNS kO.141
OS98ND io.200
10.3NS + 2.9
29.7tt c3.5
0.403* +0.138
0.580ND &O.lOb
0.404NS &0.078
0.63lND +0.152
2.064tt
1.164tt
Plasma samples taken from newly hatched ducklings (age = 0 days) and at I, 4, 7, 14, 21 and 28 days after hatching, The first number in parentheses indicates the number of pooled plasma samples analysed to determine the mean concentration, and the second number represents number of birds that contributed to each pooled blood sample. lP -c 0.05 and NS = not significant with respect to the corresponding value in newly hatched ducklings (age = day 0). tiP -c 0.01, tP < 0.05 and ND = not significantly different from the corresponding value recorded prior to exposure to stress.
Between the 1st and the 21st days after hatching, however, the magnitude of the inducible increase declined at an average daily rate of 1.64 + 0.33 ngiml (r = 0.73, P < 0.01 with 21 degrees of freedom). On the 28th day after hatching the inducible increase in plasma corticosterone concentration was the same as that observed on the 21st day and similar to that seen in the male and female adult birds; the mean inducible increases measured in the 21-, 28- and 180-day-old birds (19.0 i 2.2 ng per ml plasma, IV = 19) being only one-third of the maximum inducible response measured on the 1st day after hatching. A maximal stress-inducible increase in plasma aldosterone concentration occurred in the newly hatched ducklings, and this degree of responsiveness was sustained until at least the 4th day after hatching (Fig. 1). Between hatching and the 4th day of postnatal development, the mean inducible increase was 1.410 + 0.125 ng/ml plasma (N = 17). After the 4th day the stress-inducible increase in plasma aldosterone concentration started to decline at a mean rate of 0.05 k 0.008 nglmliday (r = 0.79, P < 0.01 with 21 degrees of freedom) and on the 21st and the 28th days after hatching no significant increase was detectable (Fig. 1). During the course of maturation, however, the stress-inducible increase in plasma aldosterone concentration was partially restored, and at 180 days of age, mean increases of 0.927 + 0.173 ng per ml plasma (N = 10) were recorded; this value was significantly higher than that recorded in the 28-day-old ducklings (P < 0.01 with 13 degrees of freedom) and represented about 60% of the value recorded at hatching. The pattern of change in stress-inducible increases in deoxycorticosterone concentrations roughly followed that of corticosterone (Fig. 1). The inducible
increase was maximal on the 1st day after hatching (1.652 f 0.099 ng per ml plasma, N = 5) and declined thereafter at 0.047 f 0.007 ng/mI/day (I = 0.79, P < 0.01 with 27 degrees of freedom) until no significant inducible response was detectable on the 21st and the 28th days. In the mature birds, however, a small but significant inducible increase in plasma deoxycorticosterone concentration was detectable in the male, but not the female, birds (Table 2).
DISCUSSION
Regular daily patterns of increase and decrease in resting plasma corticosterone concentrations have been observed in several species of birds (Boissin and Assenmacher, 1968; Chan and Phillips, 1973; ElHalawani ef at., 1973; Gorsline and Holmes, 1981). Indeed, in the duck, such a pattern of change is already established at the time of hatching (Weiss et al., 1977), and thereafter one or more zeitgebers, such as the daily photoperiod, the diurnal cycle of activity and the feeding regimen, sustain and entrain this cyclicity. There is also reason to believe that the highest plasma concentrations of corticosterone that occur during the diurnal cycle reflect rates of hormone synthesis that may be close to maximal for the adrenal steroidogenic tissue (El-Halawani et al., 1973). Thus, if a bird is exposed to a particular stressor when the resting level is maximal, the stressinduced increase in cortocosterone concentration may be minimal whereas the response may be maximal during the nadir. It is for these reasons, therefore, that the birds used in the present experiments were maintained on a standardized feeding and watering regime, were exposed to a constant daily photoperiod and were subjected to stress at a time close to the
W. N. HOLMESet al.
406
Table 2. The plasma corticosteroid concentrations (mean It SEM) in 160.day old mature male and female mallard ducks immediately before and after their exposure to a 30 min period of gentle immobilization stress
Males (5)
Corticosterone _--__ Stress ___^_~ 8.1 30.2tt 22.5 k3.8
Females (5)
9.8 f 3.0
Combined (W
8.8 f2.0
Prestress
Aldosterone _---_--Prestress stress
Deoxycorticosterone Prestress
stress
0.239 kO.042
1.341tt i0.273
0.431 *0.107
0.9w rtO.132
27.2tt + 2.6
0.265 kO.042
0.931tt io.131
0.520 fO.115
0.755ND kO.144
28.9tt f2.4
0.250 kO.037
1.177t.t *0.191
0.472 +0.080
0.837t rto.099
ttP< 0.01, fP x 0.05and ND = not significant wilh respect to the corresponding value recorded before exposure to stress
estimated nadir that occurs at about middle of the light phase (Gorsline and Holmes, 1981). The rapid and profound increases in the plasma corticosteroid concentrations that occurred when the newly hatched ducklings were subjected to stress indicate that the mechanisms which regulate the synthesis and release of these hormones are already well-established at the end of embryogenesis. This is in marked contrast to the domestic chicken which shows no stress-induced rise in plasma corticosterone
Corticosterone 50
40 30 20 IO fi u
0
1
4
7
14
21
28
180
Aldosterone 21
4
0
-a
E
2
1
4
7
14
21
28
180
Deoxycorticosterone 21
0
1
4
7
14
21
28
180
Age in days after hatching
Fig. 1. Stressor-induced increases in the plasma corticosteroid concentrations (mean k SEM) in mallard ducklings during the first month of post-natal development, and in mature male and female birds (combined values) derived from the same stock and maintained in the laboratory for 180 days prior to experimentation.
concentration until about one week after hatching (Wise and Frye, 1973). The adrenal steroidogenic tissue in the neonate chicken, however, responds readily to exogenous ACTH (Wise and Frye, 1973). Thus, as in mammals, the failure of the neonate chicken to respond to stress may reflect an immature functional status of the hypothalamo-pituitary axis rather than steroidogenic incompetence (Jailer, 1950; Levine et al., 1967; Milkovic and Milkovic, 1959, 1963). In this respect, it is interesting to note that the incubation period of the chicken is only 21 days while that of the mallard duck is 26 days. Thus, the chicken develops the capacity to respond maximally to a stressor at a time during post-natal development that corresponds to almost exactly to the developmental age of the neonate mallard duck. There is evidence to suggest that both the sympatho-m~ullary and the hypothalam~pituitary-adrenal axes are SimuitaneousIy stimulated when birds are exposed to any one of a variety of stressors (Freeman, 1983; Harvey et al., 1984; Rees et al., 1984). Thus, increases in the circulating concentrations of catecholamines, as well as corticosteroids, characterize short-term responses to stress (Brown and Nestor, 1973; El-Halawani et al, 1973; Buckland et al., 1974; Howard, 1974; Davison, 1975; Edens and Siegel, 1975; Zachariasen and Newcomer, 1975; Jurani et al., 1978). Furthermore, it has been suggested that stress-induced increases in plasma catecholamine concentrations may act to augment the stress-induced adrenal steroidogenesis. For example, intravenously administered epineph~ne, norepinephrine, P-adrenergic agonists and a-adrenergic antagonists have each been shown to potentiate the effects of both endogenous and exogenous ACTH while an a-adrenergic agonist had the opposite effect (Freeman and Manning, 1979; Rees et al., 1985). The manner in which catecholamines may act to cause increases in plasma corticosteroid concentrations, however, is not clear. One explanation may be that catecholamines increase blood flow through the adrenal glands (Harrison and Hoey, 1960) and thereby facilitate the diffusion of hormone from the steroidogenic cells. Such an effect would certainly be consistent with the very rapid increase in plasma corticosterone that occurs when birds are subjected to stress (Beuving and Vonder, 1978; Harvey et al., 1980). Another possibility is that potentiation of the hypothalamo-hypophyseal axis may occur through stimulation of /I-adrenergic receptors located in either the pituitary, as in mammals, (Tilders et ai., 1980) or the adrenal gland, as has been suggested for
Adrenal steroidogenic responsiveness
the domestic chicken (Rees et al., 1985). Studies in vitro have also suggested that direct stimulation of /3-adrenergic receptors present in cultured bovine adrenal cells may stimulate aldosterone release (McKenna et al., 1980; De Lean et al., 1984). The interpretation of these results, however, does not acknowledge the possibility that the release of aldosterone occurred following adrenergic stimulation of an intracellular renin-angiotensin system associated with the cultured cells (Ryan, 1967; Inagami et al., 1984). Also, we have been unable to identify any effect of epinephrine and nor-epinephrine on corticosteroid hormone release from superfused adrenal tissue taken from neonate mallard ducklings (Al-Ghawas, Holmes and Cronshaw, unpublished data) and Benyamina et al. (1987) have reported a failure to detect any stimulatory effect of these compounds on superfused frog adrenal tissue (unpublished data, in Benyamina et al., 1987). Finally, catecholamine-induced increases in plasma corticosterone concentrations have been attributed to a direct aminergic action on the hypothalamohypophyseal axis; in mammals fl-receptors located in the pituitary are believed to mediate this action (Axelrod and Reisine, 1984) but in birds there is some evidence for their presence in the adrenal gland (Rees et al., 1985). Clearly, the control mechanisms that regulate the stress-induced changes in adrenal steroidogenic activity are complex and have still to be defined unequivocally. For the bird, however, the experimental evidence does not seem to favor a hypothesis based on a direct stimulation of adrenal steroid hormone synthesis by catecholamines released as part of the stress reflex.
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